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LYMPHOID NEOPLASIA
Cytokinetically quiescent (G0/G1) human multiple myeloma cells are susceptible
to simultaneous inhibition of Chk1 and MEK1/2
Xin-Yan Pei,1 Yun Dai,1 Leena E. Youssefian,1 Shuang Chen,1 Wesley W. Bodie,1 Yukie Takabatake,1 Jessica Felthousen,1
Jorge A. Almenara,1 Lora B. Kramer,1 Paul Dent,2 and Steven Grant1,2
1Division
of Hematology/Oncology, Department of Medicine, Virginia Commonwealth University and the Massey Cancer Center, Richmond, VA; and
of Biochemistry, Virginia Commonwealth University, the Massey Cancer Center, and the Institute of Molecular Medicine, Richmond, VA
2Department
Effects of Chk1 and MEK1/2 inhibition
were investigated in cytokinetically quiescent multiple myeloma (MM) and primary
CD138ⴙ cells. Coexposure to the Chk1
and MEK1/2 inhibitors AZD7762 and selumetinib (AZD6244) robustly induced apoptosis in various MM cells and CD138ⴙ
primary samples, but spared normal
CD138ⴚ and CD34ⴙ cells. Furthermore,
Chk1/MEK1/2 inhibitor treatment of asynchronized cells induced G0/G1 arrest and
increased apoptosis in all cell-cycle
phases, including G0/G1. To determine
whether this regimen is active against
quiescent G0/G1 MM cells, cells were cultured in low-serum medium to enrich the
G0/G1 population. G0/G1–enriched cells
exhibited diminished sensitivity to conventional agents (eg, Taxol and VP-16)
but significantly increased susceptibility to Chk1 ⴞ MEK1/2 inhibitors or Chk1
shRNA knock-down. These events were
associated with increased ␥H2A.X
expression/foci formation and Bim upregulation, whereas Bim shRNA knockdown markedly attenuated lethality. Immu-
nofluorescent analysis of G0/G1–enriched or
primary MM cells demonstrated colocalization of activated caspase-3 and the quiescent (G0) marker statin, a nuclear envelope
protein. Finally, Chk1/MEK1/2 inhibition increased cell death in the Hoechst-positive
(Hstⴙ), low pyronin Y (PY)–staining (2N Hstⴙ/
PYⴚ) G0 population and in sorted small
side-population (SSP) MM cells. These findings provide evidence that cytokinetically
quiescent MM cells are highly susceptible to
simultaneous Chk1 and MEK1/2 inhibition.
(Blood. 2011;118(19):5189-5200)
Introduction
Multiple myeloma (MM) is an accumulative disorder of mature
plasma cells that is almost universally fatal. MM treatment has been
revolutionized by novel agents such as immunomodulatory drugs
(eg, lenalidomide) and proteasome inhibitors (eg, bortezomib). One
barrier to successful MM treatment is it is a low-growth-fraction disease
before the late phase supervenes and that MM cells can rest in a
quiescent, nonproliferative state with ⬍ 5% of cells actively cycling.1-3
Moreover, low proliferation of tumor cells, including MM cells, may
contribute to resistance to conventional or novel targeted agents.1,4,5
Cellular defenses against DNA damage are mediated by multiple checkpoints that permit cell-cycle arrest, DNA repair, or, if
damage is too extensive, apoptosis.6,7 Checkpoint kinases (Chk1 and
Chk2) play key roles in this DNA-damage response network.8,9 In
contrast to Chk2, which is inactive in the absence of DNAdamaging stimuli, Chk1 is active in unperturbed cells and is further
activated by DNA damage or replicative stress.10 Chk1 activation
occurs even in nonproliferating cells.11 Given its critical role in the
DNA-damage response, Chk1 represents an attractive target for
therapeutic intervention. Previous studies have shown that pharmacologic Chk1 inhibitors abrogate cell-cycle arrest in transformed
cells exposed to DNA-damaging agents, triggering inappropriate
G2/M progression and death through mitotic catastrophe.12
Dysregulation of the Ras/Raf/MEK/ERK cascade in transformed cells, including MM cells,13 has prompted interest in the
development of small-molecule inhibitors. Multiple agents target
the dual specificity kinases MEK1/2, which sequentially phosphorylate ERK1/2, leading to activation.14 The MEK1/2 inhibitor
PD184352 (CI-1040)15 has been supplanted by other MEK1/2
inhibitors with superior PK/PD profiles, such as selumetinib
(AZD6244/ARRY142886).14,16 AZD6244 has shown significant in
vivo activity in a MM xenograft model system,17 and trials of
AZD6244 in MM are under way.
