Bcl-2/Bcl-xL inhibition increases the efficacy of MEK inhibition alone and in combination with PI3 kinase inhibition in lung and pancreatic tumor models

Article abstract of DOI:10.1158/1535-7163.MCT-12-0949

Although mitogen-activated protein (MAP)-extracellular signal-regulated kinase (ERK) kinase (MEK) inhibition is predicted to cause cell death by stabilization of the proapoptotic BH3-only protein BIM, the induction of apoptosis is often modest. To determine if addition of a Bcl-2 family inhibitor could increase the efficacy of a MEK inhibitor, we evaluated a panel of 53 non-small cell lung cancer and pancreatic cancer cell lines with the combination of navitoclax (ABT-263), a Bcl-2/Bcl-x_L (BCL2/BCL2L1) antagonist, and a novel MAP kinase (MEK) inhibitor, G-963. The combination is synergistic in the majority of lines, with an enrichment of cell lines harboring KRAS mutations in the high synergy group. Cells exposed to G-963 arrest in G1 and a small fraction undergo apoptosis. The addition of navitoclax to G-963 does not alter the kinetics of cell-cycle arrest, but greatly increases the percentage of cells that undergo apoptosis. The G-963/navitoclax combination was more effective than either single agent in the KRAS mutant H2122 xenograft model; BIM stabilization and PARP cleavage were observed in tumors, consistent with the mechanism of action observed in cell culture. Addition of the phosphatidylinositol 3-kinase (PI3K, PIK3CA) inhibitor GDC-0941 to this treatment combination increases cell killing compared with double- or single-agent treatment. Taken together, these data suggest the efficacy of agents that target the MAPK and PI3K pathways can be improved by combination with a Bcl-2 family inhibitor.

Full text of DOI:10.1158/1535-7163.MCT-12-0949

Published OnlineFirst March 8, 2013; DOI: 10.1158/1535-7163.MCT-12-0949
Molecular
Cancer
Therapeutics
Chemical Therapeutics
Bcl-2/Bcl-xL Inhibition Increases the Efficacy of MEK
Inhibition Alone and in Combination with PI3 Kinase Inhibition
in Lung and Pancreatic Tumor Models
Nguyen Tan1, Maureen Wong1, Michelle A. Nannini1, Rebecca Hong1, Leslie B. Lee1, Stephen Price2,
Karen Williams2, Pierre Pascal Savy2, Peng Yue1, Deepak Sampath1, Jeffrey Settleman1,
Wayne J. Fairbrother1, and Lisa D. Belmont1
Abstract
Although mitogen-activated protein (MAP)–extracellular signal-regulated kinase (ERK) kinase (MEK)
inhibition is predicted to cause cell death by stabilization of the proapoptotic BH3-only protein BIM, the
induction of apoptosis is often modest. To determine if addition of a Bcl-2 family inhibitor could increase the
efficacy of a MEK inhibitor, we evaluated a panel of 53 non–small cell lung cancer and pancreatic cancer cell
lines with the combination of navitoclax (ABT-263), a Bcl-2/Bcl-xL (BCL2/BCL2L1) antagonist, and a novel
MAP kinase (MEK) inhibitor, G-963. The combination is synergistic in the majority of lines, with an enrichment
of cell lines harboring KRAS mutations in the high synergy group. Cells exposed to G-963 arrest in G1 and a
small fraction undergo apoptosis. The addition of navitoclax to G-963 does not alter the kinetics of cell-cycle
arrest, but greatly increases the percentage of cells that undergo apoptosis. The G-963/navitoclax combination
was more effective than either single agent in the KRAS mutant H2122 xenograft model; BIM stabilization and
PARP cleavage were observed in tumors, consistent with the mechanism of action observed in cell culture.
Addition of the phosphatidylinositol 3-kinase (PI3K, PIK3CA) inhibitor GDC-0941 to this treatment com-
bination increases cell killing compared with double- or single-agent treatment. Taken together, these data
suggest the efficacy of agents that target the MAPK and PI3K pathways can be improved by combination with
a Bcl-2 family inhibitor. Mol Cancer Ther; 12(6); 853–64. ꢀ2013 AACR.
prevent the cancer cell from changing its dependency to
Introduction
another signaling pathway. There is considerable preclin-
ical evidence supporting this approach, for example, in
the setting of combining inhibitors of mitogen-activated
protein (MAP)–extracellular signal-regulated kinase (ERK)
kinase (MEK; MAP kinase kinase, MAP2K) with inhib-
itors of PI3 kinase (phosphatidylinositol 3-kinase, PIK3CA)
or (2–7) BRAF (8–10), and these combinations are begin-
ning to show efficacy in the clinic (11). A third approach to
enhancing the efficacy of targeted therapy is to simulta-
neously target downstream proteins that protect tumor
cells from apoptosis and thus increase overall cell killing
(12–14). This strategy relies on increasing cancer cell cyto-
toxicity cells to decrease the probability that a cell will sur-
vive to develop resistance. In addition, altering upstream
dependencies will be of no avail because the signaling
pathways ultimately converge on these downstream apop-
totic pathways. Here, we have explored the combination
of an MEK kinase inhibitor and a Bcl-2/Bcl-xL inhibitor to
evaluate the potential utility of this combination treatment
strategy.
