Evaluation and Clinical Analyses of Downstream Targets of the … · Predictive Biomarkers and Personalized Medicine Evaluation and Clinical Analyses of Downstream Targets of the

Predictive Biomarkers and Personalized Medicine

Evaluation and Clinical Analyses of Downstream Targets ofthe Akt Inhibitor GDC-0068

Yibing Yan1, Violeta Serra6, Ludmila Prudkin7, Maurizio Scaltriti12, Sumati Murli2, Olga Rodr��guez6,Marta Guzman6, Deepak Sampath3, Michelle Nannini3, Yuanyuan Xiao4, Marie-Claire Wagle1, Jenny Q. Wu1,Matthew Wongchenko1, Garret Hampton1, Vanitha Ramakrishnan2, Mark R. Lackner1, Cristina Saura8,10,Desamparados Roda11, Andr�es Cervantes11, Josep Tabernero6,9,10, Premal Patel5, and Jos�e Baselga6,12,13

AbstractPurpose: The oncogenic PI3K/Akt/mTOR pathway is an attractive therapeutic target in cancer. However,

it is unknown whether the pathway blockade required for tumor growth inhibition is clinically achievable.

Therefore,we conductedpharmacodynamic studieswithGDC-0068, anATP competitive, selectiveAkt1/2/3

inhibitor, in preclinical models and in patients treated with this compound.

Experimental Design:We used a reverse phase protein array (RPPA) platform to identify a biomarker

set indicative of Akt inhibition in cell lines and human-tumor xenografts, and correlated the degree of

pathway inhibition with antitumor activity. Akt pathway activity was measured using this biomarker set

in pre- and post-dose tumor biopsies from patients treated with GDC-0068 in the dose escalation

clinical trial.

Results: The set of biomarkers of Akt inhibition is composed of 10 phosphoproteins, including Akt and

PRAS40, and is modulated in a dose-dependent fashion, both in vitro and in vivo. In human-tumor

xenografts, this dose dependency significantly correlated with tumor growth inhibition. Tumor biopsies

from patients treated with GDC-0068 at clinically achievable doses attained a degree of biomarker

inhibition that correlated with tumor growth inhibition in preclinical models. In these clinical samples,

compensatory feedback activation of ERK andHER3was observed, consistent with preclinical observations.

Conclusion: This study identified a set of biomarkers of Akt inhibition that can be used in the clinical

setting to assess target engagement. Here, it was used to show that robust Akt inhibition in tumors from

patients treated with GDC-0068 is achievable, supporting the clinical development of this compound in

defined patient populations. Clin Cancer Res; 19(24); 6976–86. �2013 AACR.

IntroductionThe PI3K/Akt/mTOR signaling pathway plays a critical

role in cell growth, survival, and metabolism (1). Its aber-

rant activation, either bymutations of thePIK3CA gene, lossof PTEN expression or amplification/mutation of upstreamreceptor tyrosine kinases (RTK), is frequent in humancancers and may drive tumor growth as well as limit sen-sitivity to targeted therapies (2–7). Therefore, pharmaco-logic inhibitors of the PI3K/Akt/mTOR pathway are enter-ing the clinic at a rapid pace. Although early-phase clinicalstudies have shown that a subset of patients derives benefitfrom treatment with these agents, a number of factors couldpotentially limit their clinical activity. These include treat-ment of diagnostically unselected patient populations,insufficient target inhibition, and/or the activation of com-pensatory pathways leading to resistance (8–11). Therefore,approaches are needed that enable a comprehensive mea-surement of both target inhibition and activation of com-pensatory pathways in tumors treated with a targeted ther-apy that can be translated to the clinical setting (12). Inaddition, there is a need to identify the dose and schedulefor each agent that result in meaningful target and pathwayinhibition in patients. These parameters would have clearimplications for the clinical development of targeted ther-apies, and for future rational combination strategies toimprove efficacy.

Authors' Affiliations: 1Oncology Biomarker Development, 2Portfolio Man-agement and Operations, 3Translational Oncology, 4Biostatistics, and5Exploratory Clinical Development, Genentech Inc., DNA Way, South SanFrancisco, California; 6Experimental Therapeutics, 7Molecular Pathology,8Breast Cancer and Melanoma, 9Gastrointestinal and Endocrine TumorsGroups, and 10Medical Oncology Service, Vall d'Hebron Institute of Oncol-ogy, Barcelona; 11Department of Hematology and Medical Oncology, Insti-tute of Health Research INCLIVA, University of Valencia, Valencia, Spain;and 12Human Oncology and Pathogenesis Program and 13Department ofMedicine, Memorial Sloan-Kettering Cancer Center, New York, New York

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

Yibing Yan and Violeta Serra contributed equally to this work.

Current address for S. Murli: University of Chicago Comprehensive CancerCenter, 5841 S Maryland Avenue, Chicago, IL 60637.

