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Assessment of the In Vivo Activity of PI3K andMEK Inhibitors in Genetically Defined Models ofColorectal CancerMeera Raja1, Matt Zverev1, Katja Seipel1,2, Geraint T.Williams3, Alan R. Clarke1, andPaul H.S. Shaw1

Abstract

The objective of tailoring medicines for cancer patients accord-ing to themolecular profile of their diseaseholds great promise forthe improvement of cancer therapy. Nevertheless, this approachhas been limited, in part, due to the lack of predictive andinformative preclinical studies. Herein, we describe an assessmentof the therapeutic potential of targeting PI3K/mTOR and MAPKsignaling in genetically definedmousemodels of colorectal cancermirroring disease subtypes targeted for novel therapy in theFOCUS4 trial. Our studies demonstrate that dual PI3K/mTORinhibition is highly effective in invasive adenocarcinoma modelscharacterized by combinatorial mutations in Apc and Pten; Apcand Kras; and Apc, Pten and Kras. MEK inhibition was effective in

the combinatorial Apc and Kras setting, but had no impact ineither Apc Pten mutants or in Apc Pten Kras triple mutants.Furthermore, we describe the importance of scheduling for com-bination studies and show that although no additional benefit isgained in Apc Pten mice, combination of PI3K/mTOR andMAPKinhibition leads to an additive benefit in survival in Apc Krasmiceand a synergistic increase in survival in Apc Pten Krasmice. This isthe first study using robust colorectal cancer genetically engi-neered mouse models to support the validity of PI3K/mTOR andMEK inhibitors as tailored therapies for colorectal cancer andhighlight the potential importance of drug scheduling in theclinic. Mol Cancer Ther; 14(10); 2175–86. �2015 AACR.

IntroductionThe notion of stratifying therapy according to the molecular

profile of a given tumor is considered tohold great promise for theimprovement of therapeutic outcome. Despite this, failures ofcertain novel targeted therapeutic agents in clinical trials havelimited the progress of this approach. This is largely attributed to alack of accurate guidance frompreclinical studies and it is thoughtthat the increased use of reliable genetically engineered mousemodels (GEMM) may lead to improvements in predicting ther-apeutic response (1). The FOCUS 4 clinical trial is currently thelargestmolecularly stratifiedmultisite randomized trial to includetargeted anticancer strategies against PIK3CA-mutant or PTEN(phosphatase and tensin homologue)-deficient and KRAS orBRAF-mutant metastatic colorectal cancer (for More Information:http://www.focus4trial.org/). Our preclinical study has thereforemirrored some of the major tumor genetic backgrounds of theFOCUS 4 trial using GEMMs and matched novel targeted ther-apeutic agents to help determine the rationale for treatment

combinations likely to ultimately improve patient outcome. Thegenetically engineeredmousemodels used in this study faithfullyrecapitulate molecular pathways involved in the initiation andprogression of tumorigenesis and are therefore powerful tools forthe assessment of novel agents and proof-of-principle studies formolecularly targeted therapies.

Mutational activation of the KRAS oncogene is found in 40% to50% of patients with colorectal cancer and, similarly, mutationsactivating the closely associated PI3K pathway occur in 40% ofcolorectal cancer cases (2). Both are known to be associated withpoor prognosis and also predict poor outcome to conventionalchemotherapy (3, 4). Moreover, KRAS and PI3K pathway muta-tions are established predictors of nonresponse to anti-EGFR(epidermal growth factor receptor)–targeted therapies in colorec-tal cancer (5, 6). This has led to a surge in the development oftargeted agents against PI3K and MAPK pathways and theirassessment in preclinical studies. A number of recent studies haveevidenced the benefits of targeting these pathways using potentagents such as dual PI3K/mTOR and MEK1/2 inhibitors as singleagents and in combination therapy using robust in vivomodels ofhuman cancer (7–10). For colorectal cancer however, these eva-luations have been restricted to cell-based assays and xenotrans-plantation studies that carry certain limitations, such as the lack ofan intact immune system or tumor microenvironments (11). Toaugment these approaches, we have used three GEMMs of colo-rectal cancer, each bearing combinatorial mutations in Apc, Pten,and Kras (12–14), to evaluate the benefits of targeted agentsagainst the relevant pathways activated in these tumors. Despitemany advantages, one argument against the use of GEMMS aspreclinical models is the impracticability of primary bowel resec-tion prior to palliative drug therapy. However, for many patientswith metastatic colorectal cancer, the primary site of the disease

1European Cancer Stem Cell Research Institute, Cardiff University,Cardiff, United Kingdom. 2University Hospital Bern, Bern, Switzerland.3School of Medicine, Cardiff University, Cardiff, United Kingdom.

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

Corresponding Author: Alan Clarke, European Cancer Stem Cell ResearchInstitute, Cardiff University, Hadyn Ellis Building, Maindy Road, Cardiff CF244HQ, United Kingdom. Phone: 44-29-208-74609; Fax: 44-29-208-74116; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-15-0223

�2015 American Association for Cancer Research.

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will often remain in situ in the absence of attributable symptoms;an approach being examined in the SYNCHRONOUS trial (15).Given this, we would argue that the GEMMs used in this studydirectly serve to reflect patients with colorectal cancer.

Mutations in genes that encode components of PI3K/AKT andRAS/MEK/ERK signaling frequently occur in colorectal cancer andcoexist in approximately a third of all cases (16). In addition,sequencing analysis reveals that mutations in adenomatous poly-posis coli (APC), KRAS, and PI3K are themost frequently observedgenetic events in human colorectal cancer (17). Loss of the tumor-suppressor protein PTEN has been shown to occur in 12% to 80%of human colorectal cancer cases (18–20). Moreover, PTEN loss isassociated with poor prognosis and predicts nonresponse to anti-EGFR agents currently available for KRAS wild-type colorectalcancer patients (21, 22). In light of this, we used three previouslyestablished and described GEMMs with combinatorial mutationsinApc andPten;ApcandKras; andApc,Pten, andKras (12–14).Wehave used these models of intestinal tumorigenesis, all of whichlead to invasive intestinal adenocarcinoma, to investigate theeffects of PI3K/mTOR inhibition using NVP-BEZ235; MEK1/2inhibition using MEK162 (Novartis Pharma); and combinatorialtherapy using both NVP-BEZ235 and MEK162, as stratified ther-apeutic strategies. Although it is widely known that TP53 muta-tions also contribute to the pathogenesis of human colorectalcancer, this study aimed to directly emulate current clinical trialsevaluating PI3K and MAPK pathway inhibitors by using only therelevant pathway status as a stratified approach to targeted therapy.

