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Small Molecule Therapeutics

Intermittent High-Dose Scheduling of AZD8835,a Novel Selective Inhibitor of PI3Ka and PI3Kd,Demonstrates Treatment Strategies forPIK3CA-Dependent Breast CancersKevin Hudson1, Urs J. Hancox1, Cath Trigwell1, Robert McEwen1, Urszula M. Polanska2,Myria Nikolaou1, Pablo Morentin Gutierrez2, Alvaro Avivar-Valderas1, Oona Delpuech2,Phillippa Dudley1, Lyndsey Hanson1, Rebecca Ellston1, Alys Jones1, Marie Cumberbatch1,Sabina C. Cosulich2, Lara Ward1, Francisco Cruzalegui1, and Stephen Green1

Abstract

The PIK3CA gene, encoding the p110a catalytic unit of PI3Ka,is one of the most frequently mutated oncogenes in humancancer. Hence, PI3Ka is a target subject to intensive efforts inidentifying inhibitors and evaluating their therapeutic potential.Here, we report studies with a novel PI3K inhibitor, AZD8835,currently in phase I clinical evaluation. AZD8835 is a potentinhibitor of PI3Ka andPI3Kdwith selectivity versus PI3Kb, PI3Kg ,and other kinases that preferentially inhibited growth in cells withmutant PIK3CA status, such as in estrogen receptor–positive(ERþ) breast cancer cell lines BT474, MCF7, and T47D (sub-mmol/L GI50s). Consistent with this, AZD8835 demonstratedantitumor efficacy in corresponding breast cancer xenograft mod-els when dosed continuously. In addition, an alternativeapproach of intermittent high-dose scheduling (IHDS) wasexplored given our observations that higher exposures achieved

greater pathway inhibition and induced apoptosis. Indeed,using IHDS, monotherapy AZD8835 was able to induce tumorxenograft regression. Furthermore, AZD8835 IHDS in combi-nation with other targeted therapeutic agents further enhancedantitumor activity (up to 92% regression). Combination part-ners were prioritized on the basis of our mechanistic insightsdemonstrating signaling pathway cross-talk, with a focus ontargeting interdependent ER and/or CDK4/6 pathways or alter-natively a node (mTOR) in the PI3K-pathway, approaches withdemonstrated clinical benefit in ERþ breast cancer patients. Insummary, AZD8835 IHDS delivers strong antitumor efficacy ina range of combination settings and provides a promisingalternative to continuous dosing to optimize the therapeuticindex in patients. Such schedules merit clinical evaluation. MolCancer Ther; 1–13. �2016 AACR.

IntroductionThe PI3K-AKT-mTOR pathway is a critical regulator of many

cellular processes including proliferation, survival, and transfor-mation, and is one of the most frequently deregulated signalingnetworks inhuman cancer (1). ThePI3K family of lipid kinases arekey components of this pathway, in particular, the Class I PI3Ksubfamily which comprise PI3Ka, PI3Kb, and PI3Kd (Class IA)and PI3Kg (Class IB).

Different PI3K isoforms have been shown to play key roles indistinct tumor types, often linked with genetic alterations. Inparticular, PI3Ka is a commonly deregulated oncoprotein in

human cancer (1). Activating somatic point mutations in thePIK3CA gene (encoding p110a catalytic subunit of PI3Ka) havebeen widely reported across many tumor types and these muta-tions often display a transforming gain-of-function activity (2–6).Additional reported mechanisms of deregulation of PI3Kainclude amplification of the PIK3CA gene (7, 8), mutations ina PI3K regulatory subunit (9) or activating events elsewhere in thesignaling pathway. Hence PI3Ka is a target subject to intensiveefforts in identifying inhibitors and evaluating their therapeuticpotential. Thus far, clinical activity observed has been moderate(10, 11), although more promising data have recently beenreported with more selective PI3K-inhibitors such as BYL719(alpelisib; refs. 12, 13) and GDC0032 (taselisib; ref. 14).

In this report, we describe preclinical studies with AZD8835, anovel dual inhibitor of PI3Ka and PI3Kd, currently in phase Iclinical evaluation. A particular focus of our preclinical studieswas to evaluate intermittent high-dosing schedules (IHDS) as analternative to a commonly applied default of continuous dailydosing. Several considerations motivated us to explore this path.First, continuous dosing may achieve suboptimal pathway inhi-bition in tumors, because PI3K-inhibitor clinical dose and expo-sure on continuous dosing schedules are capped by normaltissue toxicities such as hyperglycemia, diarrhea, and rash (12,13, 15). Also, additional dose reduction may be required when

1AstraZeneca Pharmaceuticals, Oncology iMed, Macclesfield, Che-shire, United Kingdom. 2AstraZeneca Pharmaceuticals, CRUK-CI Li KaShing Centre, Cambridge, United Kingdom.

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

CorrespondingAuthor:KevinHudson, AstraZeneca Pharmaceuticals, OncologyiMed, Alderley Park, Macclesfield, Cheshire, SK10 4TG, UK. Phone: 44-1625-516193; Fax: 44-1625-519749; E mail: [email protected]

doi: 10.1158/1535-7163.MCT-15-0687

�2016 American Association for Cancer Research.

MolecularCancerTherapeutics

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PI3K-inhibitors are dosed continually in combination with othertherapies (16). So, an intermittent schedule that allows higherdoses may permit greater target inhibition and also allow for abeneficial recovery period from dose-limiting physiological sideeffects such as rash. Second, it is reported widely across biologicsystems, including the PI3K-pathway, that continuous exposureand pathway inhibition lead to pathway reactivation via pertur-bation of pathway feedback mechanisms, leading to reducedeffect over time (17, 18). In contrast, intermittent dosing mayallow a "reset" of pathway signaling, allowing repeat apoptoticresponse on re-exposure to compound.

