in vivo activity of combined pi3k/mtor and mek inhibition€¦ · 15/07/2011 · pathway limits...

Preclinical Development

In Vivo Activity of Combined PI3K/mTOR and MEK Inhibitionin a KrasG12D;Pten Deletion Mouse Model of Ovarian Cancer

Kathryn M. Kinross1,2,3, Daniel V. Brown1,2,3, Margarete Kleinschmidt1,2,3, Susan Jackson2,3,James Christensen8, Carleen Cullinane2,3,7, Rodney J. Hicks2,3,4,6,Ricky W. Johnstone2,7, and Grant A. McArthur1,2,3,5,6,7

AbstractThe phosphatidylinositol 3-kinase (PI3K)/Akt pathway is commonly dysregulated in human cancer,

making it an attractive target for novel anticancer therapeutics. We have used a mouse model of ovarian

cancer generated by KrasG12D activation and Pten deletion in the ovarian surface epithelium for the preclinical

assessment of a novel PI3K/mTOR inhibitor PF-04691502. To enable higher throughput studies, we devel-

oped an orthotopic primary transplant model from these mice and evaluated therapeutic response to PF-

04691502 using small-animal ultrasound and FDG-PET imaging. PF-04691502 inhibited tumor growth at

7 days by 72% � 9. FDG-PET imaging revealed that PF-04691502 reduced glucose metabolism dramatically,

suggesting FDG-PET may be exploited as an imaging biomarker of target inhibition by PF-04691502. Tissue

biomarkers of PI3K/mTOR pathway activity, p-AKT (S473), and p-RPS6 (S240/244), were also dramatically

inhibited following PF-04691502 treatment. However, as a single agent, PF-04691502 did not induce tumor

regression and the long-term efficacy was limited, with tumor proliferation continuing in the presence of drug

treatment. We hypothesized that tumor progression was because of concomitant activation of the mitogen-

activated protein kinase pathway downstream of KrasG12D expression promoting cell survival and that the

therapeutic effect of PF-04691502 would be enhanced by combinatory inhibition of MEK using PD-0325901.

This combination induced striking tumor regression, apoptosis associated with upregulation of Bim and

downregulation of Mcl-1, and greatly improved duration of survival. These data suggest that contempora-

neous MEK inhibition enhances the cytotoxicity associated with abrogation of PI3K/mTOR signaling,

converting tumor growth inhibition to tumor regression in a mouse model of ovarian cancer driven by

PTEN loss and mutant K-Ras. Mol Cancer Ther; 10(8); 1–10. �2011 AACR.

Introduction

The phosphatidylinositol 3-kinase (PI3K) pathway isan essential regulator of cellular proliferation, survival,metabolism, and growth acting through disparate down-stream effectors including the AKT andmTOR pathways.Genomic changes leading to activation of this pathway,such as mutation or loss of PTEN, activating mutations oramplification of PI3K or activatingmutations in upstreamreceptor tyrosine kinases such as epidermal growth factor

receptor and ERBB2 are common in human cancers,making this pathway an attractive target for novel antic-ancer therapeutics (1). Multiple small-molecule inhibitorshave been targeted to different kinases throughout thepathway, including AKT inhibitors (MK-2206), mTORinhibitors (everolimus, temsirolimus, AZD8055, OSI-027), PI3K inhibitors (GDC-0941, PX866, BKM120), anddual PI3K/mTOR inhibitors [BEZ235; reviewed in(2, 3)]. Recently, PF-04691502, a novel dual PI3K/mTORinhibitor, has been described (4) and has been enteredin phase I clinical trials for the treatment of solid can-cers. PF-04691502 is orally bioavailable and is a highlypotent ATP-competitive kinase inhibitor of class 1 PI3Ksand mTOR. As yet, minimal characterization of the pre-clinical predictors of the efficacy of this drug has beenreported.

Despite evidence of improving outcomes, ovarian can-cer remains one of the leading causes of gynecologicalcancer deaths because of its propensity to present with anadvanced clinical stage (5). These tumors commonlyexhibit activation of the PI3K/Akt pathway via PIK3CAmutation (6), loss of PTEN, PTEN mutation (7), orincreased expression of mir-214 targeting PTEN (8). Cur-rent therapeutic strategies for ovarian cancer involve

Authors' Affiliations: 1Molecular Oncology Laboratory, 2Cancer Thera-peutics Program, 3Translational Research Laboratory, 4Centre for CancerImaging, 5Division of Cancer Medicine, Peter MacCallum Cancer Centre,St Andrews Place, East Melbourne; 6Department of Medicine, St Vincent'sHospital, University of Melbourne, Fitzroy; 7Department of Pathology,University of Melbourne, Parkville, Australia; and 8Pfizer Global Researchand Development, La Jolla, California

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

Corresponding Author: Dr. Kathryn Kinross, Peter MacCallum CancerCentre, St Andrews Place, East Melbourne, VIC 3002. Phone: 61-3-96561806; Fax: 61-3-96561411; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-11-0240

�2011 American Association for Cancer Research.