Corresponding Author: Jos�e Baselga, Memorial Sloan-Kettering CancerCenter, 1275 York Avenue, New York, NY 10065. Phone: 1-212-639-8000;Fax: 1-212-794-3182; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-13-0978

�2013 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 19(24) December 15, 20136976

on February 9, 2020. © 2013 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst October 18, 2013; DOI: 10.1158/1078-0432.CCR-13-0978

http://clincancerres.aacrjournals.org/

Among the different nodes in the PI3K/Akt/mTOR path-way, Akt1/2/3 occupy a central hub with distinct cellularfunctions (13), and their pharmacologic blockade down-regulates signalingmediated by diverse upstream activatingevents (14). Two classes of inhibitors are currently inclinical development: catalytic, ATP-competitive inhibitors(such as GDC-0068) that preferentially target the activekinase, and allosteric inhibitors (such as MK-2206) thatbind to the Akt plekstrin-homology domain, resulting in aconformational change that prevents Akt membrane local-ization and subsequent activation (15, 16). GDC-0068, apotent, orally bioavailable, selective pan-Akt inhibitor, wasidentified by structure-guided drug design (17). The enzy-matic IC50 values of GDC-0068 range from 5 to 30 nmol/Lfor the three isoforms of Akt (18). Cancer cell lines withactivated Akt signaling (e.g., via PTEN loss, PIK3CA muta-tions orERBB2 amplification) are sensitive toAkt inhibition(19, 20). Thepreferential bindingofGDC-0068 to the activeconformation of Akt likely contributes to this enhancedsensitivity (21).To identify pharmacodynamic biomarkers that measure

the optimal inhibitory effects of GDC-0068 in cell lines andxenograft models, we used a reverse-phase protein array(RPPA) platform (22) to conduct a focused search within apanel of 100 validated proteins involved in oncogenicsignaling pathways.We identified tenphosphoproteins thatconstituted a set of core biomarkers of GDC-0068 pharma-cologic activity. The coordinate regulation of these biomar-kers in tumor cells from patients treated with GDC-0068confirmed substantial Akt pathway suppression at well-tolerated doses in a phase I study. The identification ofbiologically active doses of GDC-0068 has enabled thedesign of rational starting doses for phase Ib combinationtrials. In addition, we observed increased feedback activa-tion of HER3 and ERK in clinical samples following GDC-

0068 treatment, providing clinical validation of resultsfrom tumormodels (23, 24). This approach could thereforebe used to monitor pharmacodynamic response to targetedtherapy in tumor biopsies and lead to rational drugcombinations.

Materials and MethodsCell lines, cell culture, and treatments

All cell lines were purchased fromAmerican Type CultureCollection and cultured as described in its database. Thetrastuzumab-resistant cell line BT474-Tr was obtained bychronic exposure to trastuzumab as described (25). GDC-0068 was provided by Genentech and rapamycin waspurchased at Sigma. For in vitro experiments, both reagentswere diluted in dimethyl sulfoxide (DMSO).

Reverse phase protein arraysCell and xenograft tumor sampleswere directly lysed in T-

PER (PIERCE) based lysis buffer containing protease inhi-bitors and phosphatase inhibitors. Paired (pre- and post-treatment) tumor biopsies from patients were flash-frozenin optimum cutting temperature (OCT formulation, SakuraFinetek) and sectioned to 6 mm. Proteins were extractedfrom the tissue sections with T-PER–based lysis buffer andcombined for the assay. Protein lysates were printed onnitrocellulose-coated glass slides, with each sample spottedon four different locations. A set of assay control sampleswas also printed on each slide. Total protein concentrationin each spot was assessed by Sypro staining. The slides wereblocked and then stained against 100 total protein andphospho-specific RPPA-validated antibodies. The intensityof specific antibody staining was subtracted from the inten-sity of secondary antibody alone and was then normalizedto total protein concentrations. The specific intensity isnormalized to the median intensity of all signals on theslides to remove potential spatial effects. For methods andoverview of RPPA, see ref. (26).

Formore informationonWesternblot analysis and immu-nohistochemistry (IHC), see Supplementary information.

Tumor modelsMice were maintained and treated in accordance with

institutional guidelines of Vall d’Hebron University Hospi-tal or Genentech Institutional Animal Care and Use Com-mittee. Six-week-old female athymic nude-Foxn1nu micewere purchased from Harlan Laboratories or NCR nudefemale mice from Taconic. Mice were housed in air-filteredlaminar flow cabinets with a 12-hour light cycle and foodand water ad libitum. Mice were handled with aseptic pro-cedures and allowed to acclimatize to local conditions for 1week before the experimental manipulations. A 17b-estra-diol pellet (Innovative Research of America) was implantedsubcutaneously into each mouse 1 day before cell injectionof BT474-Tr cells. A total of 107 BT474-Tr or PC3 cells wereresuspended in PBS and injected subcutaneously into theright flank of each mouse in 200 mL of final volume.Treatments began when tumors reached an average size of125 to 350 mm3 and were thus considered as established