Materials and MethodsExperimental animals and tissue harvesting

Procedures were carried out in accordance with UK HomeOffice regulations. All mice were maintained on an outbredbackground and genotyped by PCR for appropriate alleles.Mousestrains harboring conditional deletion of Apc and Pten andoncogenic activation of Kras using the AhCreERT andVillinCreERT transgenes were induced as described previously(12, 13). For long-term survival studies, mice were aged until adefined treatment start point, at which they were randomized toreceive either 0.5% methyl cellulose (vehicle), 35 mg/kg NVP-BEZ235 twice daily (T-D; Novartis Pharmaceuticals), 30mg/kg ofMEK162 twice daily (Novartis Pharmaceuticals), or combinationat doses described in the text. Mice were treated daily untilsymptomatic of disease or to an endpoint of 500 days afterinduction (DPI) whereupon they were harvested 4 hours afterfinal dose. Kaplan–Meier survival analysis was performed usingSPSS Statistics 18.0. For short-term pharmacodynamic experi-ments, induced mice were aged until symptomatic of disease.Mice were then administered a single dose of vehicle (equivalentvolume per weight), 35 mg/kg of NVP-BEZ235, 30 mg/kg ofMEK162, or combinations (as outlined in the text) and harvested4 hours following the final dose. Mice used for short-term experi-ments were also administered a dose of bromodeoxyuridine(BrdUrd) 2 hours prior to killing. Intestinal tissues were flushedwith water, opened longitudinally, rolled into swiss-roll struc-tures, and fixed in 10% neutral buffered formalin (Sigma) over-night before processing and embedding in paraffin wax.

Western blot analysisProteins were extracted from snap-frozen tumors and Western

blot analysis was carried out as previously described (12). Equiv-

alent protein (30 mg) from at least 3 to 6 tumors from n� 3 micewere pooled per cohort and run in triplicate. Antibodies againstpAKT Ser473 (4060), pAKT Thr308 (4056), pS6RP (4858),p4EBP1 (2855), pERK (4370), total extracellular signal–regulatedkinase (ERK; 4377) from Cell Signaling Technology and b-actin(A5216) from Sigma were used. Appropriate horseradish perox-idase (HRP)–linked secondary antibodies (GE Healthcare) andECL reagents (GE Healthcare) were used to develop blots.

Histology, immunohistochemistry, and scoringFormalin-fixed paraffin-embedded tissues were sectioned (5

mm), stained with hematoxylin and eosin (H&E) for tumornumber analysis or, used for immunohistochemistry (IHC). Totaltumor numberwas scoredblind from threeH&E-stained slides foreach sample. IHC against cleaved caspase-3 (1:200; 9664; CellSignaling Technology) and BrdUrd (1:150; C47580; BD Bios-ciences) was performed using standard methodologies. Positivestainingwas scored as a percentage of total epithelial cells per fieldof view. Typically, five fields were assessed in all tumors in aminimum of 3 mice per cohort. Error bars represent standarddeviation of the mean. Statistical analysis was performed usingthe Mann–WhitneyU test. All analysis on scoring were two-tailedtests where n equals the number of lesions scored.

ResultsDescription and validation of colorectal cancer models

Inactivation of Apc and Pten was used to mimic activation ofWnt signaling and PI3K signaling, respectively. Activation ofoncogenic Kras was used to mimic activation of MAPK signaling.Conditional mutation of these genes within the intestinal epi-thelium was achieved using tamoxifen and b-napthoflavone(BNF) for Apcf/þ Ptenf/f mice bearing the AhCreERT transgene;or tamoxifen dissolved in corn oil for Apcf/þ KrasV12DLSL/þ andApcf/þ Ptenf/f KrasV12DLSL/þ mice bearing the VillinCrERT trans-gene. These mice are referred hereon as, Apcf/þ Ptenf/f, Apcf/þ

KrasLSL/þ, and Apcf/þ Ptenf/f KrasLSL/þ, respectively. Left untreated,mice develop invasive intestinal malignancies with median sur-vivals of 100, 160, and 40 DPI, respectively. Although Apcf/þ

Ptenf/f andApcf/þPtenf/f KrasLSL/þmice develop lesionswithin thesmall intestine only, Apcf/þ KrasLSL/þ mice present with colonlesions additionally (12–14). Analysis of themolecular pathwaysin tumors harvested from these mice confirms activation ofrelevant pathways compared with Apcf/þ controls (Supplemen-tary Fig. S1), reinforcing credence of these models for therapeuticintervention. A cohort of mice for each tumor subgroup was alsoeuthanized at the relevant treatment start points to confirm thepresence of tumors with varying degrees of severity. As such,Apcf/þ Ptenf/f mice presented with adenomas and early invasiveadenocarcinomas characterized by sub-mucosal invasion, where-as Apcf/þ KrasLSL/þ and Apcf/þ Ptenf/f KrasLSL/þ mice primarilypresented with adenomas at the start of treatment (Supplemen-tary Figs. S2–S5).

Antagonism of PI3K/mTOR signaling in colorectal cancermodels

Given that PI3K signaling is increased in all three tumormodels(Supplementary Fig. S1), we first antagonized the pathway usingthe dual PI3K/mTOR inhibitor NVP-BEZ235. At 4 hours, follow-ing a single dose of NVP-BEZ235, PI3K/mTOR signaling wasreduced in Apcf/þ Ptenf/f tumors (Fig. 1A). At this time point, we

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also observed increased apoptosis (Fig. 1D; P � 0.05; Mann–Whitney U test) and reduced proliferation (Fig. 1E; P � 0.05;Mann–Whitney U test) as scoring by cleaved caspase-3 andBrdUrd positivity, respectively (Supplementary Figs. S2 andS3). Continuous daily treatment from 77 DPI was found tosignificantly increase survival of Apcf/þPtenf/fmice fromamedianof 99 to 266DPI (Fig. 2A; n� 15mice per cohort; P� 0.0001; log-rank test). Taken together, these data illustrate favorable antitu-mor activity of NVP-BEZ235 in Pten-deficient tumors.