Previously, we reported that interruption of the Ras/MEK1/2
cascade by PD184352 dramatically increased the lethality of the
multikinase and Chk1 inhibitor UCN-01.18-21 It is important to
extend these studies to more specific Chk1 and MEK1/2 inhibitors
currently in clinical trials, such as AZD776222 and AZD6244.
Moreover, the possibility exists that Chk1-inhibitor strategies
abrogating DNA-damage checkpoints might be ineffective in
cytokinetically quiescent MM cells, as is the case for more
conventional therapies.1,5 The results reported herein demonstrate
that regimens using AZD7762 and AZD6244 potently induce
MM-cell apoptosis in all phases of the cell cycle, including G0/G1.
Furthermore, this strategy selectively targets primary MM cells
while sparing their normal counterparts. Our findings indicate that,
in addition to cycling cells, cytokinetically quiescent (G0/G1) MM
cells are highly susceptible to concomitant Chk1/MEK1/2 inhibition.
Submitted February 24, 2011; accepted August 30, 2011. Prepublished online
as Blood First Edition paper, September 12, 2011; DOI 10.1182/blood-2011-02339432.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The online version of this article contains a data supplement.
© 2011 by The American Society of Hematology
BLOOD, 10 NOVEMBER 2011 䡠 VOLUME 118, NUMBER 19
Methods
Cells and reagents
The human MM cell lines NCI-H929 and U266 were purchased from
ATCC. RPMI8226 cells were provided by Dr Alan Lichtenstein (University
of California, Los Angeles). The IL-6–dependent MM cell lines ANBL-6
and KAS-6/1 were provided by Dr Robert Orlowski (The M. D. Anderson
Cancer Center, Houston, TX). BM samples were obtained with informed
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PEI et al
consent according to the Declaration of Helsinki from MM patients
undergoing routine diagnostic aspiration with approval from the Virginia
Commonwealth University institutional review board. CD138⫹ and CD138⫺
cells were isolated as described previously.19 The purity of CD138⫹ cells
was ⬎ 90% and viability ⬎ 95%. Normal BM CD34⫹ cells (M-101B) were
purchased from Lonza. The purity of CD34⫹ cells was ⬎ 95% and
viability ⬎ 80% when thawed. The MEK1/2 inhibitor AZD6244 and the
selective Chk1 inhibitor AZD7762 were provided by AstraZeneca. The
MEK1/2 inhibitor PD184352 and the selective Chk1 inhibitor CEP389123
were obtained from Upstate and Cephalon, respectively. In most cases,
parallel studies using AZD7762 and CEP3891 (and in some cases, the
prototypical Chk1 inhibitor UCN-01) in multiple MM cell lines were
performed to reduce the likelihood that off-target actions of agents or
cell-line–dependent responses might be responsible for the observed
effects. The caspase inhibitor BOC-D-fmk was purchased from Enzyme
System Products. Reagents were dissolved in sterile DMSO (final concentration ⬍ 0.1%) and stored at ⫺80°C.
Enrichment of G0/G1 cells
MM cells enriched in the G0/G1 phase were obtained by incubating H929,
8226, and U266 in low-serum medium (0.1%, 0.2%, and 0.05% FBS,
respectively)11,24 for ⱖ 42 hours before cotreatment with AZD6244/
AZD7762 or PD184352/CEP3891.