Here, reporting on preclinical studies in estrogen receptorpositive (ERþ), mutant PIK3CA (mPIK3CA) breast models, weillustrate that monotherapy IHDS with AZD8835 achieved goodantitumor efficacy. In particular, we exemplify how this schedulewas used in combination with other agents to deliver profoundantitumor regression.

Materials and MethodsCell line studies

Cells lines were grown in RPMI-1640 media, 10% FCS, 2mmol/L glutamine at 37�C/5% CO2 unless indicated otherwise.Cell lines used for core in vitro and in vivo experiments are listed,along with source, dates of acquisition, relevant molecular pro-filing details and AZD8835 antiproliferative potencies, in Sup-plementary Table S1. Cell line panel details are shown in Sup-plementary Table S2. All cell lines were authenticated via theAstraZeneca (AZ) Cell Bank using DNA fingerprinting shorttandem repeat (STR) assays. All revived cells were used within20 passages, and cultured for less than 6 months.

Compounds and formulationCompoundsAZD8835, fulvestrant, AZD9496, palbociclib, and

AZD2014 were all synthesized at AZ.For in vitro studies, compounds were prepared as 10 mmol/L

stocks in DMSO, stored under nitrogen, and dispensed resultingin final DMSO concentration less than 0.5%.

For in vivo studies, AZD8835was formulated as a suspension inHPMC/Tween [0.5% hydroxypropyl methocellulose (Methocel(Colocorn))/0.1% Polysorbate 80] and dosed per oral once (QD)or twice (BID) daily at a dose volume of 0.1 mL/10 g mouse. ForBIDdosing, AZD8835was administered 6 to 8hours apart (exceptthe glucose/insulin timecourse study in CD1 mice were 12 hoursapart). Fulvestrant was formulated in peanut oil (Sigma) anddosed three times weekly subcutaneously (s.c.) at 5 mg/mouse atdose volumeof 0.1mL/mouse. AZD9496,was formulated in 40%Polyethylene glycol (PEG)/30% Captisol and protected fromlight. It was dosed per oral QD at a dose volume of 0.1 mL/10g mouse. Where dosed in combination, it was dosed 1 hour afterthe morning dose of AZD8835. Palbociclib and AZD2014 wereformulated and dosed as for AZD8835, and coformulated whendosed with AZD8835. QD dosing was always a morning dose.

Caspase inhibitor QVD was from Sigma (SML0063).

Profiling of AZD8835 in in vitro enzyme and cellular assaysSee elsewhere for details (19). Inhibition of recombinant

PI3Ka, PI3Kb, PI3Kd, and PI3Kg was evaluated in ADP KinaseGlo-based enzyme assays at AZ. Broader kinase selectivity profilewas determined via kinase panels atUniversity ofDundee (UnitedKingdom) and DiscoveRx (KinomeScan). Cellular inhibitory

activity against each of the Class I PI3K isoforms was determinedby measuring phosphorylation of AKT protein in four differentcell lines under assay conditions where signaling was dependenton each of the individual isoforms; cell lines were BT474 (PI3Ka),MDA-MB-468 (PI3Kb), JeKo-1 (PI3Kd), and RAW264 (PI3Kg). Inaddition, selectivity against additional PI3K-related kinase (PIKK)family members, mTOR, DNAPK, ATR, and ATM, was demon-strated via bespoke assays at AZ.

Western blottingCell or ex vivo lysates were generated in ice-cold lysis buffer

containing phosphatase and protease inhibitors. Tumors werehomogenized and sonicated in ice-cold lysis buffer. Samples wereprepared in a reducing loading buffer, separated on 4% to 12%Bis-Tris Novex gels, transferred onto nitrocellulose membranes,and probed with primary antibodies overnight at 4�C (Supple-mentary Table S3). After a washing-step, membranes were incu-bated with HRP-tagged secondary antibodies (SupplementaryTable S3) for 1 to 2 hours at RT and visualized on a SyngeneChemiGenius Imager using Super-Signal West Dura Chemilumi-nescence Substrate. More details are provided in SupplementaryFile S1.

Cell-cycle analysisAZD8835 effect on cell-cycle progressionwas determined using

a Cytomics FC500 (Beckman Coulter) flow cytometer. Cells weregrown in 10 cm plates. After AZD8835 treatment, cells werewashed twice with PBS, detached with Versene (Invitrogen) andcentrifuged at 1,200 rpm for 5 minutes. Cell pellets were washedwith PBS and centrifuged at 1,200 rpm for 5 minutes beforefixation with 70% ethanol O/N at 4�C. Ethanol was removed bywashing the cells twice with PBS. DNA staining was performedusing FxCycle (Life Technologies) following the manufacturer'sinstructions. Analysis was performed using CXP analysis software(Beckman Coulter).

Cell panel proliferation assaysPharmacology data measuring cell growth inhibition by

AZD8835 were generated for a collection of 209 cancer cell lines(Supplementary Table S2). Methods were a build on those pre-viously described (20). Additional detail is provided in Supple-mentary File S1.

Cell proliferation measured by confluency (Incucyte Imager)BT474, MCF7, or T47D cells were seeded in 384-well plates at

a density of 500 to 2,000 cells per well and incubated overnight.Cells were dosed with compound(s) and cell confluency wasmeasured at 4-hour intervals over several days using the Incu-cyte platform (Essen Bioscience) following the manufacturer'sprotocol.

