Molecular
Cancer
Therapeutics
Small Molecule Therapeutics
Intermittent High-Dose Scheduling of AZD8835,
a Novel Selective Inhibitor of PI3Ka and PI3Kd,
Demonstrates Treatment Strategies for
PIK3CA-Dependent Breast Cancers
Kevin 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 human
cancer. Hence, PI3Ka is a target subject to intensive efforts in
identifying 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 potent
inhibitor of PI3Ka and PI3Kd with selectivity versus PI3Kb, PI3Kg,
and other kinases that preferentially inhibited growth in cells with
mutant PIK3CA status, such as in estrogen receptor–positive
(ERþ) breast cancer cell lines BT474, MCF7, and T47D (submmol/L GI50s). Consistent with this, AZD8835 demonstrated
antitumor efficacy in corresponding breast cancer xenograft models when dosed continuously. In addition, an alternative
approach of intermittent high-dose scheduling (IHDS) was
explored given our observations that higher exposures achieved
greater pathway inhibition and induced apoptosis. Indeed,
using IHDS, monotherapy AZD8835 was able to induce tumor
xenograft regression. Furthermore, AZD8835 IHDS in combination with other targeted therapeutic agents further enhanced
antitumor activity (up to 92% regression). Combination partners were prioritized on the basis of our mechanistic insights
demonstrating signaling pathway cross-talk, with a focus on
targeting interdependent ER and/or CDK4/6 pathways or alternatively a node (mTOR) in the PI3K-pathway, approaches with
demonstrated clinical benefit in ERþ breast cancer patients. In
summary, AZD8835 IHDS delivers strong antitumor efficacy in
a range of combination settings and provides a promising
alternative to continuous dosing to optimize the therapeutic
index in patients. Such schedules merit clinical evaluation. Mol
Introduction
human cancer (1). Activating somatic point mutations in the
PIK3CA gene (encoding p110a catalytic subunit of PI3Ka) have
been widely reported across many tumor types and these mutations often display a transforming gain-of-function activity (2–6).
Additional reported mechanisms of deregulation of PI3Ka
include amplification of the PIK3CA gene (7, 8), mutations in
a PI3K regulatory subunit (9) or activating events elsewhere in the
signaling pathway. Hence PI3Ka is a target subject to intensive
efforts in identifying inhibitors and evaluating their therapeutic
potential. Thus far, clinical activity observed has been moderate
(10, 11), although more promising data have recently been
reported 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, a
novel dual inhibitor of PI3Ka and PI3Kd, currently in phase I
clinical evaluation. A particular focus of our preclinical studies
was to evaluate intermittent high-dosing schedules (IHDS) as an
alternative to a commonly applied default of continuous daily
dosing. Several considerations motivated us to explore this path.
First, continuous dosing may achieve suboptimal pathway inhibition in tumors, because PI3K-inhibitor clinical dose and exposure on continuous dosing schedules are capped by normal
tissue toxicities such as hyperglycemia, diarrhea, and rash (12,
13, 15). Also, additional dose reduction may be required when
The PI3K-AKT-mTOR pathway is a critical regulator of many
cellular processes including proliferation, survival, and transformation, and is one of the most frequently deregulated signaling
networks in human cancer (1). The PI3K family of lipid kinases are
key components of this pathway, in particular, the Class I PI3K
subfamily which comprise PI3Ka, PI3Kb, and PI3Kd (Class IA)
and PI3Kg (Class IB).
Different PI3K isoforms have been shown to play key roles in
distinct tumor types, often linked with genetic alterations. In
particular, PI3Ka is a commonly deregulated oncoprotein in
1
AstraZeneca Pharmaceuticals, Oncology iMed, Macclesfield, Cheshire, United Kingdom. 2AstraZeneca Pharmaceuticals, CRUK-CI Li Ka
Shing Centre, Cambridge, United Kingdom.
Note: Supplementary data for this article are available at Molecular Cancer
Therapeutics Online (http://mct.aacrjournals.org/).
Corresponding Author: Kevin Hudson, AstraZeneca Pharmaceuticals, Oncology
iMed, Alderley Park, Macclesfield, Cheshire, SK10 4TG, UK. Phone: 44-1625516193; Fax: 44-1625-519749; E mail: kevin.hudson@astrazeneca.com
doi: 10.1158/1535-7163.MCT-15-0687
2016 American Association for Cancer Research.
www.aacrjournals.org
Cancer Ther; 1–13. 2016 AACR.
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Hudson et al.
PI3K-inhibitors are dosed continually in combination with other
therapies (16). So, an intermittent schedule that allows higher
doses may permit greater target inhibition and also allow for a
beneficial recovery period from dose-limiting physiological side
effects such as rash. Second, it is reported widely across biologic
systems, including the PI3K-pathway, that continuous exposure
and pathway inhibition lead to pathway reactivation via perturbation of pathway feedback mechanisms, leading to reduced
effect over time (17, 18). In contrast, intermittent dosing may
allow a "reset" of pathway signaling, allowing repeat apoptotic
response on re-exposure to compound.