Recent advances in cancer drug therapies associated
with targeting oncogenic "drivers" have shown tremen-
dous potential for improving clinical benefit. However, a
major limitation to the overall benefit from targeted ther-
apy is the development of drug resistance. Resistance can
occur because of mutations that render the drug target
insensitive to the inhibitor or when cancer cells change
their dependency on the pathway that is targeted. In the
first example, resistance can be overcome by developing
new drugs that effectively inhibit resistance-associated
mutants, as in the example of dasatinib and nilotinib,
which are effective on BCR/ABL mutants that confer
resistance to imatinib (1). Another approach is to target
multiple signaling pathways simultaneously, and thus
Authors' Affiliations: 1Genentech, Inc., South San Francisco, California;
and 2Argenta Discovery Ltd., Harlow, Essex, United Kingdom
Note: Supplementary data for this article are available at Molecular Cancer
Therapeutics Online (http://mct.aacrjournals.org/).
MEKtransduces signals downstream ofthe EGFR, RAS,
and RAF proteins, and activating mutations in any of
these proteins can lead to enhanced MEK activity. MEK
activation in turn produces prosurvival signals via inhi-
bition of the proapoptotic BH3-only proteins BIM and
Corresponding Author: Lisa D. Belmont, Genentech, Inc., 1 DNA Way,
South San Francisco, CA 94080. Phone: 650-467-7462, Fax: 650-742-
5179; E-mail: belmont.lisa@gene.com
doi: 10.1158/1535-7163.MCT-12-0949
ꢀ2013 American Association for Cancer Research.
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Tan et al.
A
B
Navitoclax
GDC-0941
G-963
C
D
Percent inhibition
Bliss score
A427
G-963 + Navitoclax at
0 µmol/L
100
80
60
40
20
0
3.3
1.1
0.01 µmol/L
0.04 µmol/L
0.12 µmol/L
0.37 µmol/L
1.1 µmol/L
3.3 µmol/L
30
20
100
80
60
40
20
0
0.37
0.12
0.04
0.01
0
10
0
−10
−20
0.0001 0.001
0.01
0.1
1
G-963 (µmol/L)
G-963 (µmol/L)
G-963 (µmol/L)
E
1,200
1,000
800
600
400
200
0
−200
−400
−600
K-Ras
mutation
EGFR
activating
mutation
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Published OnlineFirst March 8, 2013; DOI: 10.1158/1535-7163.MCT-12-0949
Bcl-xL Inhibition Enhances MEK Inhibitor Efficacy
BAD and stabilization of the antiapoptotic Bcl-2 family
member, MCL1. MEK activates ERK1/2 (MAPK1,
MAP3K2), which subsequently phosphorylates BIM on
Ser69, priming it for phosphorylation by p90RSK, and
ultimately resulting in its ubiquitination and proteasomal
degradation (15). In addition, ERK1/2-mediated phos-
phorylation of FOXO3A promotes its degradation and
thus inhibits FOXO3A-dependent transcription of BIM
(16). ERK1/2 also directly activates p90RSK, which inac-
tivates BAD. RSK phosphorylates BAD on Ser112 facili-
tating its binding to 14-3-3, which acts to sequester and
inhibit its proapoptotic activity. Activation of ERK1/2 is
reported to stabilize the antiapoptotic protein, MCL1 (17).
Thus, the inhibition of MEK is expected to promote
apoptosis by increasing BIM levels (both by transcription-
al activation and protein stabilization), by activating BAD
via reducing inhibitory phosphorylation, and by decreas-
ing levels of MCL1 through promotion of its proteasomal
degradation (Fig. 1A).
Bcl-2 family members sequester and inhibit the "exe-
cutioner" proteins BAX and BAK (BAK1), which form
pores in the mitochondrial outer membrane and allow
cytochrome C release (18). The antiapoptotic Bcl-2 family
members include Bcl-2, Bcl-xL, MCL1, BCL2A1, and Bcl-w
(BCL2L2). Of these, Bcl-2, Bcl-xL, and MCL1are expressed
in solid tumors. Inhibition of MEK neutralizes Bcl-2 and
Bcl-xL by increasing the levels of functional BIM and BAD,
and it neutralizes MCL1 by destabilizing MCL1 protein
and by stabilizing its antagonist, BIM. However, the
activation of BAD is vulnerable step in this mechanism
because compensatory activation of the PI3K pathway can
inhibit BAD via phosphorylation on Ser136. Addition of
navitoclax, a "BAD mimetic" that is not subject to the
phosphorylation events that inactivate BAD (19) could
potentially overcome mechanisms of resistance that rely
on inhibition of BAD. Furthermore, the MEK inhibitor/
navitoclax combination may increase efficacy in cases
where MEK inhibition alone results in less than complete
blockade of Bcl-xL and Bcl-2 activities.
and pancreatic ductal adenocarcinoma derived cell lines
with the aim of (1) determining the prevalence of MEK
inhibitor/navitoclax synergy in cancer lines that frequent-
ly harbor activating KRAS mutations, (2) identifying pre-
dictive biomarkers for the combination in these cancers,
and (3) evaluating combination mechanism of action. In
addition, we evaluated the combination mechanism of a
triple combination of navitoclax with a MEK inhibitor and
a PI3K inhibitor.
Materials and Methods
Chemicals
Z-VAD-FMK (Promega), necrostatin-1 (Tocris Biosci-
ence), Navitoclax (Abbott Laboratories), G-963, and GDC-
0941 (Genentech, Inc.) were dissolved in dimethyl sulf-
oxide (DMSO; Sigma-Aldrich).