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Published OnlineFirst June 1, 2011; DOI: 10.1158/1535-7163.MCT-11-0240

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platinum- or taxane-based chemotherapeutics, which aretypically effective initially, but often fail because ofacquired resistance attributed to diverse molecularcauses, one of which is increased activity of the PI3Kpathway (9–11). These findings indicate that ovariancancer patients are likely to benefit from the developmentof PI3K pathway inhibitors.

A number of genetically engineered mouse modelsof ovarian cancer have now been developed that canbe used to aid preclinical drug development (12). In thisstudy, we have used an ovarian cancer mouse modeldriven by KrasG12D activation and Pten deletion (13),which leads to upregulation of the PI3K pathway andthus, is an ideal in vivomodel for assessing efficacy of PF-04691502. Our findings show that inhibition of PI3K/mTOR alone in this tumor type can inhibit tumor growth.However, concomitant activation of the Ras/MAPKpathway limits the long-term therapeutic benefit. Toovercome this, we found that the combination of PF-04691502 with the MEK inhibitor PD-0325901 (14) ledto dramatic tumor regression and long-term survival oftumor-bearing mice. These studies show that PF-04691502 and PD-0325901 can be safely used in combina-tion in mice and induce robust antitumor response incancers bearing KrasG12D mutations and Pten deletions.

Materials and Methods

Transgenic miceWe obtained LSL-KrasG12D heterozygous mice (B6.129-

Krastm4Tyj) from the Mouse Models of Human CancerConsortium [National Cancer Institute (NCI)] and Ptendel

mice (c;129S4-Ptentm1Hwu/J) from Jackson Laboratories.All LSL-KrasG12D/þ;Ptendel/del (referred to as KrasG12D;Ptendel) mice were maintained on a mixed (Black6; BalbC;129) background. All BalbCnu/nu mice were obtainedfrom the Animal Research Centre or WEHI. All in vivoexperiments were carried out with adherence to theNational Health and Medical Research Council Austra-lian Code of Practice for the Care and Use of Animals forScientific Purposes and with approval from the PeterMacCallum Cancer Centre Animal ExperimentationEthics Committee.

Adenoviral-Cre recombination in ovaryOvarian surface epithelium was infected with replica-

tion-deficient recombinant adenovirus expressing Crerecombinase (15), a kind gift from Walter Thomas’Laboratory, Baker Institute, Australia, by methods pre-viously described (13, 16, 17). Briefly, ovulation wassynchronized by i.p. injection of 5U of pregnant mareserum gonadotropin (Intervet), followed by i.p. injectionof 5U human chorionic gonadotropin (Intervet) 48 hourslater. 1.5 days later, mice were anesthetized with an i.p.injection of ketamine and xylazine (86 and 17 mg/kg,respectively, in saline), and a dorsal excision was madeto expose the ovary. A 32-gauge needle and Hamiltonsyringe was used to inject 2.0 � 108 optical particle units

of Adenoviral-Cre (AdCre) in 5 mL (precipitate pre-pared in 1:1 Dulbecco’s Modified Eagle’s Medium andPBS/virus mix; supplemented with 0.2 mol/L CaCl2)into the ovarian bursa. The ovary was returned to thebody and the incision sealed with sutures and staples.Mice were monitored for ovarian tumor growth bysmall animal ultrasound (Vevo 770, Visual Sonics) andpalpation.

Orthotopic transplant of ovarian tumorsTumors were harvested from KrasG12D;Ptendel mice and

dissected into approximately 1 mm diameter pieces andstored in 10% DMSO:90% fetal calf serum at �80�C.Tumor pieces were thawed and washed in 3 changesof cold PBS and dissected further under a dissectingmicroscope to equal sizes approximately 0.1 mm in dia-meter. Female BalbCnu/nu mice were anesthetized withketamine/xylazine mix and the left ovary was exposed asdescribed above. A small incision was made in the ovar-ian bursa and a tumor piece placed between the bursallining and the ovary using fine forceps. The ovary wasreturned to the body and the incision sealed with suturesand staples. Mice were monitored for ovarian tumorgrowth by ultrasound and palpation.