We next evaluated the effect of NVP-BEZ235 exposure in theKras-mutant setting, given that Kras is known to activate PI3Ksignaling through direct interaction with p110a (23), and ourconfirmatory observation of this activation (Supplementary Fig.S1) in Apcf/þ KrasLSL/þ colon tumors. Western blot analysis ofshort-term treatment of NVP-BEZ235 revealed modest inhibitionof AKT signaling at Ser473 and Thr308, but substantial inhibition

of signaling downstream of mechanistic target of rapamycin(mTOR) complex 1 as assessed by levels of pS6RP (Fig. 1B).These molecular changes were accompanied by increased apo-ptotic signaling through cleaved caspase-3 (Fig. 1D; P � 0.05;Mann–Whitney U test; Supplementary Fig. S2), suggesting asignificant antitumor effect despite variable inhibition of PI3Ksignaling. In the long-term setting, NVP-BEZ235 treatment from100 DPI significantly increased survival of Apcf/þ KrasLSL/þ micefrom amedian of 153 to 343 DPI (Fig. 2B; n� 12 per cohort; P�0.0001; log-rank test), indicating considerable dependence ofKras-mutant tumors on PI3K/mTOR signaling. These data high-light the potential benefit of targeting PI3K signaling in Kras-mutant colorectal cancers.

Mutations in KRAS and those activating PI3K signaling coexistin a third of all colorectal cancers. In our Apcf/þ Ptenf/f KrasLSL/þ

mousemodel, although neither activation ofMAPK signaling nor

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Figure 1.NVP-BEZ235 leads to differential inhibition of PI3K/mTOR signaling across tumor models and activation of apoptosis. Western blot analysis of tumor lysates fromApcf/þ Ptenf/f small intestinal tumors (SIT; A), Apcf/þ KrasLSL/þ colon polyps and SITs (B), and Apcf/þ Ptenf/f KrasLSL/þ SITs (C) exposed to vehicle or 35-mg/kgNVP-BEZ235 for 4 hours. Immunoblotting with antibodies against effectors of the PI3K/mTOR pathway revealed a marked reduction in signaling across thegenotypes. Cleaved caspase-3 (D) and BrdUrd (E)-positive cells per microscopic field were scored from short term (4 hours) vehicle (blue bar) and NVP-BEZ235(red bar)–treated tumors. A significant increase in cleaved caspase-3 staining was observed for all genotypes and a significant reduction in BrdUrd-positivecells was observed in Apcf/þ Ptenf/f tumors. Data represent average of five different fields per tumor; n � 3 mice per cohort; error bars, standard deviation.(� , P � 0.05; Mann–Whitney U test).

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hyper-activation of PI3K signaling was observed in intestinaltumors (Supplementary Fig. S1),mice have a significantly reducedlifespan and present with more tumors at death (13). Despite theincreased number, Apcf/þ Ptenf/f KrasLSL/þ mice predominantlypresent with noninvasive adenomas similar to Apcf/þ KrasLSL/þ

mice (Supplementary Figs. S5A, S6A, and S7A), and as reflected inBrdUrd scoring of vehicle-treated tumors (Fig. 1e). Short-termexposure to NVP-BEZ235 resulted in minimal perturbation ofpAKT473 and pAKT308 levels, but pS6RP and p4EBP1 weresuppressed as similarly observed in the Apc Pten and Apc Krasmodels. Our data therefore show that concurrent activation ofboth PI3K and Kras diminishes the ability of NVP-BEZ235 toreduce signaling immediately downstream of PI3K (Fig. 1C).Nevertheless, levels of apoptosis as scored through cleaved

caspase-3 were found to be increased (Fig. 1D; P � 0.05;Mann–Whitney U test; Supplementary Fig. S2). Long-term inter-vention using NVP-BEZ235 from 22DPI in this tumormodel wasfound to significantly increase survival of mice from a median of40 to 104 DPI (Fig. 2C; n � 13 per cohort; P � 0.0001; log-ranktest).

Despite the favorable effects on survival in all three tumormodels, NVP-BEZ235 only reduced the total number of lesions inApcf/þ Ptenf/f mice as assessed at the time of death (Fig. 2D).However, such tumors were of increased size as assessed by totalarea (Supplementary Fig. S4A) and an increased proportion were"advanced invasive adenocarcinomas", defined as showing inva-sion through themusclewall (Supplementary Fig. S4B). Althoughthese observations may indicate a phenotype of resistant tumor

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Figure 2.Continuous NVP-BEZ235 treatment extends survival of all tumor models and reduces tumor number in Apcf/þ Ptenf/f mice. The Kaplan–Meier survival analysis ofApcf/þ Ptenf/f (A), Apcf/þ KrasLSL/þ (B), and Apcf/þ Ptenf/f KrasLSL/þ (C) mice on vehicle (blue line) or 35-mg/kg NVP-BEZ235 (green line) twice daily by oralgavage revealed significantly increased survival across all genotypes (median survivals: Apcf/þPtenf/f vehicle¼99days vs. NVP-BEZ235¼ 266DPI, Apcf/þKrasLSL/þ

vehicle ¼ 153 vs. NVP-BEZ235 ¼ 343 DPI, Apcf/þ Ptenf/f KrasLSL/þ vehicle ¼ 40 vs. NVP-BEZ235 ¼ 104 DPI; P � 0.0001, log-rank test) Treatment start points:Apcf/þ Ptenf/f ¼ 77DPI, Apcf/þ KrasLSL/þ ¼ 100DPI, Apcf/þ Ptenf/f KrasLSL/þ ¼ 22DPI. D, tumors at death were scored from H&E stained intestinal sectionsfor each cohort. A significant reduction in tumor number was observed for Apcf/þ Ptenf/f mice on NVP-BEZ235 (N-B) treatment compared with vehicle (V).� , P � 0.05; n � 12 per cohort, Mann–Whitney U test.