Cell culture, antibodies, and reagents
Cell lines were obtained from the American Type Cul-
ture Collection or Deutsche Sammlung von Mikro-
organismen und Zellkulturen (DSMZ), expanded, and
stored at early passage in a central cell bank. Lines were
authenticated by short tandem repeat (STR) and geno-
typed upon reexpansions. Cells were grown in RPMI
1640 medium supplemented with 10% FBS and 2
mmol/L glutamine (Invitrogen) and passaged no more
than 20 times after thawing. Antibodies directed against
the following proteins were used: phospho-ERK, ERK1/
2, phospho-RSK, RSK1, phospho-BAD, BIM, phospho-
BIM, Bcl-xL, MCL1, p21, p27, PARP, phospho-S6 (Cell
Signaling Technology), BAD (Santa Cruz Biotechno-
logy), g-H2AX (Millipore), MCL1 (BD Biosciences), BIM
(Enzo Life Sciences), b-actin (Sigma-Aldrich), and HRP-
conjugated horse anti-mouse and goat anti-rabbit anti-
bodies (Vector Laboratories).
Biochemical assays, Western blotting, and
immunoassays
Activity on purified MEK1 was measured with a
homogenous time resolved fluorescence (HTRF) assay.
Selectivity was measured at 1 mmol/L using a Z’-Lyte
Kinase Assay Kit using Invitrogen’s SelectScreen Kinase
Profiling Service. To examine protein expression, cells
were lysed in ice-cold RIPA buffer (Cell Signaling Tech-
nology) containing 1 mmol/L Peflabloc, Phosphatase
Inhibitor Cocktail 1 and 2 (Sigma-Aldrich), and complete
EDTA-free protease inhibitor tablet (Roche). Equal
amounts of protein were subjected to SDS-PAGE (4–
12% Bis-Tris; Invitrogen), and protein levels were
evaluated by Western blotting. Solid-phase microspot
Navitoclax has been shown to enhance the activity of
chemotherapeutic agents (20–24). However, studies on its
ability to enhance targeted therapy have been limited.
Cragg and colleagues showed BIM-dependent combina-
tion efficacy of a MEK inhibitor and ABT-737, a com-
pound with similar biochemical activity to navitoclax,
in several BRAF mutant models (12). Konopleva and
colleagues showed similar combination efficacy in AML
models (25). In this work, we set out to determine the
effectiveness of a navitoclax/MEK inhibitor combination
in a large panel of non–small cell lung cancer (NSCLC)
Figure 1. Navitoclax enhances the activity of G-963 in NSCLC cell lines. A, MAPK and PI3K pathway regulation of intrinsic apoptosis. B, chemical structures
of navitoclax (ABT-263), G-963, and GDC-0941. C, general synthetic strategy for G-963: ethyl glycolate, NaH, DMF, 0ꢀC to rt, 16 hours (86%; a); TfNPh2,
DIPEA, DME, 95ꢀC, 35 minutes (80%; b); Pd2(dba)3, XantPhos, K3PO4, 105ꢀC, 24 hours (93%; c); AgBF4, ICl, DCM, ꢁ50ꢀC, 1 hour (86%; d); 1 mol/L
NaOH, EtOH, 65ꢀC, 1.5 hours (e); H2C ¼ CH-O(CH2)2ONH2, HOBT, EDCI, DIPEA, THF, rt, 18 hours (60% over 2 steps; f); 1 mol/L HCl, MeOH, EtOH,
rt, 1.5 hours (91%; g). D, example of synergistic activity of navitoclax and G-963 in A427 cells. Left, G-963 dose–response curves in the presence of various
doses of navitoclax; middle, heat map of growth inhibition; and right, heat map of Bliss scores. E, average Bliss sums and mutational status of EGFR and
KRAS across the NSCLC panel. For EGFR mutation status, ꢂ, DEL.745KELREA > K; þ, 790T > M, 858L > R. Error bars, SD.
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Tan et al.
immunoassays from Meso Scale Discovery (MSD) were
also used to determine protein expression.
weight change (%) ¼ [(weightday
ꢁ weightday 0)/
new
weightday 0] ꢃ 100. Tumor sizes and mouse body weights
were recorded twice weekly over the course of treatment,
and the mice were observed daily. A linear mixed model-
ing approach was used to analyze the repeated measure-
ment of tumor volumes and body weight from the same
animals over time (27). This approach addresses both
repeated measurements and modest dropouts because of
any nontreatment-related death of animals before study
end. Cubic regression splines were used to fit a nonlinear
profile to the time courses of log2 tumor volume at each
dose level. These nonlinear profiles were related to dose
within the mixed model. Tumor growth inhibition as a
percentage of vehicle control (% TGI) was calculated as
the percentage of the area under the fitted curve (AUC) for
the respective dose group per day in relation to the
vehicle, using the following formula: % TGI ¼ 100 ꢃ (1 ꢁ
AUCdose/AUCveh). Data were analyzed using R, version
2.8.1 (R Development Core Team 2008; R Foundation for
Statistical Computing), the mixed models were fitted
within R using the nlme package, version 3.1-89, and
plotted using Prism 5 software (GraphPad Software, Inc.).
For pharmacodynamic studies, tumor samples were
immediately frozen in liquid nitrogen. Tumors were dis-
sociated in cell extraction buffer and lysates analyzed by
Western blotting.
Combination matrices
Cells were seeded in 384-well plates at 3,000 cells/well.
After 24 hours, cells were treated with navitoclax (dose
range of 14 nmol/L–3.3 mmol/L) and G-963 (dose range of
1.5 nmol/L–3.3 mmol/L) in a 7 ꢃ 9 matrix. Each treatment
was done in quadruplicate wells. Cells were treated for 72
hours, and ATP was determined with CellTiter-Glo (Pro-
mega). The experiment was done at least thrice, and the
data are presented as an average of all runs. G-963 IC50
values were calculated from the 9 single-agent doses
using a 4 parameter fit with XLFit (IDBS). The Bliss
expectation was calculated with the equation (A þ B) ꢁ
A ꢃ B, where A and B are the fractional growth inhibitions
induced by agents A and B at a given dose. The difference
between Bliss expectation and observed growth inhibition
induced by the combination of agents A and B is the "Bliss
excess" (26). For triple combinations, the experiment was
done exactly as that of the double combination study, with
or without the addition of GDC-0941 (1 mmol/L) to all
wells. Two independent runs of the triple combinations
were done on each cell line.