In vivo drug studies2-amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-

(6-methoxypyridin-3-yl)-4-methylpyrido[2,3-d]pyrimi-din-7(8H)-one (PF-04691502; ref. 4) was prepared in0.5% methylcellulose. Mice were dosed daily at either7.5 or 10 mg/kg in a volume of 2.5 mL/g of mouse bodyweight by oral gavage. N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4iodophenyl)amino]-Benzamide(PD-0325901; ref. 18) was prepared in 0.5% hydroxypro-pyl-methylcellulose (Biochemika fluka), 0.2% Tween80 (Sigma), and mice were dosed daily at either 1.5 or10 mg/kg in a volume of 2.5 mL/g of mouse body weightby oral gavage. For chemical structures, see Supplemen-tary Figure S1.

In vivo imagingThe volume of the left ovarian mass was monitored by

ultrasound imaging at the beginning of therapy and againat various intervals to monitor change in tumor burden(Vevo 770, Visualsonics; fixed with a 707b or 704 scan-head). FDG-PET imaging was conducted as previouslydescribed (19) using a dedicated small-animal PET scan-ner (Mosaic, Philips).

Histology and immunohistochemistryTumors were fixed in 10% neutral-buffered formalin

and paraffin embedded. Sections were stained withhemotoxylin and eosin for morphological examination.Immunohistochemistry was conducted following antigenretrieval by boiling slides in antigen target retrievalsolution pH 9 (DAKO) in a pressure cooker for 3 minutes.Antibodies used were phosphorylated (p)-AKT (S473,CS3787), p-RPS6 (CS2211) and p-ERK (CS4370) from Cell

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Signaling Technology and bromodeoxyuridine (BrdU;347580; BD Biosciences). For BrdU analysis, mice wereinjected with 100 mg/kg BrdU (Sigma) i.p. 1 hour beforesacrifice. BrdU quantitation was determined using theMetaMorph computer program (Universal Imaging),where the percentage of BrdU positive cells was countedrelative to nuclei over 5 microscopic fields at �40 mag-nification. TUNEL staining was conducted using theApopTag Peroxidase In Situ Apoptosis Detection Kitfollowing the manufacturers instructions (ChemiconInternational). All images were taken on an OlympusBX51 microscope.

Western blot analysisTumors were snap frozen in liquid nitrogen and lysed

in RIPA buffer (1 mmol/L EDTA; 1% NP40; 0.5% sodiumdeoxychlorate; 0.1% SDS; 50 mmol/L sodium fluoride; 1mmol/L sodium pyrophosphate in PBS) supplementedwith phosphatase inhibitor cocktail (Roche) for Westernblotting. Antibodies used were p-AKT (Ser473, CS9271),total AKT (CS9272), p-ERK (p44/42 MAPK, Thr202/Tyr204; CS9101), p-RPS6 (CS2215), and total S6(CS2217), each from Cell Signaling Technology, and totalERK (SC-94, Santa Cruz), Mcl-1 (600-401-394, RocklandImmunochemicals Inc.), Bim (AAP-330, ENZO LifeSciences), caspase 3 (611048, BD Biosciences), and pan-actin (clone C4, MAB1501, Millipore).

Results

Dual PI3K/mTOR inhibition with PF-04691502inhibits PI3K signaling and glucose metabolism in aKrasG12D;Ptendel mouse model of ovarian cancerTo evaluate the effects of PF-04691502 in vivo, we used

the KrasG12D;Ptendel mouse model of ovarian cancer (13)characterized by constitutive activation of KRAS anddeletion of PTENspecifically in theovarian surface epithe-lium. Female mice were superovulated and exposed toAdCre at 6 to 10 weeks old in the left ovarian bursa. Micewere monitored twice weekly by palpation and weightgain. When mice appeared to display bloating or rapidweight gain and small-animal ultrasound confirmed pre-sence of tumors, mice were recruited to therapy groups.Themedian time to recruitmentwas 17weeks post-AdCreexposure (range 9.6–41.9 weeks). Mice were randomizedinto 4groups receivingdrug (PF-04691502; 10mg/kgdailyorally) or vehicle for 2 or 7 days. To directly assess if PF-04691502 could inhibit the PI3K pathway in the tumorsthat develop in these mice, we conducted biomarkeranalysis on whole tumors following sacrifice (Fig. 1A).These tumors exhibit high basal levels of both p-AKT(Ser473) and p-RPS6, which were each reduced followingtreatmentwithPF-04691502 for 2daysor 7 days.However,the reduction in p-AKT was not as robust in mice treatedfor 7 days comparedwith 2 days. In a subset of thesemice,we used FDG-PET to noninvasively detect a metabolicresponse to PF-04691502 therapy (Fig. 1B and C). Weobserved a reduction inFDG-uptake, indicatingdecreased

glucose metabolism that correlates with the observeddecrease in PI3K pathway activation. These findings sug-gest that FDG-PET imagingmay be used as a biomarker oftarget inhibition in response to PF-04691502 therapy.