Translational RelevanceThere is a strong rationale to therapeutically target the

oncogenic PI3K/Akt/mTOR axis in cancer, yet it isunknown whether the degree of blockade of Akt signal-ing required for tumor growth inhibition is clinicallyachievable. Using reverse-phase protein arrays, we firstanalyzed the impact of a novel catalytic Akt inhibitor,GDC-0068, on a panel of 100 signaling proteins inmultiple cell lines and tumor models. We derived a setof 10 proteins as biomarkers of Akt inhibition andsubsequently studied their behavior in pre- and post-treatment tumor biopsies from patients participating inthe GDC-0068 dose-escalation phase I clinical trial. Thisapproach allowed the identification of biologicallyactive doses of GDC-0068 to support moving forwardinto phase Ib/II combination studies. In addition, sig-naling feedback observed in on-treatment biopsies pro-vided a rationale for the clinical development of Aktinhibitors in combination with targeted therapeutics.

Biomarkers Demonstrating Akt Inhibition

www.aacrjournals.org Clin Cancer Res; 19(24) December 15, 2013 6977




growing xenografts. Mice were treated once daily withvehicle (0.5% methylcellulose/0.2% Tween-80) or GDC-0068 by oral gavage. Tumors were removed 2, 9, and 24hours following 3 daily administrations and at 2 hours atend of study (11–18 days). Tumor xenografts were mea-sured with calipers three times a week and tumor volumewas determined using the formula: (length � witdth2) �(p/6). Tumor growth inhibition was calculated using thefollowing equation: TGI ¼ [1-(xtreated (final))-xtreated(day1))/(xcontrol (final)-xcontrol (day1))] � 100%, where xis mean tumor volume.

Patient samplesPatients included in the dose escalation NCT01090960

trial were biopsied before treatment initiation (pre) andafter 2/3 weeks of receiving GDC-0068 (Post). Two 18Gcore biopsies were taken at each time point. One core-biopsy was immediately embedded in OCT and snap-fro-zen for RPPA analysis. The second core-biopsywas formalinfixed and paraffin-embedded for IHC studies. Frozen biop-sies in OCT were sectioned at 6 mm thickness, and eachsection was stained briefly with hematoxylin to identify thehistorical features. Tumor cells or adjacent stromal cellswere laser-captured separately before processing for theRPPA on an Arcturus LCMworkstation (Life Technologies).The mutation status was determined as in ref. (27).

Statistical analysisThe RPPA data were processed using Partek Genomic

Suite 6.5 (Partek Inc.). Specific intensity data were logtransformed and significant changes in protein levels ineach cell linewere testedwith two-wayANOVA, consideringthe treatment time and concentration. The significantchanges in all cell lines were tested by three-way ANOVAtaking into account cell line, time, and concentration oftreatment. For the RPPA data from in vivo models andclinical biopsies, two-way ANOVA was used to identify thedifferences in subject (mouse or patient) and treatmenteffects. The significant changes were defined as more than1.6-fold with adjusted P < 0.05. The false discovery rate(FDR)was controlled by adjusting the P values with the stepup method (28).

The Dunnett test was used to calculate significance forxenograft TGI data.

The Kruskal–Wallis test was used to assess immunohis-tochemical expression differences between treatmentgroups. P values of <0.05 were considered significant. Datawere processed using PASW Statistics v17.

ResultsBiomarkers of catalytic AKT inhibition and cell survival

To identify molecular biomarkers of catalytic Akt inhi-bition, we used RPPA to analyze the effects of the Aktinhibitor GDC-0068 on the primary endothelial cell line(HuVEC), and three tumor cell lines with activated PI3K-pathway signaling (MCF-7, PC-3, and U87; Fig. 1A andSupplementary Table S1). One hundred total protein andphospho-specific antibodies of proteins, all of them pre-