Caspase assay
Cells were seeded in 96-well plates at 5,000 cells/well.
After 24 hours, cells were treated as indicated and caspase
activity was determined (Promega Caspase-Glo 3/7).
Data are presented as an average of 2 runs each done in
duplicate.
Image processing and statistics
Relative protein levels were quantified using the ImageJ
software (National Institutes of Health), and normalized
to b-actin. Statistical analysis was done using Prism 5
(GraphPad Software) to determine the r (rho) value
(Spearman ranking) and P value (2-tailed).
Flow cytometry
Adherent cells were detached with EDTA (10 mmol/L),
collected by centrifugation for 5 minutes at 300 ꢃ g,
washed with PBS, fixed in ice cold 90% methanol. Cells
were collected by centrifugation, washed, stained with
10 mg/mL propidium iodide (Sigma-Aldrich) and ana-
lyzed by flow cytometry (10,000 cells per treatment).
Results
Navitoclax enhances the activity of a MEK inhibitor
in cancer cell lines
We selected navitoclax, a dual Bcl-2/Bcl-xL inhibitor
that is the subject of clinical trials (28–31), and a novel
MEK inhibitor, G-963 (Fig. 1B), to test the combination
efficacy associated with targeting these proteins. The
general synthetic route for G-693 is shown (Fig. 1C) and
a detailed route is described in the supplementary
material (Supplementary Fig. S1). G-963 has an IC50 of
4 nmol/L (SD ¼ 1.6) on purified MEK1, and is selective for
MEK1 and MEK2 across a panel of 100 kinases (Supple-
mentary Fig. S2). G-963 was active in 39% of the lines in a
panel of 31 NSCLC cell lines, with cellular IC50 values
ranging from 3 to 600 nmol/L. The majority of responsive
cell lines (IC50 < 1 mmol/L) harbored activating mutations
in EGFR or KRAS (Table 1), consistent with reports that
activating KRAS mutations generally correlate with great-
er sensitivity to single-agent MEK inhibition (32–35). To
evaluate the potential benefit of combining an MEK
inhibitor and a Bcl-2/Bcl-xL inhibitor, we employed a
dose matrix to sample a large range of concentrations
and concentration ratios and analyzed combination
effects using the Bliss independence model. Positive Bliss
In vivo studies
Human NSCLC H2122 cells were implanted sub-
cutaneously in the hind flank of female NCR nude
mice (Taconic Farms). Tumors were monitored until
they reached a mean tumor volume of 250-350 mm3 and
distributed into groups of 8 animals before initiating
dosing. Navitoclax was dissolved in 60% Phosal 50 PG,
30% PEG 400, 10% EtOH vehicle, and dosed daily by oral
administration. G-963 was diluted in 0.5% methycellu-
lose/0.2% Tween-80 (MCT) and dosed daily by oral
administration. Tumor volume was measured in 2 dimen-
sions (length and width), using Ultra Cal IV calipers
(Model 54 10 111; Fred V. Fowler Company), as follows:
tumor volume (mm3) ¼ (length ꢃ width2) ꢃ 0.5 and
analyzed using Excel version 11.2 (Microsoft Corpora-
tion). Animal body weights were measured using an
Adventurer Pro AV812 scale (Ohaus Corporation). Per-
cent weight change was calculated as follows: body
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scores indicate combinations where the effect is greater
than additive. The heat map for A427 shows that there are
positive Bliss scores across a large range of concentrations
for both compounds (Fig. 1D). Heat maps for cell lines that
had intermediate or no synergy are in the supplementary
material (Supplementary Fig. S3). To summarize these
data, the Bliss scores for all dose combinations are added
and referred to as the Bliss sum.
We applied this combination analysis to 31 NSCLC cell
lines and 22 pancreatic cancer cell lines and found that in
the majority of cell lines the treatment combination was
associated with a greater than additive response (Table 1
and Supplementary Table S1). In the NSCLC panel, there
was a significant enrichment for cell lines with activating
mutations in EGFR or KRAS in the high synergy set (P ¼
0.01, Fishers exact test, using a cutoff Bliss Sum ꢄ 100; Fig.
1E). Sensitivity to single-agent MEK inhibitor is not
required for synergy, although the high synergy cell
lines (Bliss sum ꢄ100) are enriched for G-963 sensitivity
(P ¼ 0.0024; Fishers exact test).
BIM stabilization are comparable following combina-
tion treatment compared with the single-agent G-963. At
early time points, MCL1 levels increase in navitoclax
treated cells and decrease in G-963–treated cells, and the
effect is balanced out in the combination treatment set-
ting, similar to an effect previously observed in AML
models with ABT-737 and MEK inhibition (25). Overall,
signaling downstream of MEK does not seem to be im-
pacted by navitoclax, but there is enhanced PARP cleav-
age in all cell lines treated with the combination compared
with either single-agent (Fig. 2B).