PF-04691502 inhibits tumor growth in anorthotopic transplant model of KrasG12D;Ptendel

ovarian cancerBecause of the long latency, abdominal location and

complication of peritoneal spreading and ascites oftenmaking identification of these tumors and quantificationby ultrasound difficult, we did not find evaluation ofprimary tumors in this mouse cancer model to be readilyamenable to larger preclinical studies. To enable higher

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Figure 1. PF-04691502 inhibits the PI3K pathway and FDG uptake in micewith spontaneous KrasG12D;Ptendel ovarian adenocarcinomas. KrasG12D;Ptendelmice were allowed to develop ovarian tumors post-AdCre and thentreated with PF-04691502 for biomarker and serial FDG-PET imaginganalyses. A, Western blot of tumor p-AKT (Ser473) and p-RPS6 levels in 3representative mice per group treated with vehicle (�) or 10 mg/kgPF-04691502 (þ) for 2 or 7 days (as shown). B, serial FDG-PET scansof a representative vehicle or drug-treated mouse showing maximum-intensity-projection images (top), transaxial images (bottom), andstandardized uptake value (SUV; maximum tumor pixel intensity) of thetumor region. Arrows indicate the tumor region. C, SUVs from serialFDG-PET scans, with each line representing an individual mouse.

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throughput studies, we isolated tumors arising fromtransgenic mice and used them as a source for primary,orthotopic transplantation into multiple recipients.BalbCnu/nu recipients were used because of the mixedbackground of the original tumor-bearing transgenicmice.These tumorswere storedas tumorpieces ina frozentumor bank and before transplant were thawed, dissectedinto small pieces approximately 0.1 mm diameter, andsurgically inserted under the bursa of the left ovary. Twoindependent tumors, B84 and B87, were typically used inexperiments.Recipientsdeveloped tumorsgreater than50mm3 by 2 weeks following transplant with a take rate ofapproximately 75%. These tumors were each resistant tocisplatin (4 mg/kg weekly) and docetaxel (15 mg/kgweekly) upon initial therapy (Supplementary Fig. S2).To confirm that we could use ultrasound imaging toaccurately monitor growth of these transplanted tumors,we initially correlated the volumes obtained using ultra-sound imaging with the ex vivo tumor weight followingdissection of the tumor and found a high correlationusing these 2 measurements (Supplementary Fig. S3).

To assess the efficacy of PF-04691502, we transplantedtumors and began therapy on day 13 posttransplantationwhen tumors reached greater than 50 mm3. The averagetumor size was 172 � 24 mm3 (SEM), with a range of 54.3

to 327.7 mm3. Mice were randomized to receive eitherdrug or vehicle daily by oral gavage for 7 days andultrasound and FDG-PET scans were conducted on days0, 2, and 7 of drug treatment. PF-04691502 inhibitedtumor growth rate significantly at 2 days (1.85 � 0.18-fold tumor growth in vehicle-treated vs. 1.07 � 0.14-foldtumor growth in drug-treated mice; Student’s t testP < 0.01) and 7 days (4.31 � 0.63-fold tumor growth invehicle-treated vs. 2.14 � 0.65-fold tumor growth indrug-treated mice; Student’s t test P < 0.05). Tumorweights measured postmortem on day 7 posttherapy alsoshowed a significant lower tumor mass in treated mice(0.78 � 0.13 g in vehicle-treated vs. 0.38 � 0.06 g in drug-treated mice; Student’s t test P < 0.05; Fig. 2C). FDG-PETimaging revealed that PF-04691502 reduced glucosemetabolism dramatically at 48 hours, and this reductionwas maintained for the 7-day treatment duration (Fig. 2Dand E).