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growth in response to chronic NVP-BEZ235 dosing, this inter-pretation is confounded by the animals' longevity—treated micesurvived much longer than controls (median, 266 vs. 100 days).In contrast, analysis of tumors arising in Apcf/þ KrasLSL/þ micerevealed that while NVP-BEZ235 treatment had no effect on thetotal burden of colon tumors, it was associated with a significantreduction in the total tumor area of small intestinal tumors, albeitwithout any change in the total number of lesions present or intheir invasive characteristics (Supplementary Figs. S5A, S5B, S6A,and S6B). The later was also the case for Apcf/þ Ptenf/f KrasLSL/þ

lesions following long-term NVP-BEZ235 exposure (Supplemen-tary Fig. S7A). Interestingly, histologic examination showed thatmany of the intestinal tumors in the treated Apcf/þ Ptenf/f

KrasLSL/þ cohort contained areas of incipient or frank ulceration

(Supplementary Fig. S7B and S7C). A smallminority of thesewereulcerating adenocarcinomas, but most were noninvasive adeno-mas showing a spectrum of degenerative changes that wereinterpreted to represent progressive tumor destruction/regressionleading to loss of mucosal barrier integrity and ulceration.

MEK inhibition throughMEK162 inmodels of colorectal cancerGiven that activation of oncogenic Kras reduced the ability of

NVP-BEZ235 to completely inhibit PI3K signaling, we next inves-tigated the consequences of inhibitingMAPK signaling though theMEK1/2 inhibitor MEK162 in all three tumor models.

In Apcf/þ Ptenf/f mice, short-term exposure toMEK162 reducedlevels of pERK and surprisingly also pAKT308 (Fig. 3A), highlight-ing the complexity of targeting closely associated signaling

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Figure 3.MEK inhibition reduces MAPK signaling but results in differential modulation of PI3K signaling across the tumor models. Western blot analysis of tumor lysates fromApcf/þ Ptenf/f small intestine tumors (SITs; A), Apcf/þ KrasLSL/þ colon polyps and SITs (B), and Apcf/þ Ptenf/f KrasLSL/þ SITs (C) exposed to vehicle or 30-mg/kgMEK162 for 4 hours. Immunoblotting with antibodies against the MAPK effector pERK revealed decreased signaling across the genotypes. An increasein PI3K/mTOR signaling was observed in some Apcf/þ KrasLSL/þ tumors; however, a reduction in signaling was observed in Apcf/þ Ptenf/f KrasLSL/þ tumors.Cleaved caspase-3 (D) and BrdUrd-positive cells (E) per microscopic field were scored from short-term (4 hours) vehicle (blue bar) and MEK162 (red bar)-treatedtumors. A significant increase in cleaved caspase-3 staining was observed in Apcf/þ KrasLSL/þ colon polyps and Apcf/þ Ptenf/f KrasLSL/þ SITs. A reduction inBrdUrd-positive cells was observed in Apcf/þ Ptenf/f tumors, and a significant increase in BrdUrd-positive cells in Apcf/þ Ptenf/f KrasLSL/þ tumors. Data representaverage of five different fields per tumor; n � 3 mice per cohort; error bars, standard deviation. � , P � 0.05, Mann–Whitney U test.






cascades. Although acute MEK inhibition significantly reducedproliferation, (Fig. 3E; P � 0.05; Mann–Whitney U test; Supple-mentary Fig. S3), there was no effect on survival of Apcf/þ Ptenf/f

mice when administered MEK162 continuously from 77 DPI(Fig. 4A, median survival: vehicle ¼ 99 days vs. MEK162 ¼101 DPI; n � 15; P � 0.05, log-rank method)

In Apcf/þ KrasLSL/þ mice, short-term MEK162 exposureincreased apoptosis through cleaved caspase-3 in Apcf/þ

KrasLSL/þ colon tumors (Fig. 3d; P� 0.05; Mann–WhitneyU test)and abolished detectable ERK signaling (Fig. 3B). Interestingly,Apcf/þ KrasLSL/þ colon tumors displayed variable alterations inPI3K/mTOR signaling following MEK inhibitor treatment asassessed by Western blot analysis of pAKT308 and pS6RP, sug-gesting heterogeneous compensatory activation of the closely

associated PI3K signaling cascade (Fig. 3B) and may reflect inter-tumor heterogeneity. Long-term MEK162 administration from100 DPI significantly increased survival of mice from amedian of153 to 287 days (Fig. 4b; P � 0.0001; n � 12; log-rank test).Although median survival of Apcf/þ KrasLSL/þ mice exposed toNVP-BEZ235 is approximately 60 days (343 DPI; Fig. 2b) longer,the difference is not statistically significant (P � 0.05; log-ranktest), indicating equipotent effects of NVP-BEZ235 and MEK162with regard to long-term survival in this genetic setting.

Short-term exposure to MEK162 in Apcf/þ Ptenf/f KrasLSL/þ

tumor-bearing mice led to a reduction in ERK signaling (Fig.3C), an induction of apoptosis (Fig. 3D; P� 0.05;Mann–WhitneyU test; Supplementary Fig. S2) and also a reduction in PI3K/mTOR signaling indicated by reduced pAKT308, pS6RP, and

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Figure 4.Long-term MEK inhibition only is beneficial for Apc Kras mice. The Kaplan–Meier survival analysis of Apcf/þ Ptenf/f (A), Apcf/þ KrasLSL/þ (B), and Apcf/þ

Ptenf/f KrasLSL/þ (C) mice on vehicle (blue line) or 30-mg/kg MEK162 (red line) twice daily by oral gavage revealed significantly increased survival ofApcf/þ KrasLSL/þ mice only (median survivals: Apcf/þ Ptenf/f vehicle ¼ 99 days vs. MEK162 ¼ 101 DPI, Apcf/þ KrasLSL/þ vehicle ¼ 153 vs. MEK162 ¼ 287 DPI;P � 0.0001, log-rank test, Apcf/þ Ptenf/f KrasLSL/þ vehicle ¼ 40 vs. MEK162 ¼ 36 DPI). Treatment start points: Apcf/þ Ptenf/f ¼ 77DPI, Apcf/þ KrasLSL/þ ¼ 100DPI,Apcf/þ Ptenf/f KrasLSL/þ ¼ 22DPI. Tumors at death were scored from H&E-stained intestinal sections for each cohort (D). A significant reduction in colonand small intestinal tumor number was observed for Apcf/þ KrasLSL/þ mice on MEK162 treatment (M) compared with vehicle (V). � , P � 0.05; n � 12 per cohort,Mann–Whitney U test.