viously validated in the RPPA platform and broadly repre-senting the major oncogenic pathways, were evaluated.The platform allowed quadruple measurements of all end-points in multiple dilutions in a single experiment. Themultiple RPPAmeasurements enabled analysis of variance(ANOVA) determination across the concentration-depen-dent inhibition profiles. Throughout the experiments, weobserved that changes above 1.6-fold over the mean of thecontrol group were consistently 2-fold above the highestSD of control signals across all technical repeats. Therefore,the significant change was defined as more than 1.6-foldchanges over themean control groupwith an FDR adjustedP value of <0.05. In this unbiased analysis, a set of tenbiomarkers were consistently modulated in all cell linesand were thus taken to constitute a core biomarker set ofcatalytic Akt inhibition (Fig. 1B and Supplementary TableS2). Among them, Akt S473, Akt T308, and PRAS40 T246phosphorylation levels were found to be regulated at thelowest concentration of GDC-0068 tested (0.01 mmol/L)after 3-hour treatment in all four cell lines. Phosphoryla-tion of Akt (S473 and T308) increased after treatment withGDC-0068, an event previously described for ATP com-petitive Akt inhibitors (17, 29). The modulation of pAktand pPRAS40, together with other proteins comprising thebiomarker signature of Akt inhibition, showed similardose-dependent effects by Western blot analysis at both6 and 48 hours in the three cancer cell lines analyzed byRPPA as well as in breast cancer cell lines without intrinsicactivation of the PI3K pathway (Fig. 1C, SupplementaryFig. S1, and data not shown). The effects observed on themajority of the biomarkers comprising the signature werecomparable by RPPA andWestern blot analyses but certainbiomarkers, such as eIF4G and mTOR, were more clearlydephosphorylated in the RPPA analyses. By placing theseidentified biomarkers in the context of known biochemicalinteractions of the Akt pathway, our approach reveals thatGDC-0068 causes both accumulation of pAkt due toinhibitor-induced stabilization of an Akt active-like con-formation that restricts phosphatase accessibility (17, 29),as well as a decrease in the phosphorylation of severaldirect Akt substrates i.e., PRAS40, GSK3b, mTOR, eIF4G,ATP citrate lyase, and FOXO3a (Fig. 1D). Downstream ofmTOR, we also observed a profound reduction of phos-phorylated ribosomal protein S6 (pS6). We conclude thatthis group of proteins constitutes the main effectors ofpharmacologic Akt inhibition by GDC-0068.

We next examined the modulation of the 100 biomarkerpanel in relation to the survival of cells treated with GDC-0068 at 3 and 24 hours posttreatment. Pearson (linear) andSpearman (nonlinear) correlations of the individual bio-markers revealed that each cell line was represented by adistinct biomarker set, but the core 10-biomarkers wererepresented across all cell lines (Supplementary Table S3).We next queried whether the 10-biomarker set correlatedwith cell survival in each cell line upon treatmentwithGDC-0068 (30), for which the RPPA data was subjected toprincipal component analysis (PCA), providing a low-dimension representation of the biomarkers in response

Yan et al.

Clin Cancer Res; 19(24) December 15, 2013 Clinical Cancer Research6978




to the treatment (Supplementary Fig. S2A). The PCA bio-marker profile derived from the ten core markers wasdistinct for each cell line and correlated well to fractionalsurvival even though each cell line responded differently toGDC-0068 (Supplementary Fig. S2B). These results collec-tively suggest that the 10 core markers reflect the mainbiologic effect of GDC-0068 in modulating cell survival ina cell-specific manner.

Selective effects on cell signaling by Akt inhibitionwithGDC-0068Thebroadproteomic profiling approach allows adetailed

characterization of the pharmacodynamic consequences ofAkt inhibition. To test the specificity of our biomarker set,we compared it with the one elicited by allosteric mTORC1inhibition. For this comparison, we chose rapamycin sincemTOR is a clinically validated target. The phosphorylationevents significantly regulated in all four cell lines by either

agent or by both agents are depicted in Fig. 2A. Both GDC-0068 and rapamycin inhibited the common Akt effectorsmTOR, eIF4G, and S6 proteins. However, GDC-0068 addi-tionally modulated pFOXO3a, pGSK3b, and pATP citratelyase levels, highlighting the prominent role of Akt inregulating the survival response to the metabolic state oftumor cells.

In addition, this 100-biomarker RPPAprofiling followingGDC-0068 treatment revealed feedback activation of HER3and ERK signaling following Akt blockade at early timepoints after exposure (Fig. 2B). Increased expression andphosphorylation of HER3 after treatment with GDC-0068were confirmed byWestern blotting (Fig. 2C). The observedcrosstalk in signaling pathways and activation of upstreamsignaling following Akt inhibition by GDC-0068 is consis-tent with observations with other PI3K/Akt/mTOR inhibi-tors (23, 24). The specificmodulation of Akt T308 and S473by GDC-0068 was not coincident with the previously

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AFigure 1. A profile of Akt inhibitionon cell signaling by the ATP-competitive inhibitor GDC-0068. A,spectrum of changes in proteinphosphorylation in four cell linestreatedwithGDC-0068 as revealedby RPPA. The results were fromlysates of cells treated with GDC-0068 at 0.03, 0.1, 0.3, 1, and 3mmol/L for 3 hours in culture.Intensities of RPPA signalnormalized with total proteinconcentration were plotted as thelog2 ratio to the DMSO control ineach cell line. Blue, the negativeintensity showing a decreaseversus control. Red, the positiveintensity showing an increaseversus control. B, Venn diagramshows a number of significantlymodulated cell signaling events ineach cell line by one-way ANOVAover five concentrations of GDC-0068. The 10 events at theintercept of the four cell lines areconsidered as the biomarker coremodulated by Akt inhibition. C, the10 biomarkers were validated byWestern blot analysis. PC3, U87,andMCF7.1 cells were treatedwithGDC-0068 (0, 0.2, 1, and 5 mmol/L)for the indicated times. D, anetwork diagram of core nodesinhibited by GDC-0068.Significance was defined by two-way ANOVA (P < 0.05; adjusted forFDR). The nature of interactionsbetween each node was derivedfrom the database on theexperimental evidence ofinteractions as compiled by theString-db.org. Blue, decrease overcontrol. Red, increase over control.Scale, the intensity ratio to controlDMSO treatment. Straight line,direct phosphorylation. Dottedlines, indirect phosphorylation.