BLOOD, 10 NOVEMBER 2011 䡠 VOLUME 118, NUMBER 19
Cell-cycle analysis
Cell-cycle analysis of DNA content after drug treatment by propidium
iodide (PI) staining was performed by flow cytometry (BD Biosciences)
using Modfit Version LT2.0 software as described previously.25
Distribution of apoptotic cells in cell-cycle phases
Caspase-3 activation/DNA content analysis were performed by dualparameter flow cytometry to determine apoptotic cells within specific
cell-cycle phases.26 Cells were stained with a 1:100 dilution of Alexa Fluor
488 (AF 488)–conjugated anti-cleaved (activated) caspase-3 for 1 hour at
4°C. DNA was stained with PI as described in “Cell-cycle analysis.” In
these studies, subdiploid (late apoptotic) cells, which cannot be related to a
specific cell-cycle compartment, were gated out and not included in this
analysis.
Colocalization of statin and activated caspase-3
Dual statin and activated caspase-3 expression was quantified by flow
cytometry to determine apoptotic cells within statin-positive populations
(G0/G1 phase).27 After being fixed in 70% cold ethanol, blocked with 5%
normal goat serum, and permeabilized with 0.5% Tween 20, cells were
incubated with an anti-statin mAb (S-44) or control IgG, followed by
secondary AF 599–conjugated goat anti–mouse IgG (Invitrogen). Cells
were then stained with anti-cleaved (activated) caspase-3 Ab and analyzed
by flow cytometry.
Assessment of apoptosis and cell death
For a discussion of our assessment of apoptosis and cell death, please see
supplemental Methods (available on the Blood Web site; see the Supplemental Materials link at the top of the online article).
Immunofluorescent staining
RNA interference and stable transfection
In situ coexpression of statin and activated caspase-3 or ␥H2A.X foci
formation was performed by immunofluorescent staining.23,27 Cells fixed on
slides were rehydrated, blocked with 5% normal goat serum, and permeabilized with 0.5% Tween 20. Cells were incubated with an anti-statin mAb or
control IgG, followed by secondary AF 599–conjugated goat anti–mouse
IgG. After washing, slides were stained with either AF 488–conjugated
anti-cleaved (activated) caspase-3 Ab or AF 488–conjugated anti-␥H2A.X
Ab (Cell Signaling Technology), respectively. Images were captured with
an Olympus BX40 fluorescence microscope and analyzed with RS Image
Version 1.7.3 software (Roper Scientific Photometrics).
For a discussion of RNA interference and stable transfection, please see
supplemental Methods.
Multiparameter flow cytometric analysis of G0 phase cells
Clonogenic assays
Colony-forming ability was evaluated using a soft agar cloning assay
described previously.19
Western blot analysis
Samples were prepared from whole-cell pellets. Proteins (20 ␮g) were
separated on precast SDS-PAGE gels (Invitrogen) and electrotransferred
onto nitrocellulose membranes. Blots were probed with primary Abs:
anti-Bim (Calbiochem); anti–phospho-p44/42 (Thr202/Tyr204), antiMAPK (ERK1/2) and anti-p44/42 MAPK (Cell Signaling Technology);
anti–poly adenosine diphosphate-ribose polymerase (anti-PARP; Biomol);
anti–caspase-3 (BD Biosciences); anti-cleaved (activated) caspase-3
(Cell Signaling Technology); phospho-histone H2A.X (Ser139; Upstate
Biotechnology); and anti-statin Ab S-44 (provided by Dr E. Wang,
University of Louisville, Louisville, KY). Cytosolic and mitochondrial
fractions were prepared using digitonin lysis buffer as described previously.20 Anti–cytochrome c (BD Pharmingen), anti-AIF (Santa Cruz
Biotechnology), anti–smac/DIABLO (Upstate Biotechnology), and antiBax (Santa Cruz Biotechnology) Abs were used to monitor the release of
mitochondrial pro-apoptotic factors and Bax redistribution. Blots were
reprobed with anti-actin (Sigma-Aldrich) or anti-tubulin Abs (Oncogene) to
ensure equal loading and protein transfer.
Comet assay
Single-cell gel electrophoresis assays were performed to assess single- and
double-stranded DNA breaks using a Comet Assay Kit (Trevigen) as per the
manufacturer’s instructions.21 Images were captured using fluorescence
microscopy at 20⫻/0.50.