To confirm that the PI3K/mTOR pathwaywas success-fully inhibited with PF-04691502, Western blots andimmunohistochemistry were carried out on tumor sam-ples harvested at 7 days posttreatment (Fig. 3A and B).Similar to results seen in transgenic mice treated with PF-04691502 (Fig. 1A), mice bearing transplanted KrasG12D;Ptendel ovarian tumors also showed inhibition of p-AKT

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Figure 2. Single agent PF-04691502 reduces tumor growth rate and FDG uptake in vivo. Mice were transplanted orthotopically with KrasG12D;Ptendel ovariantumors and following tumor establishment was treated with vehicle (n ¼ 5) or PF-04691502 (n ¼ 8) at 10 mg/kg daily for 7 days. A, representativeserial 3-dimensional reconstructions and volumetric quantitation of ovarian tumor regions from ultrasound imaging. B, tumor volume change determined byultrasound imaging expressed relative to that on day 0. C, Mice were all sacrificed on day 7 and tumor weights determined. D, serial FDG-PET maximum-intensity-projection (top) and transaxial (bottom) images from a representative mouse treated with either vehicle or drug. Arrows indicate tumor region.E, change in FDG uptake in response to drug. FDG uptake ratio refers to the maximum pixel intensity in the tumor region of interest on each day expressedrelative to day 0. Data is represented as mean � SEM.

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(S473) and p-RPS6 (S240/244) following PF-04691502treatment.We next examined incorporation of BrdU in these

tumors as amarker of proliferation (Fig. 3C). Surprisingly,we observed only a marginal decrease in BrdU-positivecells in PF-04691502–treated tumors (Fig. 3C), suggestingthat despite initial stasis in tumor growth at 2 days post-treatment (indicated by ultrasound volumetric analysis,Fig. 2A), in response to PF-04691502, following 7 dayscontinuous treatment, these effects had been overcome.Given the presence of the activatedKRAS in thismodel,

we hypothesized these cells may be able to survive andreinstate proliferation through increased activation of theRAS/RAF/MEK/ERK (RAS/MAPK) signaling cascadedownstream of KrasG12D. We assessed the levels of p-ERKas a readout of RAS/MAPK signaling (Fig. 3D and E).p-ERK expression was variable in this model in bothvehicle-treated and PF-04691502–treated tumors, which isconsistentwith the findings of others that in somecontextsp-ERK can be an unreliable marker of activated KRASin tumors (20–22). To determine if these tumors exploitthis pathway to limit the efficacy of PF-04691502 therapy,we next evaluated susceptibility to combined inhibitionof the RAS/MAPK and PI3K pathways using the MEKinhibitor, PD-0325901, in combination with PF-04691502.

Combined inhibition of PI3K/mTOR (PF-04691502)and MEK (PD-0325901) leads to tumor regressionin vivoWe transplanted KrasG12D;Ptendel ovarian tumors

orthotopically and following establishment of tumors

began treatment with vehicle, PF-04691502 (7.5 mg/kg,daily), PD-0325901 (10 mg/kg, daily), or combined PF-04691502 and PD-0325901 (7.5 mg/kg and 10 mg/kg,respectively). These doses were determined to be thecombined maximum–tolerated dose causing less than20% total body weight loss. Ultrasound was used tomonitor tumor growth over 7 days of therapy (Fig. 4Aand B). PF-04691502 alone led to tumor growth inhibi-tion of 55% � 10% at the lower dose of 7.5 mg/kg.Comparison to previous experiments shows greatertumor growth inhibition with 10 mg/kg daily dosingof PF-04691502 (Fig. 2B) compared with 7.5 mg/kg,indicating that the effects of PF-04691502 are dosedependent. Interestingly, PD-0325901 alone led totumor regression (>20% volume reduction) in 6/10 mice(mean volume 36.3% � 20.5 smaller on day 7 comparedwith day 0 tumor volume). Combining PF-04691502 andPD-0325901 led to a greater effect than either agentalone, with striking tumor regression (>20% volumereduction) in all 9 mice (mean volume 80.7% � 5.0smaller on day 7 compared with day 0 tumor volume).Images of representative tumors taken from each groupfollowing 7 days therapy show these differences intumor volume (Supplementary Fig. S4).

We next tested a longer term therapeutic regimen usinga low dose drug combination of 7.5 mg/kg PF-04691502and 1.5 mg/kg PD-0325901. One limitation of the clinicaldevelopment of PD-0325901 is that prolonged exposurecan cause diarrhea and ocular toxicity (23–25); therefore,using the compound at a minimally effective doserather than the maximum-tolerated dose may improve

Figure 3. Biomarker analysisfollowing PF-04691502 therapyin vivo. Tumors were analyzedfollowing 7 days of treatment withPF-04691502 at 10mg/kg daily forbiomarkers of PI3K pathwayactivity. A, Western blotting oflysates from 5 vehicle and 6PF-04691502 mice for p-AKT andp-RPS6. B, representativeimmunohistochemistry staining ofvehicle and PF-04691502–treatedtumors stained for p-AKT andp-RPS6. C, BrdU immunohisto-chemical staining of tumors wasconducted (left) and percentage ofBrdU-positive cells wasquantitated by MetaMorph (right).D and E, biomarker of MAPKpathway signaling, p-ERK, wasmeasured by Western blot andimmunohistochemistry.