observed feedback activation of Akt following rapamycintreatment, with the latter occurring at later time points (48hours after exposure, data not shown; refs. 17, 31). Thus,RPPA analyses enabled the characterization of specificdownstream signaling effects associated with the inhibitionof Akt by GDC-0068 and, furthermore, enabled the char-acterization of feedback upregulation, providing supportfor rational combination strategies.

In vivo dose responsive inhibition of core biomarkersby GDC-0068 and correlation with tumor response

In preparation for the clinical evaluation of GDC-0068,we studied the effects of Akt inhibition in cancer cellxenografts treated with GDC-0068. Catalytic Akt inhibitionresulted in dose-dependent tumor growth inhibition in twoxenograft models with increased PI3K pathway activity,ERBB2-amplified, PIK3CA-mutant BT474-Tr, and PTEN-null PC3 (Fig. 3A).Wefirst interrogated the dose-dependentmodulation of the three biomarkers that were most signif-icantly correlated to GDC-0068 dosing and in vitro anti-proliferative response in the BT474-Tr xenograftmodel (Fig.3B, Supplementary Fig. S3, and data not shown). pAkt T308and S473 levels were significantly enhanced at the two

highest doses (22 and 88 mg/kg) across all time pointsstudied (namely, three time points after the third daily doseand one time point after 18 days). Although PRAS40 T246also showed a dose-dependent pharmacodynamic modu-lation, in contrast to pAkt, it also showed a time-dependentmodulation, with greater inhibition at earlier time pointsafter drug administration (Supplementary Fig. S3). Theseobservations support the importance of appropriate selec-tion of time points for optimal clinical biopsy collection(i.e., 2–4 hours following compound administration) andhighlight the potential importance of dose and schedulewith this compound.

Analyses of the tumors by RPPA revealed that a set of 19biomarkers was significantly, dose-dependently modulat-ed by GDC-0068 in the BT474-Tr model (Fig. 3C). Eightof these were present in the in vitro-derived core biomark-er set shown in Supplementary Table S2 (pATP citratelyase and pS6 S235 were not tested). Several phosphor-ylation events in the Ras/Raf/MEK/ERK pathway showeddistinct regulatory patterns culminating in the upregula-tion of Erk T202/Y204. Furthermore, consistent with ourin vitro data, dose-dependent upregulation of HER3 wasalso observed (Fig. 3C). These data demonstrate thatGDC-0068 significantly modulates the Akt pathway invivo, as well as elicited feedback activation of the HER3and ERK pathways. The quantitative nature of RPPAmeasurements made it possible to assess the relationshipbetween biomarker changes and both dose of treatmentand tumor growth inhibition. Because of the complexrelationship between individual markers and inhibitionof tumor growth, we used a biomarker model based onthe PCA scores for the ten core biomarkers that we hadidentified in vitro (Fig. 1D and Supplementary Fig. S2).The in vivo BT474-Tr and PC3 models showed similarbiomarker behavior (Fig. 3D, PCA loading profiles) aswell as significant correlation between the biomarker setscore (PCA score) and tumor growth inhibition (%TGI).Consistent with our results in vitro, the core biomarkersreflect the effect of GDC-0068 on tumor growth in vivo.The PCA model thus provides a holistic approach tocharacterize the correlation between quantitative changesin core biomarkers and tumor growth inhibition.

Modulation of biomarkers of Akt inhibition in tumorsfrom patients treated with GDC-0068

As part of the first in human phase I clinical trial ofGDC-0068 in patients with advanced solid tumors(NCT01090960; ref. 32), we incorporated a number ofprespecified on-study tumor biopsies to determine theclinical feasibility of inhibiting Akt at levels that corre-lated with growth inhibition in preclinical models.Tumor biopsies were obtained pre-treatment and 2 to 4hours post treatment after 8 to 15 days of continuousdaily dosing starting from the 100 mg/day dose cohort,which achieved equivalent plasma exposures to the 12.5mg/kg dose in mouse xenograft models (Fig. 3A). Wecarried out RPPA analyses on protein lysates from pairedpretreatment and on-treatment biopsies taken between 2