Dual-parametric flow cytometry to monitor DNA and RNA content was used to
identify quiescent (G0) cells using the DNA dye Hoechst 33342 (Hst; Molecular
Probes) and the RNA-specific dye pyronin Y (PY; Sigma-Aldrich), respectively.28,29 To exclude G1 phase and G0 to G1 transition cells accumulating
RNA,29 G0 populations displaying Hst⫹ and low PY uptake (Hst⫹/PY⫺) were
gated as R17. To identify cells undergoing apoptosis in the Hst⫹/PY⫺ (G0)
population, 7-amino-actinomycin D (7-AAD) staining (0.5 ␮g/mL at 37°C for
30 minutes) was adapted for triparametric analysis. Hst⫹/PY⫺/7-AAD⫹ cells
were gated as R16. Flow cytometry parameter settings were as reported
previously.29 Controls for Hst/PY or 7-AAD staining alone and negative controls
(without dye) were used as compensation. Fluorescence data were obtained from
30 000 cells per sample. Flow cytometric analysis was performed using Summit
Version 4.0 software (Dako Cytomation).
Analysis of SSP cells
Small side-population (SSP) MM cells were isolated as described previously.30,31 In addition, gates were set for both the side population (SP) and
low FSC (small size) population (designated SSP) after gating out
PI-stained (dead) cells on a flow cytometer (FACSAria II; BD Biosciences),
as described in supplemental Methods. The sorted SSP cells were then
exposed to agents as indicated, after which apoptosis was determined by
monitoring activated caspase-3 by flow cytometry.
Statistical analysis
For flow cytometric analyses, values represent the means ⫾ SD for at least
3 separate experiments performed in triplicate. Significance of differences
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Chk1/MEK INHIBITOR KILLS QUIESCENT MYELOMA CELLS
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Figure 1. The MEK1/2 inhibitor AZD6244 blocks ERK1/2 activation induced by the novel Chk1 inhibitor AZD7762 in human MM cells, leading to synergistic
induction of apoptosis and diminished clonogenicity. (A) H929 cells were pretreated (24 hours) with the indicated concentrations of the MEK1/2 inhibitor AZD6244,
followed by 200nM or 300nM AZD7762 for another 48 hours, after which time cell survival was monitored with the MTT assay. **P ⬍ .01 and ***P ⬍ .002 versus values for cells
treated with AZD7762 alone. (B-C) H929 cells were sequentially treated as described in panel A with AZD6244 in either the presence or absence of the indicated concentrations
of AZD7762 (B), or with the indicated concentrations of AZD7762 in either the presence or absence of 2.5␮M AZD6244 (C). After drug treatment, the percentage of apoptotic
(annexin V⫹) cells were determined by flow cytometry. *P ⬍ .05, **P ⬍ .01, and ***P ⬍ .001 versus values for cells treated with AZD6244 or AZD7762 alone. (D) H929 cells
were incubated for the indicated intervals with 300nM AZD7762 after preadministration (24 hours) of 2.5␮M AZD6244. Cell death was then monitored by 7-AAD staining and
flow cytometry. *P ⬍ .01 and **P ⬍ .001 versus values for cells treated with AZD7762 alone. (E) H929 cells were treated with a range of AZD6244 concentrations for 24 hours
and a range of AZD7762 concentrations for another 48 hours, alone or in combination at a fixed ratio (50:1). At the end of this period, 7-AAD⫹ cells were determined by flow
cytometry. Median-dose effect analysis was used to characterize the nature of the interaction. Two additional studies yielded equivalent results. (F) H929 cells were treated with
2.5␮M AZD6244 for 24 hours ⫾ 300nM AZD7762 for another 48 hours, and then stained on Cytospin slides by TUNEL. (G) Alternatively, after being treated with the indicated
concentrations of AZD6244 for 24 hours and AZD7762 for another 48 hours, cells were washed free of drug and plated in soft agar. After incubation for 14 days, colonies,
consisting of groups of ⬎ 50 cells, were scored, and colony formation for each condition was expressed relative to untreated controls. Results represent the means ⫾ SD for
3 separate experiments performed in triplicate. **P ⬍ .005 versus single-drug treatment. (H) In parallel, H929 cells were pretreated for 24 hours with the indicated
concentrations of AZD6244 and with 300nM AZD7762 for another 48 hours, after which time Western blot analysis was performed to evaluate ERK1/2 phosphorylation and
caspase-3 cleavage. Each lane was loaded with 20 ␮g of protein; blots were stripped and reprobed with anti-tubulin Ab to ensure equal loading and transfer. Results are
representative of 3 separate experiments.
between experimental variables was determined using the Student t test.