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p4EBP1 levels (Fig. 3C). However, this was also accompanied byincreased cellular proliferation at this time point (Fig. 3e; P �0.05; Mann–Whitney U test, Supplementary Fig. S3). Long-termMEK162 exposure from 22 DPI did not deliver a significantsurvival benefit (Fig. 4c, median survival vehicle ¼ 40 days vs.MEK162 ¼ 36 days; P � 0.05; n � 13; Mann–Whitney U test),indicating that additional Pten loss negated the beneficial effect ofMEK162 observed in Apcf/þ KrasLSL/þ tumors.

Analysis of tumor burden at death following MEK162 treat-ment in all three tumor models echo the observations fromsurvival analysis, in that a significant reduction in the totalnumber of lesions was only observed in the responding Apcf/þ

KrasLSL/þ mice (Fig. 4D). Furthermore, significant alterations intumor area were only detected in the small intestine despite theincrease in invasive lesions (Supplementary Figs. S5A, S5B, S6A,and S6B). For Apcf/þ Ptenf/f mice, MEK162 had no significanteffect on tumor number, tumor area, or invasive characteristics(Fig. 4D; Supplementary Fig. S4A and S4B), whereas for Apcf/þ

Ptenf/f KrasLSL/þmice, although total tumor number appears to bereduced, the proportion of invasive lesions following MEK162treatment is slightly increased (Fig. 4D and Supplementary Fig.S7A), potentially attributable to the increased proliferation intumors observed from acute MEK inhibition.

Combinatorial therapy in models of colorectal cancerThe observations from long-term therapeutic and short-term

antitumor experiments of NVP-BEZ235 and MEK162 as singleagents, suggest combinatorial inhibition could provide addition-al therapeutic benefits in all tumormodels, in particular for Apcf/þ

KrasLSL/þ and Apcf/þ Ptenf/f KrasLSL/þ mice, as elements of cross-talk were more apparent in these settings. First, however, it wascrucial to establish an effective method of administering the twocompounds to achieve themost favorable antitumor effects. Threecombination strategies were chosen which involved administra-tion of 30 mg/kg MEK162 1 hour prior to (combo 1), after(combo 2), or at the same time (combo 3) as 35 mg/kg NVP-BEZ235. For all cohorts, mice were euthanized 4 hours followingthe final dose and samples were collected for evaluation of shortterm pharmacodynamics and antitumor activity. Probing oftumor lysates for effectors downstreamof PI3K/mTOR andMAPKsignaling revealed that Apcf/þ Ptenf/f tumors were particularlysensitive to the order of drug sequencing. In this tumor setting,combination strategy 2 (combo2) resulted inmaximal inhibitionof pERK and PI3K/mTOR effectors (Fig. 5A), as well as increasedapoptosis and reduced proliferation (Fig. 5E and F). Interestingly,MEK inhibition prior to or concurrently with NVP-BEZ235,reduced its ability to inhibit PI3K and mTOR signaling inPTEN-deficient tumors (Fig. 5A). These findings are unlikely dueto feedback activation of PI3K signaling as single-agent MEKinhibition in Apcf/þ Ptenf/f mice did not lead to compensatoryactivation of PI3K signaling in tumors (Fig. 3A).

Apcf/þ KrasLSL/þ tumors responded to the different combina-tion strategies in similar ways through comparably reduced levelsof pERK and pS6RP (Fig. 5C and S5C), and increased levels ofcleaved caspase 3 (Fig. 5e). No discernible alterations in the levelsof pAKT at either Ser473or Thr308, or in the levels of p4EBP1wereobserved (Fig. 5B and C). One potential explanation for thisapparent contradiction is that the inhibition of pS6RP may bethrough interactions between terminal ERK signaling and mTORrather than through AKT, as the level of p4EBP1 (which is alsodownstream of AKT/mTOR signaling) was unaltered. Neverthe-

less, the pharmacodynamic effects observed in Apcf/þ KrasLSL/þ

colon polyps may be due to an additive effect of combininginhibitors, given that MEK inhibition results in increased levels ofpAKT (Fig. 3b) and PI3K/mTOR inhibition in this setting pre-dominantly alters signaling downstream of mTOR (Fig. 1b),further providing rationale for long-term combination therapyin this tumor setting.

Similarly, Apcf/þ Ptenf/f KrasLSL/þ tumors displayed parallels inresponse to the varied combination strategies. Although levels ofpERK and pS6RP were found to be reduced with all three strat-egies, pAKT473 was only reduced following combo 3 (Fig. 5D). Amarked increase in apoptosis through cleaved caspase-3 wasobserved for all three strategies (Fig. 5E), indicating proapoptoticsignaling. Proliferation was increased with both combo 1 and 2,similar to our observations with single-agent MEK162, possiblysignifying an adaptive response (Fig. 5F). Nevertheless, given thatApcf/þ Ptenf/f KrasLSL/þ mice responded dramatically to single-agent NVP-BEZ235 in the long term but acutely showed onlymodest pathway inhibition, the lack of complete pathway inhi-bition from the short-term combination studies was not regardedas unfavorable. Therefore, despite some differences in response tothe combination strategies between the tumor models, combi-nation strategy 2,which appeared to bebeneficial for all cohorts interms of favorable pharmacodynamics and antitumor activity,was chosen as the strategy for long-term combination treatment.