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Figure 2. Selective modulation of signaling network by the Akt inhibitorGDC-0068. A, selective inhibition of nodes in the signaling network byGDC-0068 and rapamycin. Significant inhibited nodes in all four testedcell lines by both inhibitors are shown on the interactive network. Thecommon effectors of Akt inhibition byGDC-0068 andmTORC1 inhibitionby rapamycin are in pink; nodes preferentially inhibited by GDC-0068 arein orange and by rapamycin in green. B, concurrent with inhibition of Aktdownstream signaling events such as phosphorylation of FOXO3a andGSK3b, pErk andHER3were upregulated in cells treatedwith GDC-0068(0.03, 0.1, 0.3, 1, and 3 mmol/L for 24 hours). Intensities of proteins wereexpressed as log2 ratio to the same protein in the DMSO treated cells. C,upregulation of HER3 phosphorylation and HER3 protein in MCF7.1 andBT474Tr cells by Western blot analysis. Cells were treated with GDC-0068 (0, 0.2, 1, and 5 mmol/L) for the indicated times. QD, every day.

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Figure 3. Effective inhibition of Akt signaling and tumor xenograft growth by GDC-0068 in mouse models. A, dose-dependent inhibition of BT474Tr breastcancer and PC3 prostate cancer xenograft models by GDC-0068. B, modulation of key pharmacodynamic markers detected by IHC at 88 mg/kgof GDC-0068. C, RPPA analysis of pharmacodynamic changes in BT474-Tr xenograft tumors. Tumors were collected at 2 hours after dose after18 days of daily dosing with GDC-0068 at 11, 22, and 88 mg/kg. Relative intensities of protein phosphorylation from lysates of tumors in each dosegroup were showed as log2 ratio to the vehicle-treated group. Dots indicate the biomarker set identified in vitro. D, PCA modeling of dose-dependentchanges of key pharmacodynamicmarkers as revealed byRPPA. Loadings of the first principal componentswere plotted for the xenograft data in eachmodelon the left. On the right, the first principal component scores for the xenograft datawere regressed against the percentage of survival inhibition. The regressionslope for BT474-Tr is �0.14 and for PC3 is �0.22.






and 4 hours post-dose after 8 to 15 days of continuousdaily dosing. Figure 4A and B show the RPPA analysisfrom a total of 9 patients within the 100, 200, and 400mg/day GDC-0068 dose cohorts, which were all well-tolerated doses (32). Analysis of the on-treatment biop-sies showed a marked increase of Akt S473 and decreaseof PRAS40 T246 in patients dosed as low as 100 mg/day.The modulation of Akt S473 and PRAS40 T246 wasconfirmed by IHC in available patient samples from anindependent, concurrent biopsy (Fig. 4C and D). A poten-

tial correlation between tumor genotype (PTEN/PIK3CAstatus) and pharmacodynamic effect was not observedand also not expected, given the preclinical data demon-strating pathway inhibition in both PI3K-activated andnon-PI3K–activated models (Supplementary Table S4).The high degree of necrosis in the PTEN-null samples#301 and #402 (rising from 0% in the pretreatmentbiopsies to as high as 30% and 90% for #301 and#402, respectively) likely contributed to the apparent lackof PRAS40 T246 modulation observed by RPPA (Fig. 4B).

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Figure 4. GDC-0068 modulated Akt signaling in tumors from patients. RPPA revealed significant modulation of (A) Akt S473 and (B) PRAS40 T246 in tumorbiopsies from patients with cancer treated with GDC-0068 at three dose escalation levels (100, 200, and 400 mg/day). Tumor core biopsies frompretreatment (pre, blue) and 15 to 22 days posttreatment (post, red) were assayed in four dilutions of tumor lysates. All the technical replicates were plotted asnormalized intensities to the mean pretreatment. C, modulation of pAkt S473 and (D) of PRAS40 T246 in tumor biopsies from representative patientstreated with 100, 200, and 400 mg daily dose (post) as compared with the PRAS40 T246 levels in predose tumor biopsy. H-Score quantification is provided.Arrows indicate stroma.

Yan et al.





As there was significant variation in the percentage oftumor tissue in biopsies, which limited our ability tointerpret the RPPA data, we used laser-capture microdis-section (LCM) to enrich for tumor cells before RPPAanalysis in all patients from the 100 mg cohort, wheresufficient material remained for LCM in paired pre- andpost-treatment tumor biopsies. We first compared thebiomarker modulation in tumor versus nontumor sam-ples. While changes in Akt S473 and Akt T308 expressionwere present in both epithelial tumor and stromal com-partments, PRAS40 T246 was modulated uniquely inepithelial tumor cells, in accordance with what wasobserved by immunohistochemical staining, underscor-ing the need to enrich for the tumor cell population (Fig.5A, Fig. 4C and D, arrows and data not shown). Inaddition, increased levels of HER3 following GDC-0068 treatment was detected only in tumor cells of thebreast tumor biopsy of patient 301 and not in the stromalcomponent (Fig. 5A). The dose of 100 mg/day GDC-0068showed substantial coordinate modulation of the GDC-0068 biomarker core in epithelial tumor cells in all threepatients (Fig. 5B). Even though the percentage change ofmarkers varied from patient to patient, the average levelsof pAkt increased significantly over pretreatment levels,and the 8 other core markers were inhibited in the rangeof 41% to 69%, with an average of 56% inhibition (Fig.5B and Supplementary Table S5). In addition, GDC-0068elicited compensatory HER3 and ERK activation intumors from 2 patients. These results demonstrated that

GDC-0068 engaged the target and significantly inhibitedsignaling downstream of Akt in patient tumor samplesstarting from as low as the 100 mg daily dose.