Here, reporting on preclinical studies in estrogen receptor
positive (ERþ), mutant PIK3CA (mPIK3CA) breast models, we
illustrate that monotherapy IHDS with AZD8835 achieved good
antitumor efficacy. In particular, we exemplify how this schedule
was used in combination with other agents to deliver profound
antitumor regression.
Materials and Methods
Cell line studies
Cells lines were grown in RPMI-1640 media, 10% FCS, 2
mmol/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 profiling details and AZD8835 antiproliferative potencies, in Supplementary Table S1. Cell line panel details are shown in Supplementary Table S2. All cell lines were authenticated via the
AstraZeneca (AZ) Cell Bank using DNA fingerprinting short
tandem repeat (STR) assays. All revived cells were used within
20 passages, and cultured for less than 6 months.
Compounds and formulation
Compounds AZD8835, 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 resulting
in final DMSO concentration less than 0.5%.
For in vivo studies, AZD8835 was formulated as a suspension in
HPMC/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. For
BID dosing, AZD8835 was administered 6 to 8 hours apart (except
the glucose/insulin timecourse study in CD1 mice were 12 hours
apart). Fulvestrant was formulated in peanut oil (Sigma) and
dosed three times weekly subcutaneously (s.c.) at 5 mg/mouse at
dose volume of 0.1 mL/mouse. AZD9496, was formulated in 40%
Polyethylene glycol (PEG)/30% Captisol and protected from
light. It was dosed per oral QD at a dose volume of 0.1 mL/10
g mouse. Where dosed in combination, it was dosed 1 hour after
the morning dose of AZD8835. Palbociclib and AZD2014 were
formulated and dosed as for AZD8835, and coformulated when
dosed 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 assays
See elsewhere for details (19). Inhibition of recombinant
PI3Ka, PI3Kb, PI3Kd, and PI3Kg was evaluated in ADP Kinase
Glo-based enzyme assays at AZ. Broader kinase selectivity profile
was determined via kinase panels at University of Dundee (United
Kingdom) and DiscoveRx (KinomeScan). Cellular inhibitory
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activity against each of the Class I PI3K isoforms was determined
by measuring phosphorylation of AKT protein in four different
cell lines under assay conditions where signaling was dependent
on each of the individual isoforms; cell lines were BT474 (PI3Ka),
MDA-MB-468 (PI3Kb), JeKo-1 (PI3Kd), and RAW264 (PI3Kg). In
addition, selectivity against additional PI3K-related kinase (PIKK)
family members, mTOR, DNAPK, ATR, and ATM, was demonstrated via bespoke assays at AZ.
Western blotting
Cell or ex vivo lysates were generated in ice-cold lysis buffer
containing phosphatase and protease inhibitors. Tumors were
homogenized and sonicated in ice-cold lysis buffer. Samples were
prepared 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 (Supplementary Table S3). After a washing-step, membranes were incubated with HRP-tagged secondary antibodies (Supplementary
Table S3) for 1 to 2 hours at RT and visualized on a Syngene
ChemiGenius Imager using Super-Signal West Dura Chemiluminescence Substrate. More details are provided in Supplementary
File S1.
Cell-cycle analysis
AZD8835 effect on cell-cycle progression was determined using
a Cytomics FC500 (Beckman Coulter) flow cytometer. Cells were
grown in 10 cm plates. After AZD8835 treatment, cells were
washed twice with PBS, detached with Versene (Invitrogen) and
centrifuged at 1,200 rpm for 5 minutes. Cell pellets were washed
with PBS and centrifuged at 1,200 rpm for 5 minutes before
fixation with 70% ethanol O/N at 4 C. Ethanol was removed by
washing the cells twice with PBS. DNA staining was performed
using FxCycle (Life Technologies) following the manufacturer's
instructions. Analysis was performed using CXP analysis software
(Beckman Coulter).
Cell panel proliferation assays
Pharmacology 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 previously described (20). Additional detail is provided in Supplementary 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 was
measured at 4-hour intervals over several days using the Incucyte platform (Essen Bioscience) following the manufacturer's
protocol.
Gene expression analysis
RNA was isolated from frozen cell pellets using RNeasy MiniKit
(Qiagen-RLT Buffer), with an additional DNAse treatment step,
following the manufacturer's protocol. Reverse transcription was
performed using 50 ng of RNA with a Reverse Transcription kit
and cDNA was then preamplified (14 cycles) using a pool of
TaqMan primers, following the manufacturer's instructions (Life
Technologies). E2F, ER, or FOXO modulated genes were selected
for analysis using literature (21–23) and internal data. Sample
Molecular Cancer Therapeutics
Intermittent High-Dose Scheduling of PI3K Inhibitor AZD8835
and assay preparation of the 96.96 Fluidigm Dynamic arrays were
carried out according to the manufacturer's instructions. Data
were collected and analyzed using Fluidigm Real-Time PCR
Analysis 2.1.1 software followed by normalization and statistical
analysis as described in Supplementary File S1.