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tolerability. This schedule was well tolerated, causing noweight loss over the treatment period (data not shown).Tumors were allowed to reach a relatively large volume(average 281.6 � 19.4 mm3) before commencing therapyto better enable the evaluation of tumor regression in thecombination group. Tumor growth was monitored withultrasound volumetric analyses (Supplementary Fig. S4),and the time to reach the predefined endpoint of clinicaldeterioration necessitating sacrifice or sacrifice at atumor volume of 1.3 cm3 was determined (Fig. 4C).The low-dose PF-04691502 did not significantly improvesurvival (median survival of 7 and 8 days in vehicle andPF-04691502 groups, respectively), whereas therapy withlow-dose PD-0325901 led to a modest survival advantage(median survival of 13 days in PD-0325901 group; t testcomparison to vehicle; P < 0.001). Combined therapywithPF-04691502 and PD-0325901 led to a substantial long-term survival advantage, with median survival extendedto 37 days (P < 0.0001, Fig. 4C).

Biomarker studies were conducted on tumor bearingmice treated with vehicle, PF-04691502, PD-0325901, andthe drug combination (Fig. 5; Supplementary Fig. S5). Asexpected, treatment with PF-04691502 alone resulted indecreased p-AKT and p-RPS6, whereas PD-0325901decreased p-ERK and partially decreased p-AKT. Inter-estingly, PF-04691502 alone led to a mild reduction inexpression of the antiapoptotic protein Mcl-1 and PD-0325901 alone caused an increase in expression of theBH3-only protein, Bim. Treatment of the tumor-bearingmice with a combination of PF-04691502 and PD-0325901resulted in a total loss of detectable p-AKT, p-RPS6, and

p-ERK, and increased expression of Bim and decreasedMcl-1. The combination of PF-04691502 and PD-0325901caused an increase in cleavage and accumulation of cas-pase-3 (Fig. 5A) and increased TUNEL staining (Fig. 5B),indicating that this treatment regimen caused increasedtumor apoptosis.

Discussion

Here we present an in vivo efficacy study of the PI3K/mTOR inhibitor, PF-04691502 in a genetically engineeredmouse model of ovarian cancer driven by KrasG12D-acti-vatingmutation and Pten deletion. To enhance preclinicalstudies using this model, we transplanted primarytumors from these mice orthotopically into the ovarianbursa and used in vivo imaging to noninvasively monitortumor growth and metabolic activity over time and inresponse to treatment. Several advantages of this trans-plant model include that the tumors are transplanted aspieces rather than single cells and therefore include thecancer-associated stromal tissue; the tumors are neverused in cell culture and thus, do not undergo any geno-mic changes associated with the adhering to plastic; andthey are transplanted orthotopically, thus being exposedto appropriate microenvironmental factors. We showedthat PF-04691502 had limited long-term single agentactivity in this model, and hypothesized that this wasbecause of sustained RAS/MAPK signaling in treatedtumors. Accordingly, combination therapy using PF-04691502 and an inhibitor of MEK (PD-0325901) resultedin robust and prolonged tumor regression.

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Figure 4. Combined inhibition of PI3K/mTOR (PF-04691502) and MEK (PD-0325901) leads to regression of transplanted KrasG12D;Ptendel ovarianadenocarcinomas in vivo. Following tumor establishment, mice were treated with vehicle control (n¼ 10); PF-04691502 (7.5 mg/kg daily; n¼ 7); PD-0325901(10 mg/kg daily; n ¼ 10); or PF-04691502 and PD-0325901 (7.5 mg/kg and 10 mg/kg, respectively, daily; n ¼ 9) for 7 days. Tumor growth was monitoredby ultrasound. A, changes in volume are expressed relative to that on day 0, mean � SEM. B, Waterfall plot representing percentage change in tumorvolume (day 7/day 0) of individual mice. #, a PD-0325901-treated mouse with unchanged tumor volume. C, a separate cohort of mice-bearing establishedtumors were treated with vehicle (n ¼ 6); PF-04691502 (7.5 mg/kg daily; n ¼ 6); low-dose PD-0325901 (1.5 mg/kg daily; n ¼ 6); or PF-04691502 andlow-dose PD-0325901 (7.5 mg/kg and 1.5 mg/kg, respectively, daily; n ¼ 7) continuously. Mice were sacrificed when tumor burden became too high orsigns of distress were observed and time to sacrifice was recorded in a Kaplan–Meier survival curve.