To test whether the degree of pathway inhibitionobserved in patients is biologically meaningful, we com-pared the pattern of the inhibition of the core biomarkersfrom patients with those derived from cell lines and xeno-grafts. Unsupervised clustering of the RPPA data from allthree sources showed that patients’ data were closely clus-tered with xenograft data (Supplementary Fig. S4). Wefurther questioned if our comprehensive approach provid-ed any advantage over utilizing a consistently inhibitedprotein such as PRAS40 T246. We compared a single-bio-marker TGI projection to the biomarker set projectionusing PCA-scores (Supplementary Table S6). The individualprojections were not only highly variable but also differsubstantially from the PCA score–based projected TGI.Therefore, we propose that using a comprehensive phar-macodynamic assessment provides a more robust readoutof pathway inhibition in a tumor sample than a singlebiomarker approach. This approach may be criticallyimportant in clinical contexts in which both drug dose andgenetic background are variables. We finally applied thexenograft-derived PCA models (Fig. 3D) to estimate thecorresponding tumor growth inhibition (TGI; Table 1 basedon the biomarker changes in the 100 mg cohort patients).The degree of pathway modulation in the 100 mg cohortpatientswas equivalent to changes leading to approximately60% TGI in preclinical models.

Strom

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Figure 5. LCM of needle biopsiesenables RPPA analysis of theGDC-0068 biomarker core. A,differential tumor and stromaresponses to GDC-0068 treatmentin patients. Tumor and stroma cellswere captured separately by lasermicrodissection from frozenbiopsy sections for RPPA analysisof Akt S473, PRAS40 T246, andHER3. Pretreatment (pre, blue) and15 to 22 days posttreatment (post,red) were assayed in four dilutions.B, modulation of the Akt inhibitorbiomarker core in tumor cellscaptured with LCM from needlebiopsy taken before the first doseand after 15 to 22 days of 100 mgdaily treatment. The 10 coresignaling nodes were shown pluspErk and HER3 proteins levels.Heatmap displays the log2 ratio ofintensity in comparison to thepredose level of the same protein.Blue denotes the decrease and redthe increase as compared with thepredose level.






DiscussionOncology drug discovery has increasingly focused on the

development of targeted therapies, with considerableefforts to improve the specificity, safety, and efficacy of suchagents. A core component of this strategy is the identifica-tion and evaluation of specific biomarkers that can func-tionally confirm target engagement and detailed pathwaystatus at clinicallymeaningful doses in treated patients (33).

In this work, we demonstrate that an unbiased screeningof different in vitro and in vivo preclinical models, with 100validated antibodies against total and activated proteinsinvolved in oncogenic signaling pathways, led to the iden-tification of core biomarkers for catalytic inhibition of Akt.RPPA is a sensitive platform to measure several dozens ofphospho-epitopes from a few nanograms of protein extractand has utility for the quantitative analysis of on-treatmentbiopsies. However, a limitation of this technique is thevariable percentage ofmalignant tissue present in the biopsyand the absence of a validated method of normalization formalignant epithelial cells. Because of this, immunohis-tochemistry can serve a valuable tool to confirm one orseveral biomarkers discovered by RPPA, but has limitedmultiplexing capacity. Our data show that it is possible touse LCM to isolate the cell population of interest and thusthat LCM can be useful to overcome the challenge of sampleheterogeneity. This techniquewas also highly informative inshowing greater modulation of elements of the pathway inneoplastic cells relative to stromal cells, consistent withpreclinical data showing that more dramatic effects areobserved in tumor cell lines compared with normal cells.Although labor intensive, the powerful combination of LCMwith the sensitive detection of RPPA is required for adequateassessment of target engagement specifically in the tumortissue andoffers auniqueopportunity to study themolecularnature of tumor–stroma interactions in the clinical setting.

Our data show that the Akt pathway, as measured bymultiple biomarkers, was modulated in three patients’tumors at a dose of 100 mg per day of GDC-0068. Thedegree of biomarker changes at the 100 mg dose wasconsistent with that observed in preclinical models withapproximately 60% tumor growth inhibition. Lin and col-leagues showed that GDC-0068 single-agent doses thatexhibit 50% to 70% TGI result in tumor regressions whencombinedwith docetaxel (21).We therefore considered the

100mgGDC-0068as aminimumdose that elicitedbiologicactivity in patients and leveraged this knowledge to initiatephase Ib/II studies starting from this dose in combinationwith standard of care chemotherapies (i.e., docetaxel andmFOLFOX) and targeted agents (abiraterone), before reach-ing the single agent maximum tolerable dose of 600 mgdaily (in regimen 21 days on/7 days off; refs. 34, 35).