In vivo studies: antitumor efficacy
Studies in BT474 and MCF7 xenograft models were performed
at AZ and according to local regulations (Home Office UK), as
previously described (20). Female Swiss athymic nude mice
(swiss nu/nu– AZ UK) were transplanted s.c. with human breast
tumor cell line BT474c [derived in AZ from BT474 (ATCC HTB20) tumors passaged in mice]; mice were implanted with 0.36 mg
60-day estrogen pellet (Innovative Research of America, #SE-121)
24 hours before cell implantation. Male SCID mice (AZ UK) were
transplanted s.c. with human breast tumor cell line MCF7 (ICRF
London); mice were implanted with a 0.5-mg 21-day estrogen
pellet 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 Molecular
Imaging Inc. and according to local regulations (NIH). Female
SCID Beige mice (Harlan) were transplanted s.c. with human
breast tumor cell line T47D (ATCC HTB-133); mice were
implanted with 0.36 mg 60-day estrogen pellet 24 hours before
cell implantation. On day zero, 1 107 cells in 50% Matrigel were
injected s.c. on the right flank of the animals.
For efficacy studies, mice were randomized into groups of 8 to
15 when average tumor volume reached approximately 200 to
500 mm3. Mice were dosed for 1 to 4 weeks at defined doses and
schedules. Tumors were measured two to three times weekly by
caliper and volume calculated using elliptical formula (pi/6 width width length). Tumor growth inhibition (%TGI) from
the start of treatment was assessed by comparison of the geometric
mean change in tumor volume for the control and treated groups.
Tumor regression was calculated as the percentage reduction in
tumor volume from baseline (pretreatment) value: % Regression
¼ (1 RTV) 100 % where RTV ¼ Geometric Mean Relative
Tumor Volume. Statistical significance was evaluated using a onetailed t test. Detailed endpoint data for individual studies are
captured in Supplementary Tables S4 and S5.
Pharmacokinetics
For 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 prepared
and stored for bioanalysis at 20 C. Plasma samples were
extracted by protein precipitation in acetonitrile. Following centrifugation, the supernatants were mixed with water 1:6 (v/v).
Extracts were analyzed by high-performance liquid chromatography/mass spectrometry (MS) using a reverse phase C18 column
and a gradient mobile phase containing water/methanol/formic
acid. Compounds were quantified by MS/MS.
Ex vivo tumor pharmacodynamics
For tumor pharmacodynamics (PD) studies, animals were
randomized into groups when tumors reached a volume of
approximately 200 to 500 mm3. Tumor establishment and
dosing are described above. Tumors were harvested at the
defined time points using a randomized process. They were
flash frozen in liquid nitrogen and stored at 80 C, for subse-
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quent analysis of proteins by Western blotting, MSD or ELISA, or
gene expression profiling using Fluidigm. Alternatively the
tumor was first split and part fixed in 10% formalin buffer for
24 hours and then embedded in paraffin for IHC staining. Three
or more tumors were analyzed per timepoint. Formalin-fixed
paraffin-embedded samples were sectioned, subjected to antigen retrieval, and stained for cleaved caspase-3 (CC3) or for
phospho-H2AX (gH2AX) using primary detection antibodies
#32042 (Abcam) and #2577 (CST), respectively. More details
in Supplementary File S1 and Table S3.
Modeling of xenograft biomarker and efficacy data
Mathematical PK/PD models quantifying the magnitude and
time course of the effect of AZD8835 on tumor pAKT-T308, CC3,
and volume in mice BT474 xenograft studies were developed. Full
description of the models and data fitting approaches used are
presented in Supplementary File S2.
Results
AZD8835 selectively inhibits PI3Ka and PI3Kd
AZD8835 (Fig. 1A) is an isoform selective small-molecule
inhibitor of PI3Ka and PI3Kd. The discovery and assay profiling
of AZD8835 are described in detail elsewhere (19). In brief,
enzyme assays (Table 1) demonstrated potent inhibition of
PI3Ka and PI3Kd with relative sparing of PI3Kg, and particularly
PI3Kb. Potency against common hotspot mutant variants of
PI3Ka (H1047R and E545K) and wtPI3Ka was equivalent (Table
1). Cellular assays measuring inhibitory activity of AZD8835
against each of the Class I PI3K isoforms demonstrated rank order
sensitivity consistent with the data from isolated enzyme assays
(Table 1). Additional selectivity profiling confirmed the broader
kinase selectivity of AZD8835 (19).