Gene expression analysisRNAwas isolated from frozen cell pellets using RNeasyMiniKit

(Qiagen-RLT Buffer), with an additional DNAse treatment step,following the manufacturer's protocol. Reverse transcription wasperformed using 50 ng of RNA with a Reverse Transcription kitand cDNA was then preamplified (14 cycles) using a pool ofTaqMan primers, following the manufacturer's instructions (LifeTechnologies). E2F, ER, or FOXOmodulated genes were selectedfor analysis using literature (21–23) and internal data. Sample

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and assay preparation of the 96.96 FluidigmDynamic arrays werecarried out according to the manufacturer's instructions. Datawere collected and analyzed using Fluidigm Real-Time PCRAnalysis 2.1.1 software followed by normalization and statisticalanalysis as described in Supplementary File S1.

In vivo studies: antitumor efficacyStudies in BT474 and MCF7 xenograft models were performed

at AZ and according to local regulations (Home Office UK), aspreviously described (20). Female Swiss athymic nude mice(swiss nu/nu– AZ UK) were transplanted s.c. with human breasttumor cell line BT474c [derived in AZ from BT474 (ATCC HTB-20) tumors passaged inmice]; mice were implanted with 0.36mg60-day estrogen pellet (Innovative Research of America, #SE-121)24 hours before cell implantation. Male SCIDmice (AZ UK) weretransplanted s.c. with human breast tumor cell line MCF7 (ICRFLondon); mice were implanted with a 0.5-mg 21-day estrogenpellet 24 hours before cell implantation. On day zero, 5 � 106

cells (MCF7 or BT474) in 50%Matrigel (BD Bioscience #354234)were injected s.c. on the left flank of the animals.

Studies in T47D xenograft model were performed at MolecularImaging Inc. and according to local regulations (NIH). FemaleSCID Beige mice (Harlan) were transplanted s.c. with humanbreast tumor cell line T47D (ATCC HTB-133); mice wereimplanted with 0.36 mg 60-day estrogen pellet 24 hours beforecell implantation.On day zero, 1� 107 cells in 50%Matrigel wereinjected s.c. on the right flank of the animals.

For efficacy studies, mice were randomized into groups of 8 to15 when average tumor volume reached approximately 200 to500 mm3. Mice were dosed for 1 to 4 weeks at defined doses andschedules. Tumors were measured two to three times weekly bycaliper and volume calculated using elliptical formula (pi/6 �width�width� length). Tumor growth inhibition (%TGI) fromthe start of treatmentwas assessed by comparisonof the geometricmean change in tumor volume for the control and treated groups.Tumor regression was calculated as the percentage reduction intumor volume from baseline (pretreatment) value: % Regression¼ (1 � RTV) �100 % where RTV ¼ Geometric Mean RelativeTumor Volume. Statistical significance was evaluated using a one-tailed t test. Detailed endpoint data for individual studies arecaptured in Supplementary Tables S4 and S5.

PharmacokineticsFor pharmacokinetics (PK), blood samples were collected by

intracardiac puncture (terminal) or by capillary micro (5 mL)sampling from tail vein (in life). Plasma samples were preparedand stored for bioanalysis at �20�C. Plasma samples wereextracted by protein precipitation in acetonitrile. Following cen-trifugation, the supernatants were mixed with water 1:6 (v/v).Extracts were analyzed by high-performance liquid chromatog-raphy/mass spectrometry (MS) using a reverse phase C18 columnand a gradient mobile phase containing water/methanol/formicacid. Compounds were quantified by MS/MS.

Ex vivo tumor pharmacodynamicsFor tumor pharmacodynamics (PD) studies, animals were

randomized into groups when tumors reached a volume ofapproximately 200 to 500 mm3. Tumor establishment anddosing are described above. Tumors were harvested at thedefined time points using a randomized process. They wereflash frozen in liquid nitrogen and stored at �80�C, for subse-

quent analysis of proteins by Western blotting, MSD or ELISA, orgene expression profiling using Fluidigm. Alternatively thetumor was first split and part fixed in 10% formalin buffer for24 hours and then embedded in paraffin for IHC staining. Threeor more tumors were analyzed per timepoint. Formalin-fixedparaffin-embedded samples were sectioned, subjected to anti-gen retrieval, and stained for cleaved caspase-3 (CC3) or forphospho-H2AX (gH2AX) using primary detection antibodies#32042 (Abcam) and #2577 (CST), respectively. More detailsin Supplementary File S1 and Table S3.

Modeling of xenograft biomarker and efficacy dataMathematical PK/PD models quantifying the magnitude and

time course of the effect of AZD8835 on tumor pAKT-T308, CC3,and volume inmice BT474 xenograft studies were developed. Fulldescription of the models and data fitting approaches used arepresented in Supplementary File S2.

ResultsAZD8835 selectively inhibits PI3Ka and PI3Kd

AZD8835 (Fig. 1A) is an isoform selective small-moleculeinhibitor of PI3Ka and PI3Kd. The discovery and assay profilingof AZD8835 are described in detail elsewhere (19). In brief,enzyme assays (Table 1) demonstrated potent inhibition ofPI3Ka and PI3Kd with relative sparing of PI3Kg , and particularlyPI3Kb. Potency against common hotspot mutant variants ofPI3Ka (H1047R and E545K) and wtPI3Kawas equivalent (Table1). Cellular assays measuring inhibitory activity of AZD8835against each of the Class I PI3K isoforms demonstrated rank ordersensitivity consistent with the data from isolated enzyme assays(Table 1). Additional selectivity profiling confirmed the broaderkinase selectivity of AZD8835 (19).