Our broader survey with RPPA also identified biomarkersthat indicate upregulation of compensatory pathways. Forexample,HER3 phosphorylation and ERK activation follow-ing GDC-0068 treatment were detected in both preclinicalmodels and, for the first time to our knowledge, in patientsamples, demonstrating that previously reported compen-satorymechanisms occur in a clinical setting (23, 24). Thesefindings have clear implications for the further developmentof rational combination therapies, for instance combiningGDC-0068 with agents targeting HER3 or MEK.

In conclusion, we demonstrated that selected preclinicalmodels could be used to effectively screen and identify abiomarker core of pathway modulation that was translat-able to the clinic. RPPA following LCMwas used to providea broad profile of signaling pathway modulation in needlebiopsies and confirmed robust pathway inhibition byGDC-0068. These findings demonstrate that GDC-0068 is inhi-biting Akt, its intended mechanism of action, in tumortissue at clinically achievable doses, and support its contin-ued testing in diagnostically defined patient populationsand with rational drug combinations. This approach can inprinciple be applied to other therapeutics to effectivelyprovide the molecular foundation for subsequent stages ofclinical development.

Disclosure of Potential Conflicts of InterestS. Murli is employed as a project team leader and is a consultant/advisory

board member in Genentech, Inc. D. Sampath has ownership interest(including patents) in Genentech. M. Wongchenko has ownership interest(including patents) in Roche. J. Tabernero and J. Baselga are consultant/advisory board members of Genentech. No potential conflicts of interestwere disclosed by the other authors.

Authors' ContributionsConception and design: Y. Yan, V. Serra, M. Scaltriti, S. Murli, D. Sampath,M. Nannini, V. Ramakrishnan,M.R. Lackner, J. Tabernero, P. Patel, J. BaselgaDevelopment of methodology: Y. Yan, V. Serra, S. Murli, Y. Xiao, J.TaberneroAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): Y. Yan, V. Serra, L. Prudkin, O. Rodr��guez, M.

Table 1. Tumor growth inhibition projection

BT474-Tr PC3

Patient number PCA Score Predicted TGI PCA Score Predicted TGI

301 3.60 51% 2.85 62%302 4.18 59% 3.43 75%303 3.38 48% 2.70 59%

NOTE: The PCA scores of the paired biopsies obtained from three 100-mg cohort patients were projected in two different xenograftmodels to predict the percentage of TGI. The PCA scores were derived from the core biomarkers in Fig. 5B and projected to the twomodels as in Fig. 3D.

Yan et al.





Guzman, D. Sampath, M. Nannini, M.-C.Wagle, J.Q.Wu, C. Saura, D. Roda,A. Cervantes, J. TaberneroAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): Y. Yan, V. Serra, L. Prudkin,M. Scaltriti, S.Murli,M.Nannini, Y. Xiao,M.-C.Wagle,M.Wongchenko, V. Ramakrishnan,M.R. Lackner, D. Roda, J. Tabernero, P. Patel, J. BaselgaWriting, review, and/or revision of the manuscript: Y. Yan, V. Serra, L.Prudkin, M. Scaltriti, S. Murli, M. Nannini, Y. Xiao, G. Hampton, V.Ramakrishnan, M.R. Lackner, C. Saura, D. Roda, A. Cervantes, J. Tabernero,P. Patel, J. BaselgaAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): V. Serra, S. Murli, M. GuzmanStudy supervision: Y. Yan, V. Serra, S. Murli, V. Ramakrishnan, J. Baselga

AcknowledgmentsThe authors thank all patients who participated in this biomarker

intensive study. The authors also express gratitude to Jim Marsters,

Genentech, and Array BioPharma Akt teams for technical support andarticle discussion.

Grant SupportThis work was supported in part by a Stand Up to Cancer Dream Team

Translational Research Grant, a Program of the Entertainment IndustryFoundation (SU2C-AACR-DT0209), a European Research Council GrantAdG09 250244, an Instituto de Salud Carlos III Intrasalud Grant PSO9/00623, and a Banco Bilbao Vizcaya Argentaria (BBVA) Foundation Grant(to J. Baselga).

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 April 14, 2013; revised September 18, 2013; accepted September24, 2013; published OnlineFirst October 18, 2013.

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2013;19:6976-6986. Published OnlineFirst October 18, 2013.Clin Cancer Res Yibing Yan, Violeta Serra, Ludmila Prudkin, et al. Inhibitor GDC-0068Evaluation and Clinical Analyses of Downstream Targets of the Akt

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