In the Apcf/þ Ptenf/f setting, mice were exposed using a dailyregimen of combo 2 (NVP-BEZ235 1 hour prior to MEK162),followed 8 hours later by an additional daily administration ofNVP-BEZ235. This regimen is termed Combo R1 and was used toensure tolerability, as twice-daily combo 2 exposure was found tocause weight loss. Although combo R1 was found to significantlyincrease survival of mice compared with vehicle controls, (Figure6a, median survival: vehicle¼ 99 days vs. combo¼ 270 DPI; n�15; P � 0.0001; log-rank method) this did not provide anyadditional benefit to single agent NVP-BEZ235 (Fig. 6A, mediansurvival: NVP-BEZ235¼ 266 days vs. combo¼ 270DPI; n� 15; P� 0.05; log-rank test). Assessment of tumors at death revealedfurther similarities to single-agent NVP-BEZ235: total tumornumbers (Fig. 6D; P � 0.05; Mann–Whitney U test), tumor area(Supplementary Fig. S4A), and the proportion of invasive lesions(Supplementary Fig. S4B) were found to be similar to NVP-BEZ235, indicating that the addition of MEK162 in this settingprovided no additional benefits for survival or tumor burden.These data strongly suggest that PI3K and mTOR inhibition iseffective and sufficient for a therapeutic response in PTEN-deletedintestinal tumors.

For combination treatment experiments inApcf/þKrasLSL/þ andApcf/þ Ptenf/f KrasLSL/þ mice, the dosing regimen was furtherreduced to only a single daily combo 2 regimen (NVP-BEZ2351 hour prior to MEK162) to ensure tolerability. This is referred toas combo R2. Subsequently, additional control arms of singleagentswere undertaken to establish the full effects of combinationtreatment. In Apcf/þKrasLSL/þmice, the reduced once-daily (O-D)NVP-BEZ235 and MEK162 remained equipotent (Fig. 6b). Inaddition, NVP-BEZ235 elicited effects in a dose-dependent man-ner, but MEK162 reached maximal effects with the once-dailydosing regimen, in terms of survival benefit (Figs. 2B, 4B, and 6B).Although the reduced single-agent doses resulted in similar colontumor severity profiles, the total tumor area appears increasedcompared with higher dose regimens, suggesting that singletreatments are more permissive for colon tumor growth






(Supplementary Fig. S5A and S5B). In contrast, small intestinallesions fromonce daily–treatedmice appeared similarwith regardto tumor area and severity in comparison with tumors frommice

on twice-daily treatments, suggesting comparable sensitivities ofsmall intestinal tumors to the long-term treatments (Supplemen-tary Fig. S6A and A6B).

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Figure 5.Sequencing of combination is crucial for effective inhibition of MEK–ERK and PI3K–AKT signaling in Apc Pten tumors. Western blot analysis of tumor lysates fromApcf/þ Ptenf/f small intestinal tumors (SITs; A), Apcf/þ KrasLSL/þ colon polyps and SITs (B and C), and Apcf/þ Ptenf/f KrasLSL/þ SITs (D) mice exposed to thedifferent combination strategies. Immunoblottingwith antibodies against downstreameffectors ofMAPK and PI3K/mTORpathways revealed complete inhibition ofsignaling only with combo 2 in Apcf/þ Ptenf/f tumors. Apcf/þ KrasLSL/þ and Apcf/þ Ptenf/f KrasLSL/þ tumors were less sensitive to the order of sequencingand all combinations reduced signaling MAPK and PI3K/mTOR signaling through pERK and pS6RP, respectively. Scoring of cleaved caspase-3 (E) andBrdUrd-positive cells (F) in tumors from mice exposed to vehicle (blue bar), combo 1 (red bar), 2 (green bar), and 3 (purple bar). A significant increase incleaved caspase-3 scoring was observed with combo 2 and 3 in Apcf/þ Ptenf/f tumors, with combo 2 and 3 in Apcf/þ KrasLSL/þ colon polyps and with all threecombination strategies in Apcf/þ KrasLSL/þ and Apcf/þ Ptenf/f KrasLSL/þ SITs. A significant reduction in BrdUrd-positive cells was observed with combo 1and 2 in Apcf/þ Ptenf/f tumors and a significant increase in staining with combo 2 and 3 in Apcf/þ KrasLSL/þ and combo 1 and 2 in Apcf/þ Ptenf/f KrasLSL/þ SITs.Data represent average of five different fields per tumor, n � 3 mice per cohort, error bars represent standard deviation. � , P � 0.05, Mann–Whitney U test.

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Combination treatment in Apcf/þ KrasLSL/þ mice was found tobe well tolerated and resulted in an additive increase in mediansurvival compared with single agents (Fig. 6b, median survivals:vehicle¼ 153 days vs. NVP-BEZ235 O-D¼ 238 days vs. MEK162O-D ¼ 286 days vs. combo R2 ¼ 389 DPI, all comparisons aresignificant at P� 0.0001 except forMEK162O-D vs. NVP-BEZ235O-D P � 0.05; log-rank method). In the colon, combinationtreatment was found to result in reduced tumor number, but nosignificant change in tumor area or severity, despite the increase inelapsed time from treatment start point, potentially indicatingthat treatment results in restriction of both tumor growth and

progression (Fig. 6D and Supplementary Fig. S5A and A5B).Contrastingly, in the small intestine, combined NVP-BEZ235 andMEK162 administration resulted in reduced tumor number aswell as reduced tumor area, indicating potent effects on tumorgrowth (Fig. 6D and Supplementary Fig. S6A and S6B).