Western blot analyses (Fig. 1B) demonstrated inhibition of
PI3K-pathway signaling by AZD8835 across ERþ breast cancer cell
lines, MCF7, BT474, and T47D; these all possess a mPIK3CA gene
(E545K, K111N, and H1047R, respectively) and displayed submmol/L growth inhibition sensitivity (GI50s: 0.31 mmol/L, 0.53
mmol/L, and 0.2 mmol/L, respectively) to AZD8835 (Supplementary Table S1), consistent with PI3Ka dependency. In contrast,
AZD8835 was relatively ineffective in inhibiting PI3K-pathway
signaling (Fig. 1B) or cell growth (GI50 9.33 mmol/L; Supplementary Table S1) in MDA-MB-468 cells (wtPIK3CA/PTEN null),
consistent with the previously reported PI3Kb dependency of this
cell line (24).
Next, we measured antiproliferative activity of AZD8835 across
a pan-tissue human cancer cell line panel comprising 209 cell
lines. Unbiased analyses of the genomic markers associated with
AZD8835 sensitivity identified mutations in PIK3CA gene
(mPIK3CA) as the most highly associated predictor of positive
response. Only 12 of 177 PIK3CA WT cell lines (7.7%) were
sensitive to AZD8835, whereas 15 of 32 mPIK3CA cell lines
(46.9%) were sensitive, corresponding to an OR of 12.1 and a
P value of 1.2 107 (Fig. 1C). In addition, KRAS mutation was
demonstrated as a marker of resistance to AZD8835 (Fig. 1C and
Supplementary File S1). AZD8835 was also evaluated in a larger
cell panel comprising >900 cell lines (25) and again identified
mPIK3CA as the strongest biomarker of response (MANOVA
analysis, P ¼ 2.7 108, data not shown). Therefore, mPIK3CA
is a predictive response biomarker for AZD8835 and may potentially be used in patient selection. Overall, our data are consistent
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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 four
breast cell lines (2 h). C, GI50 waterfall plot across tumor cell line panel, indicating relationship with PIK3CA and KRAS mutation status. For analysis, sensitive
defined 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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Molecular Cancer Therapeutics
Intermittent High-Dose Scheduling of PI3K Inhibitor AZD8835
Table 1. Potency of AZD8835 vs. PI3K isoforms in enzyme and cell assays
Enzyme assay IC50
(mmol/L) and (CIR)
Enzyme assay
PI3Ka – wt
0.0062 (1.73)
PI3Ka – E545K
0.0060 (1.51)
PI3Ka – H1047R
0.0058 (1.03)
PI3Kb
0.43 (2.03)
PI3Kd
0.0057 (1.46)
PI3Kg
0.090 (1.45)
Cell PI3K-Isoform selectivity
assay: cell line
PI3Ka: BT474
PI3Kb: MDA-MB-468
PI3Kd: JEKO-1
PI3Kg: RAW264
Cell assay (pAKT) IC50
(mmol/L) and (CIR)
0.090 (1.43)
3.5 (6.27)
0.049 (1.70)
0.53 (1.33)
NOTE: Enzymes assayed by ADP Kinase Glo. Cell assays measured AKT phosphorylation. Data are geometric means of multiple IC50 determinations. Confidence
interval 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.
with previous studies demonstrating that models with mPIK3CA
and/or other markers of PI3Ka deregulation are preferentially
sensitive to PI3Ka inhibitors (26). These data also provided
further evidence for PI3Ka inhibition as primary pharmacology
for AZD8835. Given that AZD8835 preferentially displayed activity in mPIK3CA background, including ERþ breast cancer models,
such models were used in our subsequent cell culture and in vivo
studies.
We studied the mechanism behind AZD8835 growth inhibition of MCF7, BT474, and T47D cells by analyzing cell-cycle
profiles using flow cytometry. Using a moderate concentration
of 250 nmol/L AZD8835, corresponding to the GI50s for
proliferation (Supplementary Table S1), we observed an elevated G0–G1 population in BT474 and MCF7 cells, consistent
with cell-cycle arrest in G1 (Fig. 1D). This was less evident in T47D
where instead there was a small increase in sub-G0–G1 population
consistent with induction of cell death. Notably, the increase in
the sub-G0–G1 population was evident in all three cell lines at a
higher concentration (2.5 mmol/L) of AZD8835. As anticipated,
higher concentration of AZD8835 also resulted in stronger inhibition of PI3K-pathway signaling (Fig. 1B). A timecourse performed in BT474 cells illustrated that the strongest pathway
inhibition was observed at early timepoints (1–2 hours) with
subsequent partial recovery of signaling (Fig. 1E). Cleaved PARP
was also observed as a similarly early onset event (Fig. 1E),
suggesting that induction of apoptosis contributes to the cell
death phenotype, as reported for other PI3K-inhibitors (27). This
transient pathway inhibition is consistent with pathway feedback
and reactivation (17, 18) and was similarly observed in a timecourse study in MCF7 cells (Supplementary Fig. S1).
Monotherapy AZD8835 in vivo efficacy: intermittent and
continuous schedules
Our observation of pathway feedback, early onset apoptosis,
and dose-dependent cell death made us question whether continuous dosing of PI3K-pathway inhibitors provides an optimal in
vivo dosing schedule. Therefore, we were motivated to also evaluate intermittent high-dose scheduling (IHDS) of AZD8835.