Navitoclax/G-963 combination treatment results in
G1 arrest and caspase-dependent cell death
We conducted flow cytometry of single- and double-
agent–treated cells in the presence or absence of the
caspase inhibitor ZVAD-FMK or the necroptosis inhib-
itor necrostatin and analyzed DNA content. Navitoclax
alone has little effect on cells, whereas G-963 causes a G1
arrest after 24 hours and a slight increase in the sub-2N
population by 48 hours while maintaining a strong
G1 arrest (Fig. 2C and D). The combination treatment
increases the sub-2N population to 25%, compared with
navitoclax (3%) or G-963 (5%) alone, by 48 hours. The
increase in the sub-2N population is abrogated in all
drug-treated cell lines with the addition of Z-VAD-
FMK, whereas the increase in the sub-2N population
is still observed in the presence of necrostatin, which
inhibits necroptosis by inhibition of RIP kinase (36).
These data suggest that MEK inhibition leads primarily
to G1 arrest, and addition of navitoclax increases cas-
pase-dependent cell death.
To identify additional biomarkers predictive of
synergy, we measured baseline protein levels of BAD,
pS112-BAD, BIM, MCL1, Bcl-xL, BAX, BAK, puma by
Western blot (24) and pERK and pAKT using MSD
technology (data not shown), and find that the only
significant correlation with Bliss sum is baseline level of
pS112-BAD (Supplementary Fig. S4). We also investi-
gated the correlation of baseline gene expression and
synergy scores. However, we did not identify genes that
exhibited strong correlation between expression and
synergy [defined as false discovery rate (FDR) < 0.1].
For subsequent studies, we chose models harboring
KRAS mutations because of the observed enrichment
for KRAS mutation in high synergy lines.
Navitoclax enhances the efficacy of G-963 in H2122
xenografts
MEK inhibition results in increased BIM across a
panel of KRAS mutant NSCLC cell lines alone and in
combination with navitoclax
We evaluated the effect of the combination using H2122
xenografts in nude mice. Animals bearing H2122 xeno-
grafts were dosed with G-963, navitoclax, or the combi-
nation. Single-agent navitoclax was not efficacious,
whereas treatment with G-963 resulted in tumor growth
delay (74% TGI). The combination of navitoclax and G-963
increased the efficacy and resulted in 99% TGI (Fig. 3A
and B). The enhanced antitumor activity induced by
navitoclax when combined with G-963 was statistically
significant compared with single-agent G-963 treatment
based on 95% confidence intervals and nonoverlapping
individual tumor volumes between the 2 groups (Fig. 3B).
All single-agent and combination treatments were well
tolerated as indicated by minimal changes in animal body
weights (data not shown). Within 4 hours of treatment,
MCL1 protein levels decreased after treatment with G-963
alone or in combination with navitoclax and remained
suppressed for 8 hours. Increases in BIM were observed as
early as 1 hour posttreatment with G-963 and remained
elevated through 8 hours. Navitoclax had no effect on
MCL1or BIMprotein levels nordid it alterthe levels of Bcl-
xL. The combination treatment resulted in increased
PARP cleavage (Fig. 3C).
To explore the mechanism underlying the observed
synergy, we examined the consequences of MEK inhibi-
tion on the intrinsic apoptosis pathway across a panel of
KRAS mutant NSCLC cell lines (Fig. 2A). We observed a
durable suppression of ERK phosphorylation, although
p90RSK phosphorylation was reduced to varying extents.
BIM protein levels increased in response to MEK inhibi-
tion in all cell lines tested, irrespective of synergy scores.
Most cell lines exhibit a reduction in pS112-BAD at early
time points, but the kinetics of this response vary greatly
between cell lines and the effect is not durable. MCL1
levels are modestly reduced in some cell lines, but fre-
quently recover. The collective biochemical findings point
to a consistent stabilization of BIM, although the effects
on BAD and MCL1 are transient and vary between cell
lines.
We next examined cells treated with the combination of
navitoclax and the MEK inhibitor. Navitoclax did not alter
ERK or p90RSK phosphorylation, and the effects of MEK
inhibition on ERK and RSK phosphorylation as well as
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Bcl-xL Inhibition Enhances MEK Inhibitor Efficacy
A
A427
Hop18
H2122
Calu 6
A549
C
2
4
8 24 48 C 2
4
8 24 48
C
2
4
8 24 48
C
2
4
8 24 48 C 2 4 8 24 48
hours
Phospho-ERK
(p44/p42)
ERK
Phospho-RSK
(ser380)
RSK1
Bim EL
Bim L
Bim S
Phospho-Bad
(ser112)
Bad
Mcl-1
Actin
Figure 2. Cellular response to
G-963 and the G-963/navitoclax
combination. A, Western blots of
whole cell lysates from cells treated
with 2 mmol/L G-963. B, Western
blots of lysates from cells treated
with navitoclax (1 mmol/L), G-963
(2 mmol/L), or the combination. C,
DNA content assessed by flow
cytometry for A427 cells treated with
navitoclax (1 mmol/L), G-963
B
A427
24
Hop18
H2122
Calu 6
A549
4 24
4
4
24
4
24
4
24
hours
Navitoclax
G-963
Phospho-ERK
(p44/p42)
Phospho-RSK
(ser380)
RSK1
(2mmol/L), or thecombination plus or
minus Z-VAD-FMK (20 mmol/L) or
necrostatin-1 (10 mmol/L) for 24
hours; D, DNA content assessed by
flow cytometry for A427 cells treated
with navitoclax (1 mmol/L), G-963
(2mmol/L), or thecombination plus or
minus Z-VAD-FMK (20 mmol/L) or
necrostatin-1 (10 mmol/L) for 48
hours.