Median Dose Effect analysis was performed as described previously.21
Results
Novel Chk1 and MEK1/2 inhibitors synergistically promote
apoptosis in IL-6–dependent and –independent MM cells
To determine whether the specific, clinically relevant Chk1 inhibitor
AZD7762 and the MEK1/2 inhibitor AZD6244 interacted in MM cells,
IL-6–independent H929 cells were pretreated with various AZD6244
concentrations for 24 hours, followed by exposure to AZD7762 (200 or
300nM) for another 48 hours, after which time survival was monitored
using the MTT assay. Significant reductions in survival occurred with
coadministration of 500nM AZD6244 and became more pronounced at
higher concentrations (Figure 1A). Parallel results were obtained with
annexin V staining (Figure 1B). Pretreatment with minimally toxic
concentrations of AZD6244 for 24 hours significantly increased annexin
V positivity induced by 48 hours of 100-600nM AZD7762 treatment
(Figure 1C). Combined treatment markedly increased cell death, which
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PEI et al
BLOOD, 10 NOVEMBER 2011 䡠 VOLUME 118, NUMBER 19
Figure 2. The AZD7762/AZD6244 regimen induces
apoptosis in IL-6–dependent MM cells and selectively targets primary CD138ⴙ MM cells while sparing their normal hematopoietic counterparts.
(A) IL-6–dependent ANBL-6 cells were exposed to 2.5␮M
AZD6244 or 2.5␮M PD184352 for 24 hours, followed by
50nM AZD7762 for another 48 hours, after which time
cells were immunofluorescently stained with AF 488–
conjugated Ab directed against cleaved (activated)
caspase-3 to monitor caspase-3 activation. Images were
captured microscopically at 40⫻/0.65. (B) Normal cord
blood CD34⫹ cells were exposed to 5␮M
AZD6244 ⫾ 300nM AZD7762 or 4␮M VP-16 for
24 hours, after which time the percentage of apoptotic
(annexin V⫹) cells was determined by flow cytometry.
(C-D) Primary CD138⫹ MM cells (C) and their CD138⫺
counterparts (D) were isolated from the BM samples of
9 patients with MM. Cell death responses of cells exposed for 24 hours to 250nM AZD7762 ⫾ 5␮M AZD6244
were examined by trypan blue exclusion. (E) Once the
number of CD138⫹ cells was suitable, followed by drug
treatment as described in panels C and D, primary cells
were immunofluorescently stained with AF 488–
conjugated cleaved (activated) caspase-3 Ab to confirm
apoptosis. The representative images shown were captured microscopically at 40⫻/0.65. (F) Primary CD138⫹
MM cells from a MM patient were treated as described in
panel C, and then subjected to Western blot analysis to
monitor ERK1/2 phosphorylation and Bim expression.
Lanes were loaded with 10 ␮g of protein; blots were
reprobed with Abs to ␤-actin to ensure equivalent loading
and transfer.
was first discernible at 24 hours and the most pronounced 36-48 hours
after AZD7762 addition (Figure 1D). Median dose effect analysis
yielded combination index values ⬍ 1.0, indicating synergistic interactions (Figure 1E). TUNEL analysis confirmed the striking increase in
apoptosis after combined treatment (Figure 1F). Moreover, coadministration of minimally toxic AZD7762 and AZD6244 concentrations
sharply reduced clonogenicity (Figure 1G). Finally, AZD6244 pretreatment effectively blocked AZD7762-induced ERK1/2 activation and
increased caspase-3 cleavage (Figure 1H).
To determine whether these effects could be generalized, parallel
studies were performed in multiple MM cell lines. As shown in Figure
2A, pretreatment for 24 hours with AZD6244 or another MEK1/2
inhibitor, PD184352, markedly increased caspase-3 activation induced
by 48 hours of 50nM AZD7762 treatment in IL-6–dependent ANBL-6
cells. Western blot analysis revealed that MEK1/2 inhibitors blocked
AZD7762-induced ERK1/2 activation, while increasing PARP and
caspase-3 cleavage (supplemental Figure 1A). Similar interactions
occurred in multiple other MM cell lines, including IL-6–dependent
KAS-6/1 cells (supplemental Figure 1B) and IL-6–independent
RPMI8226 and U266 cells (supplemental Figure 1C-D).