Western blot analyses (Fig. 1B) demonstrated inhibition ofPI3K-pathway signaling by AZD8835 across ERþ breast cancer celllines, MCF7, BT474, and T47D; these all possess a mPIK3CA gene(E545K, K111N, and H1047R, respectively) and displayed sub-mmol/L growth inhibition sensitivity (GI50s: 0.31 mmol/L, 0.53mmol/L, and 0.2 mmol/L, respectively) to AZD8835 (Supplemen-tary Table S1), consistent with PI3Ka dependency. In contrast,AZD8835 was relatively ineffective in inhibiting PI3K-pathwaysignaling (Fig. 1B) or cell growth (GI50 9.33 mmol/L; Supplemen-tary Table S1) in MDA-MB-468 cells (wtPIK3CA/PTEN null),consistent with the previously reported PI3Kb dependency of thiscell line (24).

Next, wemeasured antiproliferative activity of AZD8835 acrossa pan-tissue human cancer cell line panel comprising 209 celllines. Unbiased analyses of the genomic markers associated withAZD8835 sensitivity identified mutations in PIK3CA gene(mPIK3CA) as the most highly associated predictor of positiveresponse. Only 12 of 177 PIK3CA WT cell lines (7.7%) weresensitive to AZD8835, whereas 15 of 32 mPIK3CA cell lines(46.9%) were sensitive, corresponding to an OR of 12.1 and aP value of 1.2 � 10�7 (Fig. 1C). In addition, KRASmutation wasdemonstrated as a marker of resistance to AZD8835 (Fig. 1C andSupplementary File S1). AZD8835 was also evaluated in a largercell panel comprising >900 cell lines (25) and again identifiedmPIK3CA as the strongest biomarker of response (MANOVAanalysis, P ¼ 2.7 � 10�8, data not shown). Therefore, mPIK3CAis a predictive response biomarker for AZD8835 and may poten-tially be used in patient selection. Overall, our data are consistent

Intermittent High-Dose Scheduling of PI3K Inhibitor AZD8835

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Figure 1.AZD8835 inhibits signaling and growth in mPIK3CA cell lines. A, AZD8835 structure. B, Western blot analyses indicating PI3K-pathway inhibition across fourbreast cell lines (2 h). C, GI50 waterfall plot across tumor cell line panel, indicating relationship with PIK3CA and KRAS mutation status. For analysis, sensitivedefined as GI50 � 1.0 mmol/L. D, cell-cycle profiles (24 hours, flow cytometry) across three breast cancer cell lines. E, BT474 Western blot time course(2.5 mmol/L AZD8835). All data representative of �2 experiments. Error bars, SEM. Significance markers: � , P < 0.05; �� , P < 0.01; �� , P < 0.001.

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with previous studies demonstrating that models with mPIK3CAand/or other markers of PI3Ka deregulation are preferentiallysensitive to PI3Ka inhibitors (26). These data also providedfurther evidence for PI3Ka inhibition as primary pharmacologyfor AZD8835. Given that AZD8835 preferentially displayed activ-ity inmPIK3CA background, including ERþ breast cancer models,such models were used in our subsequent cell culture and in vivostudies.

We studied the mechanism behind AZD8835 growth inhi-bition of MCF7, BT474, and T47D cells by analyzing cell-cycleprofiles using flow cytometry. Using a moderate concentrationof 250 nmol/L AZD8835, corresponding to the GI50s forproliferation (Supplementary Table S1), we observed an ele-vated G0–G1 population in BT474 and MCF7 cells, consistentwith cell-cycle arrest in G1 (Fig. 1D). This was less evident in T47Dwhere instead therewas a small increase in sub-G0–G1populationconsistent with induction of cell death. Notably, the increase inthe sub-G0–G1 population was evident in all three cell lines at ahigher concentration (2.5 mmol/L) of AZD8835. As anticipated,higher concentration of AZD8835 also resulted in stronger inhi-bition of PI3K-pathway signaling (Fig. 1B). A timecourse per-formed in BT474 cells illustrated that the strongest pathwayinhibition was observed at early timepoints (1–2 hours) withsubsequent partial recovery of signaling (Fig. 1E). Cleaved PARPwas also observed as a similarly early onset event (Fig. 1E),suggesting that induction of apoptosis contributes to the celldeath phenotype, as reported for other PI3K-inhibitors (27). Thistransient pathway inhibition is consistent with pathway feedbackand reactivation (17, 18) and was similarly observed in a time-course study in MCF7 cells (Supplementary Fig. S1).

Monotherapy AZD8835 in vivo efficacy: intermittent andcontinuous schedules

Our observation of pathway feedback, early onset apoptosis,and dose-dependent cell death made us question whether con-tinuous dosing of PI3K-pathway inhibitors provides anoptimal invivo dosing schedule. Therefore, we were motivated to also eval-uate intermittent high-dose scheduling (IHDS) of AZD8835.

We initially evaluated AZD8835 when dosed continuously(every day). Using a maximum well-tolerated dose (MWTD) of25 mg/kg BID, we observed good antitumor efficacy in bothBT474 (93% TGI) and MCF7 (25% regression) breast xenograftmodels in mice (Fig. 2A and B). In subsequent efficacy studies,IHDS was evaluated. Figure 2C illustrates a head to head study inthe BT474 model, where similar efficacy was achieved usingAZD8835 continuous dosing at 25 mg/kg BID (11% regression),a 2 days on/5 off schedule at the MWTD of 50 mg/kg BID (91%TGI), or a day 1 and 4 schedule at 100 mg/kg (92% TGI; in thisinitial study dosed QD). Greater tolerability of the day 1 and 4

dosing schedule in nude mice allowed a more intense MWTD of100mg/kg BID to be explored in the BT474model. This scheduleresulted in greater efficacy; 40% regression (Fig. 2D). IHDS studiesin SCID mice strains permitted a lower MWTD of 50 mg/kg BIDAZD8835 but nevertheless delivered tumor growth inhibition(see Discussion). We therefore investigated the day 1 and 4 BIDAZD8835 schedule in our subsequent in vivo efficacy studies in thecombination setting (see below).