Bim EL
Bim L
Bim S
Mcl-1
PARP
Clv PARP
Actin
C
D
A427 (24 hours)
A427 (48 hours)
100
100
80
60
40
20
0
80
60
40
20
0
Sub 2N
G2–M
S
G1
Sub 2N
G2–M
S
G1
+
+
+
Z-VAD-FMK Necrostatin-1
(20 µmol/L) (10 µmol/L)
+
Z-VAD-FMK Necrostatin-1
(20 µmol/L) (10 µmol/L)
Addition of a PI3 kinase inhibitor enhances the
activity of the navitoclax/MEK inhibitor combination
Because the combination of navitoclax and a MEK
inhibitor did not cause complete cell death (Fig. 2D), we
evaluated combined inhibition of PI3K, Bcl-2/Bcl-xL, and
MEK. Activation of the PI3K pathway reduces proapop-
totic signaling by promoting an inhibitory phosphoryla-
tion of BAD on Ser136 (5) and repressing FOXO3-driven
transcription of BIM (37). Thus, inhibition of this pathway
is expected to promote proapoptotic signaling by increas-
ing the activity of BAD and the level of BIM. We selected
an effective, clinically relevant dose of the PI3K inhibitor
GDC-0941 (Fig. 1B; refs. 38–40) and added it to all wells of
the navitoclax/G-963 combination matrix on 3 cell lines.
In all lines, the overall growth inhibition is enhanced with
the addition of GDC-0941, but the patterns of response
vary. In the A427 cell line, there is significant enhancement
of activity at the higher doses of G-963. This seems to be
largely driven by the synergy between of G-963 with the
addition of the PI3K, as seen in the G-963/GDC-0941
combination curve on the far right panel of Fig. 4A. In
H2122 cells, there is an increase in activity with the
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Tan et al.
A 2,000
B
2,500
Figure 3. H2122 xenografts exhibit
tumor stasis upon treatment with
the combination of G-963 and
navitoclax. Tumor-bearing mice
were dosed orally and daily (QD) for
28 days with 100 mg/kg of
navitoclax, 25 mg/kg of G-963 or
the combination. A, mean tumor
volume from mice treated with
vehicle (blue), G-963 (red),
1,800
1,600
1,400
1,200
1,000
800
Vehicle
G-963
Vehicle
2,000
1,500
1,000
500
G-963
Navitoclax
Combination
0
2,500
Navitoclax
Combination
2,000
1,500
1,000
500
600
400
navitoclax (yellow), or the
200
combination (green). B, tumor
volumes of individual mice with
mean tumor volume for the vehicle
control group (blue) or each drug-
treated group (solid black); dotted
blue lines represent the mean
tumor volume for the vehicle
control for comparison. C, Western
blots of H2122 tumor xenografts
after a single dose of navitoclax
(navi), G-963, or the combination
(combo) of both drugs at the
indicated times.
0
0
0
5
10 15 20 25 30 0
5 10 15 20 25 30
0
5
10
15 20
Day(s)
Treatment period
25 30
Day(s)
Day(s)
Treatment period Treatment period
C
1 hour
G-963 Combo
4 hours
Navi G-963 Combo
8 hours
Veh
Navi
Veh
Navi G-963 Combo
Mcl-1
Bcl-xL
Bim EL
Clv PARP
Actin
addition of GDC-0941 across the entire dose matrix (Fig.
4B), and the effects of G-963 and GDC-0941 seem additive,
as seen with the shift of the G-963 dose response with the
addition of GDC-941 (Fig. 4B, right). Finally, in the A549
cell line, the enhancement of activity is more pronounced
at the lower doses of G-963 (Fig. 4C). These observations
highlight the importance of measuring effects across a
range of concentrations and ratios to capture the range of
response.
on S380. There is also an increase in BIM gel mobility,
indicative of loss of phosphorylation in all G-963–treated
samples; the loss of BIM phosphorylation was confirmed
with a phospho-S69–specific antibody. Moreover, induc-
tion of p21 (CDKN1A) and p27 (CDKN1B) are observed
after 24hours inallG-963–treatedcells, consistent with the
observed G1 arrest (Fig. 5C and D). The activity of GDC-
0941 on PI3K was showed by a decrease in phospho-AKT1
(S473) in all treated cells. The addition of navitoclax did
not alter the proximal downstream effectors of MEK and
PI3K signaling. We next examined BAD phosphorylation
following treatment. Surprisingly, navitoclax causes an
increase of phosphorylation on BAD at S112 and S136.
This may reflect negative feedback loop exerting inhibi-
tion on BAD that results from the addition of the "BAD
mimetic." However, we do not have a mechanism to
account for this observation. Addition of GDC-0941 to
navitoclax/G-963 combination reduces phosphorylation
of BAD. Consistent with the observations that cells are
under stress and undergoing apoptosis following drug
treatments, we detect increased g-H2AX levels and PARP
cleavage (Fig. 5C and D).
Weevaluated the effect ofthe triple combination on cell-
cycle state and cell death by flow cytometry. There were
no significant changes in cell-cycle state or sub-2N content
at 8 hours. By 24 hours there was an increase in cells with
2N DNA content (G1) in G-963 treated cells alone or in
combinations, and a small increase in cells with sub-2N
content was evident in the navitoclax/G-963 and the triple
combination treated cells. By 48 hours there was an
increase in cells with sub-2N content in the G-963/navi-
toclax and G-963/GDC-0941 combinations, 25% in each
Addition of a PI3 kinase inhibitor to the navitoclax/
MEK inhibitor combination increases apoptosis
To explore the mechanism of navitoclax/G-963/GDC-
0941 triple combination-induced cell death, we measured
intrinsic apoptosis by caspase activation and effects on
signaling pathway components by Western blotting. The
triple combination causes strong activation of caspases 3
and/or 7 as early as 8 hours posttreatment in A427 and
H2122 cells (purple bars in Fig. 5A and B), compared with
the single- or double-agent treatments in which high
levels of caspase activation are not observed until 24 or
48 hours posttreatment. The rapid kinetics of the triple
combination might provide an advantage in vivo if expo-
sure is limited by the pharmacokinetic properties of the
compounds.