AZD6244 selectively increases AZD7762 lethality in primary
CD138ⴙ MM cells
Selectivity of the AZD7762/AZD6244 regimen was examined.
AZD7762 ⫾ AZD6244 exerted minimal toxicity toward normal
CD34⫹ cells (Figure 2B). Primary CD138⫹ MM cells isolated from
BM samples of 9 unselected MM patients were exposed for
24 hours to AZD7762 ⫾ AZD6244, after which time cell death
was monitored. For each sample, individual drug treatment induced
minimal to modest lethality, whereas combined treatment substantially increased cell death (Figure 2C). Combined treatment
induced minimal lethality in CD138⫺ BM cells (Figure 2D). A
representative sample stained for fluorescently labeled activated
caspase-3 highlights the pronounced killing of primary CD138⫹
MM cells while sparing their CD138⫺ counterparts (Figure 2E).
Western blot analysis revealed that AZD7762 induced ERK1/2
activation in primary CD138⫹ MM cells, an effect abrogated by
AZD6244 and accompanied by BimEL up-regulation (Figure 2F),
as observed in cultured MM cell lines.20 Similar phenomena were
also observed with another specific Chk1 inhibitor, CEP389123
(data not shown).
MEK1/2 inhibitors promote DNA damage induced by Chk1
inhibitors in both MM cell lines and primary CD138ⴙ MM cells
Whereas exposure of MM cell lines (eg, H929 or RPMI8226) to
AZD7762 or AZD6244 individually had minimal effects, combined treatment robustly increased the expression of ␥H2A.X
(Figure 3A), a double-strand DNA damage marker.23 Coexposure
of primary CD138⫹ MM cells to AZD7762/AZD6244 clearly
increased DNA damage, manifested by increased ␥H2A.X nuclear
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Figure 3. MEK1/2 inhibitors enhance DNA damage induced by
AZD7762 in MM cell lines and primary CD138ⴙ MM cells. (A) H929 and
RPMI8226 cells were exposed to either AZD6244 (H929, 2.5␮M; 8226,
5␮M) or PD184352 (5␮M for both lines) for 24 hours followed by the
indicated concentrations of AZD7762 (H929, 24 hours; 8226, 32 hours).
Cells were then lysed and subjected to Western blot analysis to assess
␥H2A.X (phosphorylated H2A.X at Ser139) expression. (B) Primary
CD138⫹ MM cells isolated from the BM sample of a patient with MM were
incubated with 250nM AZD7762 in the presence or absence of 5␮M
AZD6244 for 24 hours, and then immunofluorescently stained with AF
488–conjugated phospho-H2A.X (Ser139) Ab. Images were captured
microscopically at 60⫻/1.40 under oil (top panels). In parallel, a comet
assay was performed to assess DNA breaks (bottom panels), as described in “Methods.” (C) U266 and H929 cells were sequentially treated
with 2.5␮M AZD6244 for 24 hours, followed by AZD7762 (U266, 100nM;
H929, 300nM) for 28 hours in the presence or absence of 20␮M
BOC-D-fmk. After treatment, the percentage of apoptotic (annexin V⫹)
cells was determined by flow cytometry and was significantly lower
(*P ⬍ .05 and **P ⬍ .02) than the values for cells treated with AZD6244/
AZD7762 in the absence of BOC-D-fmk. (D) Alternatively, cells treated as
described in panel C were lysed and subjected to Western blot analysis
using the indicated primary Abs. For panels A and D, each lane was
loaded with 20 ␮g of protein; blots were stripped and reprobed with
anti-tubulin or anti-actin Ab to ensure equal loading and transfer. Two
additional studies yielded equivalent results.
foci (Figure 3B top panels) and a comet assay (Figure 3B bottom
panels). Similar results were obtained in primary CD138⫹ cells
exposed to CEP3891 and PD184352 (data not shown). The
pan-caspase inhibitor BOC-fmk attenuated MM cell apoptosis but
not ␥H2A.X expression (Figure 3C-D), arguing against the possibility that DNA damage resulted from apoptosis.