We initially evaluated AZD8835 when dosed continuously
(every day). Using a maximum well-tolerated dose (MWTD) of
25 mg/kg BID, we observed good antitumor efficacy in both
BT474 (93% TGI) and MCF7 (25% regression) breast xenograft
models in mice (Fig. 2A and B). In subsequent efficacy studies,
IHDS was evaluated. Figure 2C illustrates a head to head study in
the BT474 model, where similar efficacy was achieved using
AZD8835 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 this
initial study dosed QD). Greater tolerability of the day 1 and 4
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dosing schedule in nude mice allowed a more intense MWTD of
100 mg/kg BID to be explored in the BT474 model. This schedule
resulted in greater efficacy; 40% regression (Fig. 2D). IHDS studies
in SCID mice strains permitted a lower MWTD of 50 mg/kg BID
AZD8835 but nevertheless delivered tumor growth inhibition
(see Discussion). We therefore investigated the day 1 and 4 BID
AZD8835 schedule in our subsequent in vivo efficacy studies in the
combination setting (see below).
PD studies: AZD8835 IHDS induces waves of cell death
To understand the efficacy observed with IHDS, we evaluated
pharmacokinetic/pharmacodynamic (PK/PD) relationships in
both 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 and
dose/exposure dependent (Fig. 3A). In parallel studies, we
observed glucose and insulin elevation as a transient pharmacologic response to AZD8835 in mice (Supplementary
Fig. S2A and S2B), as also observed for other PI3K-inhibitors
(10, 28, 29).
Figure 3B shows an extended AZD8835 dose response in BT474
xenografts at 2-hour timepoint. This demonstrates a relationship
between exposure and pAKT reduction in tumor tissue. Furthermore, AZD8835 induced rapid-onset apoptosis (2 hours) as
measured by cleavage of caspase-3 (CC3), consistent with our
cell studies (Fig. 1B, D, and E). This CC3 elevation was confirmed
using an IHC endpoint (Fig. 3C). We also observed that in
addition to CC3 induction on day 1, CC3 was also induced when
AZD8835 was redosed on day 4 (Fig. 3D).
Interestingly, in BT474 xenografts, we observed that AZD8835
increased gH2AX (pSer139-H2AX), which tracked with an
increase in CC3 signal (Supplementary Fig. S3A). Similarly,
AZD8835 increased gH2AX (Supplementary Fig. S3B) in MCF7
xenografts (which lack caspase-3 expression; ref. 30). We suspected that the increase in gH2AX was a consequence of caspase
activation, possibly as a response to endonuclease/caspase-mediated DNA fragmentation during apoptosis (31), thereby providing an alternative apoptosis readout in MCF7. Supportive of this,
we demonstrated that the AZD8835-induced gH2AX signal in
MCF7 cells was caspase dependent by using the caspase inhibitor,
QVD (Supplementary Fig. S3C). In addition, we measured PARP
cleavage as another measure of apoptosis in MCF7 xenografts;
the response tracked that for gH2AX (Supplementary Fig. S3B
and S3C).
PK/PD efficacy modeling
The pAKT/CC3 PD and efficacy data from multiple AZD8835
monotherapy studies in the BT474 xenograft were compiled to
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A
3.0
MCF7
5.0
Control
AZD8835 25 mg/kg bid cont.
4.0
Relative tumor volume
Relative tumor volume
B
BT474
2.0
1.2
1.0
0.8
0.6
Control
AZD8835 25 mg/kg bid cont.
3.0
2.0
1.2
1.0
0.8
0.6
0
5
10
15
0
20
5
Days of treatment
C
15
20
15
20
Days of treatment
D
BT474
BT474
3.0
4.0
Relative tumor volume
Control
AZD8835 25 mg/kg bid cont.
AZD8835 50 mg/kg bid d1/d2
AZD8835 100 mg/kg qd d1/d4
3.0
Relative tumor volume
10
2.0
1.2
1.0
0.8
0.6
Control
AZD8835 100 mg/kg bid d1/d4
2.0
1.2
1.0
0.8
0.6
0.4
0.4
0
5
10
15
20
Days of treatment
0
5
10
Days of treatment
Figure 2.
Intermittent high-dose scheduling (IHDS) of monotherapy AZD8835 delivers antitumor efficacy. Continuous dosing schedule (25 mg/kg; BID) in BT474 (A) or
MCF7 (B) xenografts. C, comparison of continuous and intermittent (50 mg/kg, 2 days on/5 off, BID or 100 mg/kg, day 1 and 4, QD) dosing schedules
in BT474 xenografts. D, increased efficacy in BT474 xenografts with 100 mg/kg, day 1 and 4, BID schedule. Bars alongside timelines illustrate dosing of each agent.