PD studies: AZD8835 IHDS induces waves of cell deathTo understand the efficacy observed with IHDS, we evaluated

pharmacokinetic/pharmacodynamic (PK/PD) relationships inboth MCF7 and BT474 xenografts.

Initially, analyzing both proximal (pAKT) and downstream(pPRAS40, pS6) PI3K-pathway biomarkers in tumor tissue,we demonstrated that pathway inhibition was both time anddose/exposure dependent (Fig. 3A). In parallel studies, weobserved glucose and insulin elevation as a transient phar-macologic response to AZD8835 in mice (SupplementaryFig. S2A and S2B), as also observed for other PI3K-inhibitors(10, 28, 29).

Figure 3B shows an extendedAZD8835 dose response in BT474xenografts at 2-hour timepoint. This demonstrates a relationshipbetween exposure and pAKT reduction in tumor tissue. Further-more, AZD8835 induced rapid-onset apoptosis (2 hours) asmeasured by cleavage of caspase-3 (CC3), consistent with ourcell studies (Fig. 1B, D, and E). This CC3 elevation was confirmedusing an IHC endpoint (Fig. 3C). We also observed that inaddition to CC3 induction on day 1, CC3 was also induced whenAZD8835 was redosed on day 4 (Fig. 3D).

Interestingly, in BT474 xenografts, we observed that AZD8835increased gH2AX (pSer139-H2AX), which tracked with anincrease in CC3 signal (Supplementary Fig. S3A). Similarly,AZD8835 increased gH2AX (Supplementary Fig. S3B) in MCF7xenografts (which lack caspase-3 expression; ref. 30). We sus-pected that the increase in gH2AX was a consequence of caspaseactivation, possibly as a response to endonuclease/caspase-medi-ated DNA fragmentation during apoptosis (31), thereby provid-ing an alternative apoptosis readout in MCF7. Supportive of this,we demonstrated that the AZD8835-induced gH2AX signal inMCF7 cells was caspase dependent by using the caspase inhibitor,QVD (Supplementary Fig. S3C). In addition, we measured PARPcleavage as another measure of apoptosis in MCF7 xenografts;the response tracked that for gH2AX (Supplementary Fig. S3Band S3C).

PK/PD efficacy modelingThe pAKT/CC3 PD and efficacy data from multiple AZD8835

monotherapy studies in the BT474 xenograft were compiled to

Table 1. Potency of AZD8835 vs. PI3K isoforms in enzyme and cell assays

Enzyme assayEnzyme assay IC50

(mmol/L) and (CIR)Cell PI3K-Isoform selectivityassay: cell line

Cell assay (pAKT) IC50

(mmol/L) and (CIR)

PI3Ka – wt 0.0062 (1.73) PI3Ka: BT474 0.090 (1.43)PI3Ka – E545K 0.0060 (1.51) PI3Kb: MDA-MB-468 3.5 (6.27)PI3Ka – H1047R 0.0058 (1.03) PI3Kd: JEKO-1 0.049 (1.70)PI3Kb 0.43 (2.03) PI3Kg : RAW264 0.53 (1.33)PI3Kd 0.0057 (1.46)PI3Kg 0.090 (1.45)

NOTE: Enzymes assayed by ADP Kinase Glo. Cell assays measured AKT phosphorylation. Data are geometric means of multiple IC50 determinations. Confidenceinterval ratio (CIR) is the ratio that when multiplied by the GeoMean gives the 95% upper confidence limit. The lower 95% limit is the GeoMean divided by the CIR.






build a PK/PD/efficacy model (see Supplementary File S2). Datagenerated across studies using different doses and schedules(continuous and IHDS) were a good fit and could be explainedusing this model. In the model, AZD8835 is assumed to have adual action comprising antiproliferative and proapoptoticeffects. The antiproliferative effects are reflected by tumorpAKT-T308 levels (PI3K-pathway signaling) and proapoptoticeffects by CC3 levels. A direct response Emax-type model wasused in the PK/PD analysis of tumor pAKT-T308 versus con-centration of AZD8835 in plasma. An indirect response modelwith tumor cells divided into sensitive and insensitive cells wasused to explain the CC3 effects. Results of the model-fits totumor pAKT, CC3, and Volume are shown in Supplementary FileS2. The model captures a desensitization to apoptosis (CC3) oncontinuous dosing, contrasting with IHDS which producesrepeat waves of strong apoptosis induction (Fig. 3E and Sup-plementary File S2).

Selection of agents to combine with AZD8835Our initial data demonstrated some potential for AZD8835

antitumor activity in the clinic when used as monotherapy.However, it is more likely that optimal patient benefit will beachieved when using AZD8835 as a combination treatment,particularly where combination cotherapies and dosing scheduleare optimized for therapeutic index. Therefore, continuing withour focus on mPIK3CA, ERþ breast cancer models, our next aimwas to investigate potential combinations in vitro which couldthen be evaluated in in vivo efficacy studies using a backbone ofAZD8835 IHDS. With a focus on mechanisms that have demon-strated benefit in ERþ breast cancer patients (see Discussion),we evaluated two broad classes of combination partner forAZD8835: "inter-pathway," combining with agents targetingparallel but interconnected driver pathways (ER, CDK4/6); or"intra-pathway," combining with an agent targeting downstreamin the PI3K-pathway.