Because the differences in response to single-, double-,
and triple-agent combinations were most pronounced at
8 hours, we conducted Western blotting of pathway pro-
teins at 2, 8, and 24 hours posttreatment to the capture the
relevant time points. MEK inhibition with G-963 alone
and in all tested combinations promotes loss of ERK1/2
phosphorylation and decreased p90RSK phosphorylation
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Published OnlineFirst March 8, 2013; DOI: 10.1158/1535-7163.MCT-12-0949
Bcl-xL Inhibition Enhances MEK Inhibitor Efficacy
A
B
C
+ GDC-0941 (1 µmol/L)
A427
100
80
60
40
20
0
100
80
60
40
20
0
3.3
1.1
G-963
GDC-0941 (1 µmol/L)
Combination
0.37
0.12
0.04
0.01
0
−5 −4 −3 −2 −1
G-963 concentration (Log)
0
1
G-963 (µmol/L)
G-963 (µmol/L)
G-963 (µmol/L)
G-963 (µmol/L)
+ GDC-0941 (1 µmol/L)
H2122
100
80
60
40
20
0
100
80
60
40
20
0
3.3
1.1
G-963
GDC-0941 (1 µmol/L)
Combination
0.37
0.12
0.04
0.01
0
−5 −4 −3 −2 −1
G-963 concentration (Log)
0
1
G-963 (µmol/L)
+ GDC-0941 (1 µmol/L)
A549
100
80
60
40
20
0
100
80
60
40
20
0
3.3
1.1
G-963
GDC-0941 (1 µmol/L)
Combination
0.37
0.12
0.04
0.01
0
−5 −4 −3 −2 −1
G-963 concentration (Log)
0
1
G-963 (µmol/L)
inhibitors. Cells were treated in a G-963/navitoclax dose matrix as described and then
Figure 4. Triple combinations of MEK, PI3 kinase, and Bcl-2/Bcl-xL
overlaid with media containing DMSO or 1 mmol/L GDC-0941 as indicated. Growth inhibition across all combinations is represented as a heat map of
percent control. For the triple combination, the data are shown as a percent inhibition compared with the untreated control (bottom left). The graphs on the
right represent the dose–response curve for G-963 alone and with 1 mmol/L GDC-0941. A, A427 cells; B, H2122 cells; C, A549 cells.
double combination, compared with untreated and sin-
gle-agent treatment samples which all had less than 5%
sub-2N. The percentage of cells with sub-2N content
was highest in the triple combination with 41% sub-2N
(Fig. 5E).
cell lines tested, whereas loss of BAD phosphorylation
varied, and thus could represent vulnerability in MEK
inhibitor–dependent induction of apoptosis. BAD can
also be inhibited via activation of the PI3K pathway,
making it susceptible to "rewiring" of upstream signaling.
Navitoclax can circumvent resistance via rephosphoryla-
tion of BAD because it acts a BAD mimetic that is not
subject toinhibitory phosphorylation. Stabilization of BIM
is likely critical to the mechanism of the combination
because it binds to and inhibits the antiapoptotic activity
of MCL1, which navitoclax cannot do. Overall, our data
are consistent with a model in which the stabilization of
BIM observed after MEK inhibition is necessary for inhi-
bition of MCL1, but not sufficient to neutralize all of the
antiapoptotic activity of Bcl-2, Bcl-xL. Navitoclax alone
does not induce apoptosis because it can only neutralize
Discussion
Inhibitors of MEK and PI3K are being tested as single
agents and in combination in the clinic. Because efficacy of
targeted agents is often limited, we wanted to identify
combinations that improve efficacy. MEK inhibitor–medi-
ated cytotoxicity is enhanced by Bcl-2/Bcl-xL inhibition
across a large panel of NSCLC and pancreatic cell lines.
MEK inhibition is reported to increase cell death by
increasing BIM protein and suppressing inhibitory phos-
phorylation of BAD. Increased BIM was observed in all
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Tan et al.
A427
H2122
A
B
40K
35K
30K
25K
20K
15K
10K
5K
80K
70K
60K
50K
40K
30K
20K
10K
0
48
24
0
48
24
8
8
4
4
2
Time (hours)
2
Time (hours)
0
0
Treatments
Treatments
A427
H2122
8
C
D
2
24
2
8
24
Figure 5. Apoptosis and signaling
pathway modulation because of
combinations of MEK, PI3 kinase,
inhibitors. A427
and Bcl-2/Bcl-xL
(A) and H2122 (B) treated with
navitoclax (1 mmol/L), G-963
(2 mmol/L), GDC-0941 (1 mmol/L)
alone, or in double or triple
combinations. Caspase activation
was measured using Caspase-Glo
3/7. Western blots of A427 (C) or
H2122 (D) cells treated with the
indicated agents for 2, 8, or 24
hours. E, DNA content of A427
cells 8, 24, or 48 hours after
treatment.
E
A427
Sub 2N
G2–M
S
G1
8 hours
24 hours
48 hours
Bcl-2 and Bcl-xL, leaving MCL1 to protect the cell. How-
ever, in the combination navitoclax neutralizes Bcl-2 and
Bcl-xL, allowing the excess pools of BIM that result from
MEK inhibition to neutralize MCL1.