Error bars, SEM.
build a PK/PD/efficacy model (see Supplementary File S2). Data
generated across studies using different doses and schedules
(continuous and IHDS) were a good fit and could be explained
using this model. In the model, AZD8835 is assumed to have a
dual action comprising antiproliferative and proapoptotic
effects. The antiproliferative effects are reflected by tumor
pAKT-T308 levels (PI3K-pathway signaling) and proapoptotic
effects by CC3 levels. A direct response Emax-type model was
used in the PK/PD analysis of tumor pAKT-T308 versus concentration of AZD8835 in plasma. An indirect response model
with tumor cells divided into sensitive and insensitive cells was
used to explain the CC3 effects. Results of the model-fits to
tumor pAKT, CC3, and Volume are shown in Supplementary File
S2. The model captures a desensitization to apoptosis (CC3) on
continuous dosing, contrasting with IHDS which produces
repeat waves of strong apoptosis induction (Fig. 3E and Supplementary File S2).
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Selection of agents to combine with AZD8835
Our 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 be
achieved when using AZD8835 as a combination treatment,
particularly where combination cotherapies and dosing schedule
are optimized for therapeutic index. Therefore, continuing with
our focus on mPIK3CA, ERþ breast cancer models, our next aim
was to investigate potential combinations in vitro which could
then be evaluated in in vivo efficacy studies using a backbone of
AZD8835 IHDS. With a focus on mechanisms that have demonstrated benefit in ERþ breast cancer patients (see Discussion),
we evaluated two broad classes of combination partner for
AZD8835: "inter-pathway," combining with agents targeting
parallel but interconnected driver pathways (ER, CDK4/6); or
"intra-pathway," combining with an agent targeting downstream
in the PI3K-pathway.
Molecular Cancer Therapeutics
Intermittent High-Dose Scheduling of PI3K Inhibitor AZD8835
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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 relationship
examples 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 in
BT474, 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 dosing
schedules, derived via PK/PD modeling. All data representative of multiple experiments. Error bars, SEM. Significance markers (vs vehicle, or intra-dose
comparisons where indicated): , P < 0.05; , P < 0.01; , P < 0.001.
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Mol Cancer Ther; 2016
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Hudson et al.
First, we evaluated the effect of AZD8835 combinations with
these classes of agents in cell growth studies using an Incucyte
Imager. Anti-estrogens were selective estrogen receptor downregulators (SERDs), fulvestrant (32) or AZD9496 (33), CDK4/6
inhibitor was palbociclib (PD-0332991; ref. 34) and AZD2014
(35) provided a dual mTORC1/2 kinase inhibitor.
Consistent with our expectations, stronger growth inhibition
was observed with all combinations compared with monotherapies. This is illustrated in MCF7 cells where the combination of 0.3
mmol/L AZD8835 plus 0.1 mmol/L fulvestrant or 0.1 mmol/L
palbociclib prevented cell growth (Fig. 4A). Similar results were
observed with the alternative SERD, AZD9496 (Supplementary
Fig. S4A–S4C), and also with fulvestrant in other mPIK3CA cell
lines, BT474 and T47D (Supplementary Fig. S4A and S4B). In
addition, we confirmed that AZD8835/fulvestrant combination
activity was retained (Supplementary Fig. S4C) in two fulvestrantrefractory MCF7-derived cell lines that were generated by continuous incubation with fulvestrant (MCF7-F100-16; ref. 36) or by
long-term estrogen deprivation (MCF7-GHPED, generated at AZ).
To provide mechanistic understanding of these combination
effects, 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 general
theme was stronger pathway-biomarker suppression with combinations compared with monotherapies. Furthermore, in some
cases monotherapy induced pathway cross-talk/activation, consistent with a potential resistance mechanism, which was reversed
by the combination partner.
Some of the specific mechanistic observations for combinations of AZD8835 with SERDs, fulvestrant or AZD9496, were
observed across more than one cell line (Fig. 4B). Across all cell
lines, the combinations achieved increased suppression of
pP70S6K and/or pS6. Also, particularly in T47D and BT474 cells,
the SERDs increased pAKT which was reversed by the addition of
AZD8835. In addition, particularly in T47D cells and to a small
extent in MCF7 cells, but not in BT474 cells, these combinations
increased ERa downregulation. Finally, in T47D the SERDs
reversed AZD8835-mediated induction of progesterone receptor
(PR), an ERa transcriptional target. Collectively, these findings are
consistent with and build on previous reports with related agents
(23, 37–40).
AZD8835, when combined with palbociclib, consistently
enhanced suppression of pRb (and total Rb) and the E2F
transcriptional targets, CDC6 and E2F (E2F-1), consistent with
stronger inhibition of the Rb-pathway (Fig. 4C). AZD8835 also
suppressed palbociclib-induced increase in Cyclin-D1. Also,
particularly in T47D, PI3K-pathway biomarkers (pAKT and
pS6) were increased by palbociclib treatment but reversed by
AZD8835 (Fig. 4C). In addition, AZD8835 plus palbociclibtreated cells were subject to transcriptional profiling, using the
Fluidigm platform. We observed that the expression of E2F
transcriptional markers were suppressed more with the combination, compared with single agents, and more consistently
than for selected ER and FOXO transcriptional targets (Fig. 4D
and Supplementary Fig. S4D).