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Figure 3.AZD8835 PK/PD relationship in xenografts: higher dose achieves increased PI3K pathway inhibition and triggers rapid onset apoptosis. A, PK/PD relationshipexamples for pathway markers (MSD/ELISA endpoints) in BT474 (after 2 days AZD8835 BID dosing, i.e., 4th dose) and MCF7 (after AZD8835 single dose).B, dose response in BT474 (Western blot analysis) demonstrating early onset (2 h) apoptosis (CC3) after single AZD8835 dose. C, CC3 (2 h) after AZD8835 dose inBT474, measured by IHC. D, IHDS (dosing day 1 and 4) induces repeat waves of CC3 in BT474. E, simulation of CC3 induction in BT474 by different dosingschedules, derived via PK/PD modeling. All data representative of multiple experiments. Error bars, SEM. Significance markers (vs vehicle, or intra-dosecomparisons where indicated): � , P < 0.05; �� , P < 0.01; �� , P < 0.001.






First, we evaluated the effect of AZD8835 combinations withthese classes of agents in cell growth studies using an IncucyteImager. Anti-estrogens were selective estrogen receptor down-regulators (SERDs), fulvestrant (32) or AZD9496 (33), CDK4/6inhibitor was palbociclib (PD-0332991; ref. 34) and AZD2014(35) provided a dual mTORC1/2 kinase inhibitor.

Consistent with our expectations, stronger growth inhibitionwas observed with all combinations compared with monothera-pies. This is illustrated inMCF7 cellswhere the combinationof 0.3mmol/L AZD8835 plus 0.1 mmol/L fulvestrant or 0.1 mmol/Lpalbociclib prevented cell growth (Fig. 4A). Similar results wereobserved with the alternative SERD, AZD9496 (SupplementaryFig. S4A–S4C), and also with fulvestrant in other mPIK3CA celllines, BT474 and T47D (Supplementary Fig. S4A and S4B). Inaddition, we confirmed that AZD8835/fulvestrant combinationactivity was retained (Supplementary Fig. S4C) in two fulvestrant-refractory MCF7-derived cell lines that were generated by contin-uous incubation with fulvestrant (MCF7-F100-16; ref. 36) or bylong-termestrogendeprivation (MCF7-GHPED, generated atAZ).

To provide mechanistic understanding of these combinationeffects, we analyzed pathway biomarkers across MCF7, BT474,and T47D cells. Several consistent findings were observed,although (consistent with heterogeneity across cancer cell lines)there were also cell line-specific differences. One notable generaltheme was stronger pathway-biomarker suppression with com-binations compared with monotherapies. Furthermore, in somecases monotherapy induced pathway cross-talk/activation, con-sistent with a potential resistancemechanism, whichwas reversedby the combination partner.

Some of the specific mechanistic observations for combina-tions of AZD8835 with SERDs, fulvestrant or AZD9496, wereobserved across more than one cell line (Fig. 4B). Across all celllines, the combinations achieved increased suppression ofpP70S6K and/or pS6. Also, particularly in T47D and BT474 cells,the SERDs increased pAKT which was reversed by the addition ofAZD8835. In addition, particularly in T47D cells and to a smallextent in MCF7 cells, but not in BT474 cells, these combinationsincreased ERa downregulation. Finally, in T47D the SERDsreversed AZD8835-mediated induction of progesterone receptor(PR), an ERa transcriptional target. Collectively, thesefindings areconsistent with and build on previous reports with related agents(23, 37–40).

AZD8835, when combined with palbociclib, consistentlyenhanced suppression of pRb (and total Rb) and the E2Ftranscriptional targets, CDC6 and E2F (E2F-1), consistent withstronger inhibition of the Rb-pathway (Fig. 4C). AZD8835 alsosuppressed palbociclib-induced increase in Cyclin-D1. Also,particularly in T47D, PI3K-pathway biomarkers (pAKT andpS6) were increased by palbociclib treatment but reversed byAZD8835 (Fig. 4C). In addition, AZD8835 plus palbociclib-treated cells were subject to transcriptional profiling, using theFluidigm platform. We observed that the expression of E2Ftranscriptional markers were suppressed more with the com-bination, compared with single agents, and more consistentlythan for selected ER and FOXO transcriptional targets (Fig. 4Dand Supplementary Fig. S4D).

The combination of AZD8835 plus the dual mTORC1/2inhibitor, AZD2014, also enhanced inhibition of cell prolifer-ation (Supplementary Fig. S4E). Alongside, we observedenhanced suppression of PI3K-pathway biomarkers, includingpS6 (Supplementary Fig. S4F), as reported previously for PI3K/

mTOR inhibitor combinations (41). In addition, the combi-nation enhanced suppression of pRb (and total Rb; Supple-mentary Fig. S4F).

Collectively the above data supported progression of each ofthese classes of combinations into in vivo efficacy studies.