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PF-04691502 is a novel, orally bioavailable and highlypotentdual PI3KandmTOR inhibitor (4). Earlypreclinicaland clinical development of PI3K pathway inhibitors hasbeen focused on the identification of clinical settings inwhich single agent PI3K pathway inhibitors may be effec-tive. For example, the single agent use of the mTORC1inhibitor, temsirolimus, has proved efficacious for mantlecell lymphoma therapy (26) and is approved for thetreatment of renal cell carcinoma (27). Recently, thePI3K inhibitor, GDC-0941, has been shown to inhibittumor growth in a subset of breast cancer lines withPIK3CA mutations, HER2 amplification, or alterationsin 2 PI3K pathway components (28). Preclinically, dualinhibition of PI3K and mTOR has shown antitumor effi-cacy in the context ofPik3ca(H1047R)-driven lung (29) andbreast (30, 31) tumors andHER2-amplified breast cancers(31). However, doubt remains as to the ability of PI3Kpathway inhibitors to enable long-term tumor growthinhibition across a range of tumor types without thedevelopment of resistant tumors. In this study,KrasG12D;Ptendel ovarian tumors display high activity ofthe PI3K pathway and were hypothesized to be suscep-tible to single agent PF-04691502 therapy. Indeed, PF-04691502 led to robustPI3Kpathway inhibition and tumorgrowth delay. However, these tumors are able to over-come their dependency on this pathway, to survive andcontinue proliferating despite ongoing exposure to thedrug.The swift adaptation followingPF-04691502 therapyin theKrasG12D;Ptendel ovarian tumormodel highlights theextensive cross-talk between parallel cancer-signalingpathways and consequently, the requirement for multi-pathway inhibition for the long-term prevention of pro-

liferation or induction of cell death. Activation of theRAS/MAPK signaling pathway is a known mechanismby which tumors can become resistant to PI3K pathwayinhibition, with activating mutations in Kras conferringresistance to PI3K pathway inhibition in vitro (32, 33). Assuch, combined inhibition of PI3K and RAS/MAPK path-ways has had success preclinically. In vivo studies in LSL-KrasG12D lung cancer mice (29), breast cancer MCF7 xeno-grafts (34), PTEN-deleted/Nxk3.1-deleted androgen-independent prostate cancer–bearing mice (35), andTPO-KrasG12D;Ptendel thyroid cancer mice (36) have eachshown improved efficacy with combined PI3K and MEKpathway inhibition.

MEK inhibitors have shown promise as anticanceragents (37, 38), and one such agent, PD-0325901, showedtarget inhibition and some clinical activity in a phase Iclinical trial (24). However, a phase II clinical trial inadvanced non–small cell lung cancer was negative for itsprimary endpoint, with no patients achieving partial orcomplete responses (23, 24). These studies highlight thepossibility that MEK inhibitors, as with PI3K pathwayinhibitors, may need to be developed with alternatescheduling, appropriate patient selection criteria, or becombined with other rationally determined inhibitors tobe clinically successful. Consistent with a dual depen-dency of the KrasG12D;Ptendel ovarian tumors on both thePI3K/mTOR and the RAS/MAPK pathway, PD-0325901also had limited efficacy as a single agent. While MEKinhibition did result in greater effects compared with PF-04691502 alone, including tumor regression in somemice,these effects were short lived in the context of continuouslong-term dosing. Combined inhibition of both signaling

Figure 5. Combination PF-04691502 and PD-0325901therapy induces cell death. A,tumor-bearing mice were treatedwith vehicle, PF-04691502 (10mg/kg), PD-325901 (10 mg/kg), orPF-04691502 and PD-325901 (10mg/kg and 10mg/kg, respectively)at 0 hour and 23 hours and thensacrificed. Tumor tissue washarvested 1 hour following thesecond dose. Western blots wereconducted to evaluate targetinhibition of p-AKT, p-RPS6, andp-ERK (top) and to assessinduction of apoptosis via Bim,Mcl-1, and cleaved caspase-3protein expression (bottom). B,representative images of TUNELimmunohistochemistryconducted on tumor samplesderived from mice sacrificed 6hours following 1 dose of drugs atconcentrations as in A.

A

p-AKT

Total AKT

p-RPS6

Total RPS6

p-ERK

Total ERK

Bim

Mcl-1

Cleaved casp3(17 kDa)

PD-′901 (10 mg/kg)

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PF-′502 (10 mg/kg)

TUNEL

B

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PF-′502

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ation






pathways led to improved efficacy because of theenhanced induction of apoptosis, which can be in partattributed to the combined activation of pro-apoptoticBH3-only protein Bim as a result of MEK inhibition andrepression of Mcl-1 as a result of PI3K/mTOR inhibition(35, 39). Here, the ability of high Bim and low Mcl-1expression to cooperatively initiate apoptosis in responseto PI3K pathway and MEK inhibition is consistent withsimilar observations in human leukemia cells (40) andhuman lung cancer cell lines (41).