Cotargeting Chk1 and MEK1/2 results in G0/G1 arrest of MM
cells and increased apoptosis in all phases of the cell cycle,
including G0/G1
To elucidate effects of this regimen on the cell cycle, flow cytometry was
performed in H929 cells exposed to AZD6244 for 24 hours, followed by
AZD7762 for an additional 24 hours, before the induction of extensive
apoptosis (Figure 1D). AZD6244 or AZD7762 alone modestly increased the G0/G1 population (Table 1). However, combined treatment
significantly increased the G0/G1 population by ⬎ 80% (P ⬍ .01).
Similar results were obtained in other MM cell lines such as 8226
(Table 1) and U266 (data not shown), indicating that Chk1/MEK1/2
inhibition arrests MM cells in G0/G1.
To examine effects of cell-cycle distribution on responses to
Chk1/MEK1 inhibition, dual-parameter flow cytometric analysis
of cleaved (activated) caspase-3 and cell cycle (PI staining for
DNA content) in 8226 cells was performed to monitor caspase-3
activation specifically in each cell-cycle phase after gating out
Table 1. Cell-cycle distribution of MM cells after being exposed to
AZD7762 ⴞ AZD6244 for 24 hours
G0G1
S
G2M
H929
Ctrl
56.6 ⫾ 2.6
18.6 ⫾ 1.4
21.2 ⫾ 1.9
AZD7762 (200nM)
65.0 ⫾ 2.0
13.3 ⫾ 0.6*
18.0 ⫾ 1.4
AZD7762 (300nM)
66.8 ⫾ 1.7
13.0 ⫾ 0.4*
21.2 ⫾ 1.7
AZD6244 (1.5␮M)
63.4 ⫾ 1.5
19.3 ⫾ 0.7
19.0 ⫾ 0.8
6244 ⫹ 7762 (200nM)
81.3 ⫾ 1.3†
9.1 ⫾ 0.5†
10.1 ⫾ 1.0†
6244 ⫹ 7762 (300nM)
80.5 ⫾ 1.3†
9.5 ⫾ 0.5†
10.9 ⫾ 1.2†
8226
Ctrl
58.5 ⫾ 2.0
29.1 ⫾ 2.2
12.6 ⫾ 1.0
AZD6244 (3␮M)
71.0 ⫾ 2.7*
17.5 ⫾ 2.0
10.5 ⫾ 1.7
AZD7762 (300nM)
61.4 ⫾ 0.7
26.7 ⫾ 1.2
12.0 ⫾ 0.5
6244 ⫹ 7762
79.6 ⫾ 1.2†
11.7 ⫾ 1.02*
7.7 ⫾ 0.8
Values represent the means ⫾ SD of triplicate determinations.
*P ⬍ .05 and †P ⬍ .01 ⫽ significantly greater or less than values for untreated
cells.
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BLOOD, 10 NOVEMBER 2011 䡠 VOLUME 118, NUMBER 19
Figure 4. The Chk1/MEK1/2 inhibitor strategy kills MM cells in G0/G1 phase. (A) RPMI8226 cells cultured in 10% FBS were sequentially exposed to 1.5␮M AZD6244 for
24 hours, followed by 400nM AD7762 for another 24 hours or 5nM Taxol as a control. The distribution of apoptotic cells in various cell-cycle phases was then determined by flow
cytometry combining staining for cleaved (activated) caspase-3 (y-axis) and DNA content (PI; x-axis). The representative results are shown to indicate caspase-3 activation in
specific populations of cell-cycle phases, including G0/G1, S, and G2M. Values indicate the -fold increases (treated vs untreated control) of cells displaying caspase-3 activation
within each phase of the cell cycle after gating out the subdiploid population. Inset shows the corresponding results of cell-cycle analysis. Taxol-treated cells were incubated
with IgG instead of anti-cleaved caspase-3 Ab to demonstrate the specificity of the immunostaining. (B) H929 cells were cultured in low- or high-serum–containing medium
(0.1% vs 10% FBS) for 42 hours, and cell-cycle profiles were analyzed by flow cytometry. (C) After being cultured in 0.1% or 10% FBS medium for 42 hours, H929 cells were
exposed to various agents, including the Chk1 inhibitors AZD7762 (300nM) and UCN-01 (150nM), the microtubulin-stabilizing agent Taxol (5nM), and the topoisomerase
inhibitor VP-16 (4␮M) for 24 hours. Cell death was then assessed by 7-AAD staining and flow cytometry and was significantly greater than (**P ⬍ .01) or less than (#P ⬍ .02)
values for cells cultured in 10% FBS. (D) U226 cells were stably transfected with constructs encoding shRNA against human Chk1 (shChk1) or scramble control shRNA (shCtrl)
and clones selected with G418. Western blot analysis demonstrates down-regulation of Chk1 expression in 2 shChk1 clones (C4 and E7) compared with shCtrl cells (inset).