In vivo efficacy studies applying IHDSAZD8835 in combinationNext, we performed combination in vivo efficacy studies, apply-

ing AZD8835 IHDS (day 1 and 4 BID). As previously demon-strated (Fig. 2D), AZD8835dosed at 100mg/kgday 1 and4BID inthe BT474/nude-mice xenograft model again induced tumorregression (31 and 36%: Fig. 5A). Notably, AZD8835 combinedas a doublet with fulvestrant or palbociclib increased tumorregression (59 and 54%, respectively) compared with singleagents (Fig. 5A and Supplementary Table S5). We also performedsimilar studies in the MCF7 xenograft model, grown in SCIDmice, where the tolerated dose (MWTD) of AZD8835 was lower(50 mg/kg BID day 1 and 4). Again, a combination benefit witheither fulvestrant or palbociclib was seen (Fig. 5B and Supple-mentary Table S5). Moreover, when the agents were used in atriplet combination, thereby simultaneously targeting three driverpathways, a strikingly profound tumor regression (92%) wasobserved (Fig. 5B and Supplementary Table S5).We also observedstronger in vivobiomarkermodulationwith combinations relativeto monotherapies for a subset of PD endpoints (Fig. 5C), con-sistent with cell studies (Fig. 4B and C). Superior efficacy withcombinations was also observed when fulvestrant was replacedwith an alternative SERD, AZD9496 (Supplementary Fig. S5A andTable S5), or with fulvestrant in an additional xenograft model,T47D (Supplementary Fig. S5B and Supplementary Table S5).Using an intra-pathway approach, the combination of AZD8835with the dual mTORC1/2 inhibitor, AZD2014, also resulted inimproved efficacy compared with monotherapy. This was partic-ularly evident in the BT474 model (in nude mice) where tumorregression was achieved with a triplet combination incorporatingfulvestrant, despite a tolerability requirement for AZD8835 dosereduction (Supplementary Fig. S6A and Supplementary Table S5).In the MCF7 model (SCID mice), this combination was less welltolerated so the overall efficacy achieved with the lower MWTDwas relatively moderate; nevertheless a positive combinationeffect was still observed (Supplementary Fig. S6B and S6C andSupplementary Table S5).

DiscussionHere, we describe pharmacologic studies with a novel PI3Ka/d

inhibitor, AZD8835, in mPIK3CA, ERþ breast cancer models.In particular, we have explored the potential of intermittent

high-dose scheduling, here termed IHDS, as an alternative tocontinuous dosing.Weweremotivated to explore this path giventhat clinical dose and exposure of PI3K inhibitors is capped bynormal tissue toxicities (12, 13, 15) which may result in sub-optimal pathway inhibition in tumors. Indeed tolerabilityissues observed on continuous dosing of PI3K pathway inhibi-tors are driving a shift towards more intermittent scheduling,albeit to date these are less radical shifts than illustrated in thismanuscript.

We demonstrated that we could achieve higher dose in in vivostudies when applying IHDS, compared with continuous dosing.This was particularly the case in nudemice (BT474model), wheregreater tolerability allowed the IHDS concept to be tested

Hudson et al.





Figure 4.Selection of combination partners for AZD8835 in ERþ breast cell lines. A, growth inhibition by combinationswith fulvestrant or palbociclib inMCF7 (Incucyte platform).Enhanced inhibition of signaling pathways (24 h, Western blot analysis) via AZD8835 (300 nmol/L) plus SERDs (fulvestrant or AZD9496, 100 nmol/L; B), orAZD8835 (300 nmol/L) plus palbociclib (30 or 300 nmol/L; C), in three cell lines. D, mRNA profiling heatmap showing increased inhibition (fold change) of E2Ftranscriptional targets (black bar) when AZD8835 (0.1–1 mmol/L) combined with 30 nmol/L palbociclib (MCF7, 24 h). All data are mean of three experiments.






maximally (100 mg/kg AZD8835 day 1 and 4 BID dosing sched-ule) and here outperformed continuous dosing in efficacy studies(see Fig. 2A vs. Figs. 2D/5A). In contrast, in SCID strains (MCF7,T47D efficacy studies) intermittent scheduling allowed a relative-ly modest doubling of dose (to 50 mg/kg AZD8835) comparedwith that achievable using continuous dosing; even so, despitenear halving of accumulative weekly dose relative to continuousdosing, monotherapy AZD8835 IHDS nevertheless delivered

tumor growth inhibition (MCF7 - Fig. 5B and SupplementaryFig. S5A; T47D - Supplementary Fig. S5B) so this encouraged us toprogress into combination studies. Another notable feature ofBT474 xenografts is a relatively slow growth rate compared withsome xenograft models, although arguably this is more represen-tative of growth rates of clinical tumors. Growth of such tumors isless likely to "take off" during the nondosing days characteristic ofintermittent schedules.

Figure 5.Combination of AZD8835 with fulvestrant and/or palbociclib results in tumor regression in ERþ breast xenografts. Efficacy studies applying combination ofAZD8835 (IHDS; dose indicated, day 1 and 4 BID) with fulvestrant (F; schedule: 5 mg, day 1, 3, 5, QD) and/or palbociclib (P; schedule: 50 mg/kg, continuous, QD)in BT474 (A) or MCF7 (B) xenografts. Bars alongside timelines illustrate dosing of each agent. C, PD studies in BT474 or MCF7 xenografts showing enhancedcombination effect on pathways (PI3K, CDK4/6) or apoptosis (CC3). Fulvestrant dosed day previous to AZD8835 to allow plasma exposure to reach steadystate. MCF7 tumors harvested 2 hours after (co)dose. Error bars, SEM. Significance markers (vs vehicle, or intra-dose comparisons where indicated): � , P < 0.05;�� , P < 0.01; �� , P < 0.001.

Hudson et al.