Finally, combination treatment (combo R2) administered toApcf/þPtenf/f KrasLSL/þmice also resulted in an increase in survivalwhen compared with vehicle cohorts (Fig. 6C, median survivals:vehicle¼40days vs. combo¼125DPI;P�0.0001; log-rank test).Analysis of tumors at death indicated no difference in the numberof lesions (Fig. 6D), but did also uncover the presence of tumor

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Figure 6.Long-term evaluation of combination treatment in Apc Pten, Apc Kras, and Apc Pten Kras mice. The Kaplan–Meier survival analysis of Apcf/þ Ptenf/f (A),Apcf/þ KrasLSL/þ (B), and Apcf/þ Ptenf/f KrasLSL/þ (C) mice on vehicle (blue line) or combination (purple line) with control NVP-BEZ235 (green line) andMEK162 (red line) treatments. Administration of combination treatment significantly increased survival of Apcf/þ Ptenf/f mice compared with vehicle, howevernot compared with single-agent NVP-BEZ235. For Apcf/þ KrasLSL/þ mice, combination treatment prolonged longevity and resulted in an additive increasein survival when compared with single-agent MEK162 and NVP-BEZ235. In Apcf/þ Ptenf/f KrasLSL/þ mice, combined treatment synergistically increased survival ofmice compared with NVP-BEZ235 and MEK162 alone. (Median survivals: Apcf/þ Ptenf/f vehicle ¼ 99 days vs. combo R1 ¼ 270 DPI; P � 0.0001, log-rank test;Apcf/þKrasLSL/þ vehicle¼ 153 vs. comboR2¼ 389DPI;P�0.0001, log-rank test; Apcf/þPtenf/f KrasLSL/þ vehicle¼40vs. combo¼ 125 DPI;P�0.001, log-rank test.)Treatment start points: Apcf/þ Ptenf/f ¼ 77DPI, Apcf/þ KrasLSL/þ ¼ 100DPI, Apcf/þ Ptenf/f KrasLSL/þ ¼ 22DPI. Scoring of tumors from H&E-stained sections ofintestines (D) revealed a significant reduction in tumor number in those receiving combination therapy (C) compared with vehicle (V) in the Apcf/þ Ptenf/f

and Apcf/þ KrasLSL/þ cohorts (� , P � 0.05; n � 12 per cohort, Mann–Whitney U test).






ulceration suggestive of signs of tumor destruction/regression, asdescribed previously (Supplementary Fig. S7B and S7C). Despitethis, proportionally more invasive lesions characterized by sub-mucosal invasion were observed in the combination-treatedcohort, which may be due to either resistant tumor growth orsimply the increase in time elapsed (Supplementary Fig. S7A).Nevertheless, combination therapy resulted in a synergistic benefitin survival when compared with single-agents NVP-BEZ235 andMEK162, as when reduced to once-daily administration NVP-BEZ235 no longer elicited any survival benefit in Apcf/þ Ptenf/f

KrasLSL/þ mice (Fig. 6C; median survival: vehicle ¼ 40 days vs.NVP-BEZ235 O-D ¼ 36 DPI, P � 0.05; log-rank test). These datafurther suggest dose-dependent effects of reduced NVP-BEZ235similar to those seen in the Apcf/þ KrasLSL/þ mice. Furthermore,analysis of tumors at the end of treatment revealed that propor-tionally more superficially invasive carcinomas were present inNVP-BEZ235 once daily–treated mice in comparison with vehicleandNVP-BEZ235 twice-dailymice. Together, thesedata suggest thedose of NVP-BEZ235 used for therapeutic antagonism is critical.

DiscussionAlthough the promise of stratified medicine is currently far

from fulfilled, support for the notion of tailoring medicines for aspecific patient population remains strong, for example as evi-denced by the development of imatinib to specifically treatchronic myeloid leukemia and gastrointestinal stromal tumors.Here, we use robust and reliable GEMMs to assess the effect ofgenetic variation on drug response, in the context of murineintestinal neoplasia, with likely relevance to human colorectalcancer to inform clinical trials such as the FOCUS 4 trial. Giventhat mutations activating PI3K/AKT and RAS/MEK/ERK signalingcoexist in a third of all colorectal cancers, the efficacy of the dualPI3K/mTOR inhibitor NVP-BEZ235 and the MEK1/2 inhibitorMEK162 as single agents and in combination was addressed inthree autochthonous tumor models with differing combinatorialmutations in Apc, Pten, and Kras.

We have shown that dual PI3K and mTOR inhibition usingNVP-BEZ235 is beneficial therapeutically in the Apcf/þ Ptenf/f

setting in particular, but also for Apcf/þKrasLSL/þ andApcf/þPtenf/f

KrasLSL/þmice, evidenced by increasedmedian survivals (Fig. 2A–C). Thismaybe attributable to the role of PI3K/mTOR signaling ina number of crucial cellular processes, including cell survival andproliferation through activation of Akt. Phosphorylation andactivation of Akt through PDK1 at Thr308 and at Ser473 bymTORcomplex 2 (TORC2) leads to subsequent activation of TORC1signaling which regulates protein synthesis through activation ofribosomal protein S6 kinases and the eukaryotic initiation factor4E binding protein (4E-BP1; refs. 24, 25). In addition, Akt hasinhibitory effects on negative regulators of the cell cycle, includingp27 (kip1; ref. 26) and p21 (cip1; ref. 27), but also elicitsinhibitory effects on GSK-3, thus activating c-Myc and cyclinD1 that promote progression of the cell cycle (28). PI3K signalingis also critical for regulating cell death through inhibition of theproapoptotic factors Fas, Bim, and Bad (29–31) and regulatingautophagy and cellular metabolism, processes implicated inmediating increased longevity. A number of these properties ofPI3K signalingwere evaluated immediately after exposure toNVP-BEZ235 in our three models to determine the pharmacodynamicand antitumor effects of NVP-BEZ235. Here, Apcf/þ Ptenf/f micedisplayed substantial sensitivity to NVP-BEZ235 as complete

pathway inhibition was coupled with favorable increases inapoptotic signaling and reduced proliferation (Fig. 1A, D, andE). Interestingly, Kras-mutant tumors exhibited greater sensitivityfor TORC1 inhibition than PI3K inhibition (Fig. 1B), which maybe a result of promiscuous Kras activation of PI3K signalingthrough interactions with P110 (23). Furthermore, this effect wasexacerbated in the presence of concurrent Pten deletion andsuggests that oncogenic Kras prevents the ability of NVP-BEZ235to completely reduce AKT signaling (Fig. 1c). If indeed this effect isattributable to the presence of oncogenic Kras signaling, thishighlights the potential advantages of MEK inhibition in thesetumor settings.