The combination of AZD8835 plus the dual mTORC1/2
inhibitor, AZD2014, also enhanced inhibition of cell proliferation (Supplementary Fig. S4E). Alongside, we observed
enhanced suppression of PI3K-pathway biomarkers, including
pS6 (Supplementary Fig. S4F), as reported previously for PI3K/
OF8 Mol Cancer Ther; 2016
mTOR inhibitor combinations (41). In addition, the combination enhanced suppression of pRb (and total Rb; Supplementary Fig. S4F).
Collectively the above data supported progression of each of
these classes of combinations into in vivo efficacy studies.
In vivo efficacy studies applying IHDS AZD8835 in combination
Next, we performed combination in vivo efficacy studies, applying AZD8835 IHDS (day 1 and 4 BID). As previously demonstrated (Fig. 2D), AZD8835 dosed at 100 mg/kg day 1 and 4 BID in
the BT474/nude-mice xenograft model again induced tumor
regression (31 and 36%: Fig. 5A). Notably, AZD8835 combined
as a doublet with fulvestrant or palbociclib increased tumor
regression (59 and 54%, respectively) compared with single
agents (Fig. 5A and Supplementary Table S5). We also performed
similar studies in the MCF7 xenograft model, grown in SCID
mice, where the tolerated dose (MWTD) of AZD8835 was lower
(50 mg/kg BID day 1 and 4). Again, a combination benefit with
either fulvestrant or palbociclib was seen (Fig. 5B and Supplementary Table S5). Moreover, when the agents were used in a
triplet combination, thereby simultaneously targeting three driver
pathways, a strikingly profound tumor regression (92%) was
observed (Fig. 5B and Supplementary Table S5). We also observed
stronger in vivo biomarker modulation with combinations relative
to monotherapies for a subset of PD endpoints (Fig. 5C), consistent with cell studies (Fig. 4B and C). Superior efficacy with
combinations was also observed when fulvestrant was replaced
with an alternative SERD, AZD9496 (Supplementary Fig. S5A and
Table 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 AZD8835
with the dual mTORC1/2 inhibitor, AZD2014, also resulted in
improved efficacy compared with monotherapy. This was particularly evident in the BT474 model (in nude mice) where tumor
regression was achieved with a triplet combination incorporating
fulvestrant, despite a tolerability requirement for AZD8835 dose
reduction (Supplementary Fig. S6A and Supplementary Table S5).
In the MCF7 model (SCID mice), this combination was less well
tolerated so the overall efficacy achieved with the lower MWTD
was relatively moderate; nevertheless a positive combination
effect was still observed (Supplementary Fig. S6B and S6C and
Supplementary Table S5).
Discussion
Here, 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 to
continuous dosing. We were motivated to explore this path given
that clinical dose and exposure of PI3K inhibitors is capped by
normal tissue toxicities (12, 13, 15) which may result in suboptimal pathway inhibition in tumors. Indeed tolerability
issues observed on continuous dosing of PI3K pathway inhibitors are driving a shift towards more intermittent scheduling,
albeit to date these are less radical shifts than illustrated in this
manuscript.
We demonstrated that we could achieve higher dose in in vivo
studies when applying IHDS, compared with continuous dosing.
This was particularly the case in nude mice (BT474 model), where
greater tolerability allowed the IHDS concept to be tested
Molecular Cancer Therapeutics
Intermittent High-Dose Scheduling of PI3K Inhibitor AZD8835
Figure 4.
þ
Selection of combination partners for AZD8835 in ER breast cell lines. A, growth inhibition by combinations with fulvestrant or palbociclib in MCF7 (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), or
AZD8835 (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 E2F
transcriptional 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.
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Mol Cancer Ther; 2016
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Hudson et al.
Figure 5.
þ
Combination of AZD8835 with fulvestrant and/or palbociclib results in tumor regression in ER breast xenografts. Efficacy studies applying combination of
AZD8835 (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 enhanced
combination effect on pathways (PI3K, CDK4/6) or apoptosis (CC3). Fulvestrant dosed day previous to AZD8835 to allow plasma exposure to reach steady
state. 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.
maximally (100 mg/kg AZD8835 day 1 and 4 BID dosing schedule) 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 relatively modest doubling of dose (to 50 mg/kg AZD8835) compared
with that achievable using continuous dosing; even so, despite
near halving of accumulative weekly dose relative to continuous
dosing, monotherapy AZD8835 IHDS nevertheless delivered
OF10 Mol Cancer Ther; 2016
tumor growth inhibition (MCF7 - Fig. 5B and Supplementary
Fig. S5A; T47D - Supplementary Fig. S5B) so this encouraged us to
progress into combination studies. Another notable feature of
BT474 xenografts is a relatively slow growth rate compared with
some xenograft models, although arguably this is more representative of growth rates of clinical tumors. Growth of such tumors is
less likely to "take off" during the nondosing days characteristic of
intermittent schedules.