In addition to exploring the effects of combining 2targeted therapies, it is advantageous to exploit pre-clinical models to understand the effect of standardchemotherapies in combination with targeted therapeu-tics, given the established role of standard chemotherapyin ovarian cancer. Each of our KrasG12D;Ptendel ovariancancer transplanted clones showed intrinsic resistance tocurrent ovarian cancer platinum- and taxane-based che-motherapeutics, cisplatin, and docetaxel, respectively.When we combined PF-04691502 (5 mg/kg, daily) withcisplatin or docetaxel, we only observed very minimaltumor growth inhibition (Supplementary Fig. S2).Attempts at dose escalation to increase efficacy werelimited by toxicity of the combined drugs. Notably,we observed greater tumor growth inhibition withhigh dose PF-04691502 as a single agent (10mg/kg, daily)compared with the low doses achievable (5mg/kg, daily)when used in combination with either cytotoxic agent.This may be an important observation going forwardin clinical development, where PF-04691502 may be sui-table for patients subsequent to failing platinum andtaxane therapy, but would only be suitable in combina-tion with those drugs if efficacious PF-04691502 drugconcentrations can be achieved without adverse toxiceffects. However, more extensive analysis of in vivomod-els with various levels of chemotherapy resistanceand various dosing schedules would need to be carriedout before we understand how widely applicable thisfinding is.

The use of molecular imaging such as FDG-PET tononinvasively monitor drug responses is aiding thedevelopment and testing of novel anticancer com-pounds (42). It is well described that the PI3K/AKTpathway is a major driver of glucose metabolism incancer cells (43), therefore FDG-PET is likely to be anappropriate method to identify response to inhibitors ofthis pathway. Here, we show that high basal FDG-PETsignal observed in the KrasG12D;Ptendel ovarian tumorswas greatly reduced in response to PF-04691502, corre-sponding with inhibition of p-AKT and p-RPS6 protein

expression in ex vivo biomarkers analyses following1 week of therapy. However, there was discordancebetween the dramatic inhibition of glucose uptake com-pared with the lack of effect on BrdU incorporation andthe minimal long-term effects of single agent PF-04691502. This is most likely explained by FDG-PETmetabolic activity corresponding only to glucoseuptake, and it does not measure a cells ability to pro-liferate or generate energy by alternate metabolic path-ways such as via glutaminolysis (44). This has also beenobserved recently with mTOR inhibitors, where inhibi-tion of PI3K/AKT pathway signaling and glucose meta-bolismwas independent of tumor proliferation (45), andhighlights the need to develop other PET tracers thatcorrelate with drug target inhibition, tumor viability,and tumor proliferation. Despite the limitations of FDG-PET response to reflect the antitumor efficacy of PF-04691502, importantly, it does reflect successful targetpathway inhibition and may therefore be a valuablebiomarker of kinase inhibition during clinical develop-ment of this drug.

In conclusion, we have found PF-04691502 to be apotent inhibitor of the PI3K pathway in an in vivo mousemodel of ovarian cancer driven by KrasG12D and Ptendeletion, and this pathway inhibition may be imaged viaFDG-PET. While there may be clinical scenarios in whichsingle agent activity of PF-04691502 is efficacious, in thismodel driven by mutant Kras, at least, the therapeuticbenefit of PF-04691502 alone is limited. Importantly, theefficacy of PF-04691502 can be enhanced by combinedinhibition of MEK using PD-0325901.

Disclosure of Potential Conflicts of Interest

J. Christensen: employee and shareholder, Pfizer.

Acknowledgments

The authors thank Kerry Ardley, Titaina Potdevin, and EkaterinaBogatyreva for their technical assistance in conducting animal experi-ments, ultrasound imaging, and PET imaging, respectively.

Grant Support

This work was supported by a grant to G.A. McArthur and R.J. Hicksfrom Pfizer.

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 March 31, 2011; accepted May 6, 2011; published OnlineFirstJune 1, 2011.

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Published OnlineFirst June 1, 2011.Mol Cancer Ther Kathryn M. Kinross, Daniel V. Brown, Margarete Kleinschmidt, et al.

Deletion Mouse Model of Ovarian CancerPten;G12DKras Activity of Combined PI3K/mTOR and MEK Inhibition in a In Vivo

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