Of the proteins investigated for correlative biomarker
studies, only the baseline level of pS112 BAD revealed
significant correlation with synergy scores observed for
the navitoclax/G-963 combination treatment. These find-
ings suggest that cells with higher levels of phospho-BAD
are less likely to benefit from the addition of navitoclax
because there is a sufficiently large pool of phosphory-
lated (inactive) BAD that can be activated upon MEK
inhibition to saturate the binding sites on Bcl-xL and
Bcl-2. Although there is preclinical evidence and mecha-
nistic rationale for larger pools of S112 phospho-BAD
acting as a negative predictor for synergy, the dynamic
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Molecular Cancer Therapeutics
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Published OnlineFirst March 8, 2013; DOI: 10.1158/1535-7163.MCT-12-0949
Bcl-xL Inhibition Enhances MEK Inhibitor Efficacy
range of baseline phospho-BAD is small and quantitative
evaluation of phosphorylated epitope in clinical samples
is likely to be challenging. The best correlation of navito-
clax/MEK inhibitor synergy was single-agent sensitivity
to MEK inhibition. Thus, the same predictive biomarkers
relevant for MEK inhibitors, that is activation of the RAS/
RAF/MEK pathway, could potentially be used to enrich
for response to the combination with navitoclax. An MEK-
dependent transcriptional profile could provide the most
accurate predictor of pathway activation (41). However,
this is challenging to implement as a companion diagnos-
tic. We observed enrichment in activating KRAS muta-
tions the high synergy cell lines and propose that KRAS
mutant cancers represent a molecularly defined subset
of cancers that may be more likely to respond to a MEK
inhibitor navitoclax combination.
The RAS/RAF/MEK/ERK signaling cascade plays an
important role in controlling cell proliferation and regu-
lating the cell cycle. ERK activation throughout G1 and
ERK translocation to the nucleus are required for entry to
S phase (42–44). Navitoclax does not alter MEK inhibitor-
induced G1 arrest observed at 24 hours, but strongly
increases cell death at 48 hours, suggesting that the cells
die after entering G1. The observed cell death is dependent
upon caspase activity, but not the extrinsic apoptosis and
necrosis kinase, RIP1, consistent with our observation of
caspase 3/7 activation and PARP cleavage.
Bcl-xL inhibitors can enhance efficacy of GDC-0941 in
breast cancer models (13, 14), this is the first study to
explore the triple combination of navitoclax with both a
PI3K and an MEK inhibitor. The addition of the PI3K
inhibitor, GDC-0941, to the combination suppresses AKT
activation, resulting in increased cytotoxicity as measured
by decreased ATP caspase3/7 activation, g-H2AX induc-
tion, increased sub-2N DNA content, and PARP cleavage.
In conclusion, we show the potential benefit of codos-
ing with navitoclax to enhance the activity of a MEK
inhibitor, especially in cancers with KRAS mutations.
This benefit could be further improved with the addi-
tion of a PI3K inhibitor. Although this work was under
review, 2 studies independently confirmed the value of
combining a MEK inhibitor with navitoclax (45, 46). In
light of the broad scope of current clinical evaluation
underway with agents targeting the MEK, PI3K and
Bcl-2/Bcl-xL pathways, these findings provide a ratio-
nale for clinical testing of previously unrecognized
therapeutic combinations.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Authors' Contributions
Conception and design: N. Tan, S. Price, D. Sampath, W. J. Fairbrother,
L. D. Belmont
Development of methodology: N. Tan, S. Price
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): N. Tan, M. Wong, M. A. Nannini, R. Hong, L. B.
Lee, K. Williams
Analysis and interpretation of data (e.g., statistical analysis, biostatis-
tics, computational analysis): N. Tan, M. Wong, M. A. Nannini, L. B. Lee,
K. Williams, P. Yue, D. Sampath, L. D. Belmont
Writing, review, and/or revision of the manuscript: N. Tan, S. Price,
D. Sampath, J. Settleman, W. J. Fairbrother, L. D. Belmont
Administrative, technical, or material support (i.e., reporting or orga-
nizing data, constructing databases): N. Tan, P. Pascal Savy
Study supervision: N. Tan, M. A. Nannini, D. Sampath
The navitoclax/MEK inhibitor combination effect
observed in vitro was validated in vivo using a human
NSCLC H2122 xenograft model. In contrast to each single
agent, we observed a combination treatment effect char-
acterized by tumor stasis when navitoclax was coadmi-
nistered with G-963. We propose that the combination of
both agents increased apoptosis in the H2122 NSCLC
xenograft model because of antagonism of Bcl-xL by
navitoclax and G-963–induced stabilization of BIM result-
ing in neutralization of MCL1.
Finally, we show that the triple combination of G-963/
GDC-0941/navitoclax has increased activity compared
with the double combinations. There is tremendous in-
terest in combining MEK inhibitors with PI3K inhibitors
to combat resistance via alternate signaling that sup-
presses apoptosis. However, the activation of apoptosis
can be incomplete even with the combination of these
kinase inhibitors. Although studies suggest that Bcl-2/
Acknowledgments
The authors thank S. Selvaraj, M. Yu, and R. Neve for providing and
authenticating cell lines; M. Belvin and the Abbott Bcl-2 team for helpful
input.
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 September 26, 2012; revised March 4, 2013; accepted March 4,
2013; published OnlineFirst March 8, 2013.
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Molecular Cancer Therapeutics
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Published OnlineFirst March 8, 2013; DOI: 10.1158/1535-7163.MCT-12-0949
Bcl-2/Bcl-xL Inhibition Increases the Efficacy of MEK Inhibition
Alone and in Combination with PI3 Kinase Inhibition in Lung and
Pancreatic Tumor Models
Nguyen Tan, Maureen Wong, Michelle A. Nannini, et al.
Mol Cancer Ther 2013;12:853-864. Published OnlineFirst March 8, 2013.
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