Given the above observations, MEK inhibition was next inves-tigated usingMEK162 as single-agent therapy in our threemodelsof intestinal tumorigenesis. MEK inhibition in Apcf/þKrasLSL/þ

mice was found to be equipotent with PI3K/mTOR inhibition interms of survival (Figs. 2B, 4B, and 6B), suggesting equivalentdependence of KRAS-mutant tumors on PI3K/AKT and RAS/ERKsignaling. This property of KRAS-mutant tumors appears to becontext dependent. Similarly to the observations we report here,independent MEK and PI3K inhibition have been shown to beeffective in KRAS melanoma models (9); however, KRAS-mutantpancreatic and lung tumors were found to be more responsive toMEK rather than PI3K inhibition (8, 10). Our data also show thatadditional PTEN deletion renders otherwise sensitive KRAS-mutant tumors nonresponsive to MEK inhibition (Fig. 4C). Thisfinding is in accordance with previous studies and providesfurther evidence for the notion that KRASmutational status aloneis not sufficient as a prognostic marker for response to MEKinhibition (32–34).Moreover, given that Apcf/þPtenf/fmice showno response to long-term MEK inhibition (Fig. 4A), mutationsactivating the PI3K pathway, such as PTEN deletion, can also beused to predict nonresponse to MEK inhibitors in the KRAS wild-type tumor setting.

Although single-agents NVP-BEZ235 andMEK162 substantial-ly improved survival of mice as described previously, it wasanticipated that combination therapy may further increase thisbenefit. Despite the attractiveness of combinatorial therapy, thereis currently little data in the literature to direct the most appro-priate dosing schedule for combination of PI3K and MEK inhi-bitors. This is crucial, as while agents may be effective as singleagents, antagonism between two agents when combined, espe-cially given cross-talk between the two pathways may be evident.Furthermore, due to overlapping sensitivities, the combinationmay result in no net clinical gain when administered jointly (35).To address some of these issues, we chose to investigate threevaried combination strategies that differed in the order of com-pound administration. Here, MEK162 was administered 1 hourprior to (combo 1), after (combo 2), or at the same time (combo3) as NVP-BEZ235 to determine whether scheduling is key toachieve concomitant pathway inhibition. Interestingly, sensitivityto the scheduling was primarily detected in Apcf/þ Ptenf/f tumors,whereby MEK162 administered prior to or at the same time asNVP-BEZ235 diminished sensitivity to complete PI3K andmTORinhibition (Fig. 5a). Given that the most favorable antitumoreffects, in terms of increased apoptosis and reduced proliferation,were also observed by this strategy (combo 2), it was surprisingthat long-term administration provided no additional benefits tosingle-agent NVP-BEZ235 (Figs. 5E and F and 6A). It is possiblethat while MEK inhibition may lead to favorable pharmacody-namic effects in combination acutely, MAPK signaling is not

Raja et al.





required for tumormaintenance inApcf/þPtenf/fmiceand thereforepathway inhibition does not lead to a synergistic effect in the longterm. In direct contrast to the Apcf/þ Ptenf/f setting, Apcf/þKrasLSL/þ

and Apcf/þ Ptenf/f KrasLSL/þ tumors in response to the short termcombination strategies displayed less effective and less variation ininhibition of both pathways (Fig. 5B–D). Nevertheless, the obser-vation that long-term combo 2 administration increased mediansurvival additively in Apcf/þKrasLSL/þ mice and synergistically inApcf/þ Ptenf/f KrasLSL/þmice indicates that while some antagonismmay be prevalent as suggested by the short-term combinationstudies, the two pathways are essential in tumor maintenance(Fig. 6B and C). These data suggest that combination therapy inKRAS-mutant settings could provide substantial benefits.

In summary, we have performed a systematic preclinical studythat effectively evaluates rational therapeutic strategies for Apc,Pten, and Kras mutant colorectal cancer with the aim of identi-fying optimal clinical strategies. Our data show that PI3K/mTORinhibition enhances survival in both Kras-mutant and Pten-mutant settings. In contrast, MEK inhibition is only effective ina Kras-mutant background and this is overridden by additionalPten mutation. Critically, we also demonstrate true synergybetween the two therapies, but only in the presence of all three(Apc, Kras, and Pten)mutations. Taken together, our data confirmthe notion that specific pathway targeting is effective, at least withthe GEMMs used here, and so support the general concept ofstratified approaches to therapy. Our data also highlight bothsynergies and limitations in the use of therapeutic combinations,which occur in a genotype specific manner. Such studies toidentify stratified approaches have been previously conductedfor lung and ovarian cancers (7, 33); however, this is the first suchstudy conducted using GEMMs for colorectal cancer. As such, ourstudies should inform human clinical trials such as the FOCUS 4trial (http://www.focus4trial.org/).

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: M. Raja, A.R. Clarke, P.H.S. ShawDevelopment of methodology: M. Raja, P.H.S. ShawAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M. Raja, M. Zverev, K. SeipelAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M. Raja, K. Seipel, G.T. Williams, A.R. Clarke, P.H.S.ShawWriting, review, and/or revision of the manuscript: M. Raja, G.T. Williams,A.R. Clarke, P.H.S. ShawAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): M. Zverev, K. SeipelStudy supervision: A.R. Clarke, P.H.S. Shaw

AcknowledgmentsThe authors thank Mark Bishop and Lucie Taylor for genotyping of animals,

Fangli Chen for microscopy assistance, Derek Scarborough and Mark Isaac forhistology services, andWolfgangHackl andNovartis Pharmaceuticals for kindlysupplying NVP-BEZ235 and MEK162.

Grant SupportM. Raja was funded by Tenovus and NISCHR, M. Zverev was funded by

Cancer Research UK, and P.H.S. Shaw was funded by Cancer Research UK(Bobby Moore Fellowship), Welsh Deanery as Walport Clinical Lecture, andNISCHR (Welsh Government).

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 18, 2015; revised July 13, 2015; accepted July 18, 2015;published OnlineFirst July 23, 2015.

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Raja et al.




2015;14:2175-2186. Published OnlineFirst July 23, 2015.Mol Cancer Ther Meera Raja, Matt Zverev, Katja Seipel, et al. Genetically Defined Models of Colorectal Cancer

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