Molecular Cancer Therapeutics
Intermittent High-Dose Scheduling of PI3K Inhibitor AZD8835
A PK/PD/efficacy model was built using monotherapy BT474
data and resulting simulations fitted well to our overall observations, as detailed in Supplementary File S2. The model
captures our observations that on continuous dosing schedule
(25 mg/kg BID) there is a reduced apoptosis signal, where the
signal is stronger following the first dose compared with the
subsequent doses which induced relatively little apoptosis. In
contrast, 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 subpopulation of cells sensitive to induction of apoptosis and days are
required to replenish such population. Alternatively, the break
in dosing may allow "reset" of the pathway signaling from
pathway feedback and reactivation which overcomes the desensitization to apoptosis induction.
We then prioritized our combination therapy strategies for
AZD8835. We considered evaluation of combinations with
anti-estrogen/anti-estrogen-receptor (ER) therapy to be of particular 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/ER
signaling pathway interactions, coupled with reports that combining PI3K-inhibitor and ER directed agents can combat resistance (37–40). Such PI3K/ER pathway directed combination
therapies have recently progressed into the clinic (15, 42, 43).
A second attractive combination opportunity for PI3K-inhibitors
was combination with CDK4/6-inhibitors, which target the Rbpathway, since in mPIK3CA tumors this combination has previously been reported as synergistic (44). Also CDK4/6 inhibitors
may combine well with anti-estrogens and help to combat
acquired resistance to ER antagonists (37, 45, 46), and this
approach has generated positive clinical data (47). Therefore
triplet combinations may also have potential and initial clinical
studies are already underway, for example, combining CDK4/6
inhibitor (LEE011) with PI3K inhibitor (BYL719) and anti-estrogen letrozole (48). Regarding intra-PI3K-pathway combinations,
an interesting combination opportunity is with mTOR inhibitors.
Again there is some precedent in this area, for example, Elkabets
and colleagues (41) reported synergy through combining a rapalogue mTORC1-inhibitor, everolimus, with PI3K inhibitor
BYL719; also there is some precedent for dual PI3K/mTOR
inhibition using mixed profile inhibitors such as GDC-0980,
dosed intermittently (49).
We initially combined AZD8835 with ER, CDK4/6, and mTOR
directed agents [SERDs (fulvestrant or AZD9496), palbociclib,
AZD2014, respectively] in in vitro studies, observing enhanced
combination activity and coupled with mechanistic data. We then
evaluated these combinations in in vivo efficacy studies using a
foundation of AZD8835 IHDS. Compelling efficacy, consistently
observed as a tumor regression response (Fig. 5A and B and
Supplementary Fig. S5A and S5B), was observed with the interpathway combinations where AZD8835 was combined with
SERDs and/or CDK4/6 inhibitors. Also notable with all these
inter-pathway combinations was that the MWTD of each agent
was the same as used in monotherapy studies, indicating minimal
combined toxicities, as illustrated in Supplementary Fig. S7.
Combination benefit was also illustrated with an "intra-pathway" combination where AZD8835 was combined with
AZD2014. Despite a requirement for significant dose reductions
(Supplementary Fig. S6), particularly in the MCF7/SCID model,
combination benefit was again observed. We anticipate that this
combination may be better tolerated in the clinic where hyperglycemia, the suspected tolerability issue in mice, could be better
managed.
We conclude that AZD8835 IHDS provides flexibility and a
promising alternative to continuous dosing with potential for an
improved therapeutic index. Such schedules and combinations
merit clinical evaluation.
Disclosure of Potential Conflicts of Interest
All 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 were
disclosed by the other authors.
Authors' Contributions
Conception and design: K. Hudson, U.J. Hancox, U.M. Polanska, P. Morentin
Gutierrez, S.C. Cosulich, L. Ward, F. Cruzalegui, S. Green
Development of methodology: K. Hudson, C. Trigwell, U.M. Polanska,
P. Morentin Gutierrez, M. Cumberbatch
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): U.M. Polanska, A. Avivar-Valderas, O. Delpuech,
P. Dudley, M. Cumberbatch
Analysis 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. Cosulich
Writing, 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. Green
Administrative, technical, or material support (i.e., reporting or organizing
data, constructing databases): U.J. Hancox, R. McEwen, U.M. Polanska,
L. Hanson
Study supervision: K. Hudson, L. Ward
Other (project leader): S. Green
Acknowledgments
The authors thank Mike Dymond for advice on statistical analysis of data and
to Amar Rahi, Emily Lawrie, and the AZ Lab Animal Sciences Group in support
of PD/efficacy studies.
The costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked advertisement in
accordance 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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