A PK/PD/efficacy model was built using monotherapy BT474data and resulting simulations fitted well to our overall obser-vations, as detailed in Supplementary File S2. The modelcaptures our observations that on continuous dosing schedule(25 mg/kg BID) there is a reduced apoptosis signal, where thesignal is stronger following the first dose compared with thesubsequent doses which induced relatively little apoptosis. Incontrast, IHDS schedules, which incorporate a break in dosing,produce repeat waves of apoptosis induction on repeat dosing(see Fig. 3D and E). We speculate that there may be a subpop-ulation of cells sensitive to induction of apoptosis and days arerequired to replenish such population. Alternatively, the breakin dosing may allow "reset" of the pathway signaling frompathway feedback and reactivation which overcomes the desen-sitization to apoptosis induction.

We then prioritized our combination therapy strategies forAZD8835. We considered evaluation of combinations withanti-estrogen/anti-estrogen-receptor (ER) therapy to be of partic-ular interest. Indeed, PIK3CA mutations are most prevalent(29%–45%) in the ERþ (luminal) subset of breast tumors (6).In addition, there is literature evidence for bidirectional PI3K/ERsignaling pathway interactions, coupled with reports that com-bining PI3K-inhibitor and ER directed agents can combat resis-tance (37–40). Such PI3K/ER pathway directed combinationtherapies have recently progressed into the clinic (15, 42, 43).A second attractive combination opportunity for PI3K-inhibitorswas combination with CDK4/6-inhibitors, which target the Rb-pathway, since in mPIK3CA tumors this combination has previ-ously been reported as synergistic (44). Also CDK4/6 inhibitorsmay combine well with anti-estrogens and help to combatacquired resistance to ER antagonists (37, 45, 46), and thisapproach has generated positive clinical data (47). Thereforetriplet combinations may also have potential and initial clinicalstudies are already underway, for example, combining CDK4/6inhibitor (LEE011) with PI3K inhibitor (BYL719) and anti-estro-gen letrozole (48). Regarding intra-PI3K-pathway combinations,an interesting combination opportunity is withmTOR inhibitors.Again there is some precedent in this area, for example, Elkabetsand colleagues (41) reported synergy through combining a rapa-logue mTORC1-inhibitor, everolimus, with PI3K inhibitorBYL719; also there is some precedent for dual PI3K/mTORinhibition using mixed profile inhibitors such as GDC-0980,dosed intermittently (49).

We initially combined AZD8835 with ER, CDK4/6, andmTORdirected agents [SERDs (fulvestrant or AZD9496), palbociclib,AZD2014, respectively] in in vitro studies, observing enhancedcombination activity and coupledwithmechanistic data.We thenevaluated these combinations in in vivo efficacy studies using afoundation of AZD8835 IHDS. Compelling efficacy, consistentlyobserved as a tumor regression response (Fig. 5A and B andSupplementary Fig. S5A and S5B), was observed with the inter-pathway combinations where AZD8835 was combined with

SERDs and/or CDK4/6 inhibitors. Also notable with all theseinter-pathway combinations was that the MWTD of each agentwas the same as used inmonotherapy studies, indicatingminimalcombined toxicities, as illustrated in Supplementary Fig. S7.

Combination benefit was also illustrated with an "intra-path-way" combination where AZD8835 was combined withAZD2014. Despite a requirement for significant dose reductions(Supplementary Fig. S6), particularly in the MCF7/SCID model,combination benefit was again observed. We anticipate that thiscombination may be better tolerated in the clinic where hyper-glycemia, the suspected tolerability issue in mice, could be bettermanaged.

We conclude that AZD8835 IHDS provides flexibility and apromising alternative to continuous dosing with potential for animproved therapeutic index. Such schedules and combinationsmerit clinical evaluation.

Disclosure of Potential Conflicts of InterestAll authors are current or former employees of AstraZeneca. K. Hudson,

U.M. Polanska and C. Trigwell have ownership interest (including patents)and are AstraZeneca shareholders. No potential conflicts of interest weredisclosed by the other authors.

Authors' ContributionsConception and design: K. Hudson, U.J. Hancox, U.M. Polanska, P. MorentinGutierrez, S.C. Cosulich, L. Ward, F. Cruzalegui, S. GreenDevelopment of methodology: K. Hudson, C. Trigwell, U.M. Polanska,P. Morentin Gutierrez, M. CumberbatchAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): U.M. Polanska, A. Avivar-Valderas, O. Delpuech,P. Dudley, M. CumberbatchAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): K. Hudson, U.J. Hancox, C. Trigwell, R. McEwen,U.M. Polanska, M. Nikolaou, P. Morentin Gutierrez, A. Avivar-Valderas,O. Delpuech, L. Hanson, R. Ellston, A. Jones, M. Cumberbatch, S.C. CosulichWriting, review, and/or revision of the manuscript: K. Hudson, U.J. Hancox,C. Trigwell, R. McEwen, U.M. Polanska, M. Nikolaou, P. Morentin Gutierrez,O. Delpuech, A. Jones, M. Cumberbatch, S.C. Cosulich, L. Ward, F. Cruzalegui,S. GreenAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): U.J. Hancox, R. McEwen, U.M. Polanska,L. HansonStudy supervision: K. Hudson, L. WardOther (project leader): S. Green

AcknowledgmentsThe authors thankMikeDymond for advice on statistical analysis of data and

to Amar Rahi, Emily Lawrie, and the AZ Lab Animal Sciences Group in supportof PD/efficacy studies.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received August 19, 2015; revised December 24, 2015; accepted January 25,2016; published OnlineFirst February 2, 2016.

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Published OnlineFirst February 2, 2016.Mol Cancer Ther Kevin Hudson, Urs J. Hancox, Cath Trigwell, et al.

-Dependent Breast CancersPIK3CATreatment Strategies for , Demonstratesδ and PI3KαSelective Inhibitor of PI3K

Intermittent High-Dose Scheduling of AZD8835, a Novel

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