Cells were then cultured in medium containing 0.05% or 10% FBS for 48 hours, after which time the percentage of apoptotic (annexin V⫹) cells was determined by flow
cytometry and was significantly greater (**P ⬍ .02 and ***P ⬍ .01) than values for cells cultured in 10% FBS. (E) H929 cells were cultured in 0.1% FBS for 26 hours, and then
treated with 1.5␮M AZD6244 for 18 hours, followed by the indicated concentrations of AZD7762 for another 24 hours. The extent of apoptosis was analyzed by annexin V
staining and flow cytometry and was significantly greater (**P ⬍ .005 and ***P ⬍ .001) than values for cells cultured in 10% FBS. (F) Alternatively, cells were subjected to
Western blot analysis using the indicated primary Abs.
subdiploid cells.26 As shown in Figure 4A, treatment with the
microtubule-stabilizing agent Taxol resulted in a 4.7-fold increase over values for untreated cells in caspase 3 activation in
G2/M cells, clearly greater than that observed in G0/G1-phase
(2.3-fold) and S-phase (2.0-fold) cells (Figure 4A), documenting the reliability of this approach. AZD7762/AZD6244 increased caspase-3 activation within all phases of the cell cycle
(eg, 4.0-, 3.3-, and 2.6-fold increases in G0/G1, S, and G2M
phase, respectively; Figure 4A). These findings argue that the
Chk1/MEK1/2 inhibitor regimen targets MM cells in each phase
of the cell cycle, including G0/G1.
G0/G1–enriched MM cells display increased susceptibility to the
AZD7762/AZD6244 regimen
The preceding findings indicated that the Chk1/MEK1/2 inhibitor
regimen arrested cultured MM cells in G0/G1, induced apoptosis in
the G0/G1 population, and was active toward primary CD138⫹ MM
cells. To validate whether, in addition to cycling MM cells, this
regimen might also be active against their cytokinetically quiescent
counterparts, H929 cells were cultured in the presence of 10%
versus 0.1% FBS, the latter to halt cell proliferation and to enrich
the G0/G1 population.11,24,32 Cells maintained in 0.1% FBS medium
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BLOOD, 10 NOVEMBER 2011 䡠 VOLUME 118, NUMBER 19
Chk1/MEK INHIBITOR KILLS QUIESCENT MYELOMA CELLS
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Figure 5. Bim plays a functional role in the lethality of
Chk1/MEK1/2 inhibition toward G0/G1–enriched MM
cells. (A) H929 cells were exposed to the indicated
concentrations of AZD6244 for 24 hours, followed by
300nM AZD7762 for an additional 24 hours, after which
time Western blot analysis was performed to monitor the
expression of Bim, including phosphorylated (slow migrating) and unphosphorylated (fast migrating) BimEL, as well
as BimL and BimS isoforms. (B) Alternatively, cytosolic
and mitochondria-enriched (pellet) extracts were subjected to Western blot analysis to monitor the release of
mitochondrial proapoptotic proteins (ie, cytochrome c,
AIF, and Smac) and translocation of Bax. Parallel blots
probed for Cox-2 (cytochrome oxidase subunit 2, a
protein of mitochondrial inner membrane) and ␤-actin are
shown to ensure equal loading and transfer for mitochondrial and cytosolic fractions, respectively. (C) Bim shRNA
(shBim, clone W12) and scramble control shRNA (shCtrl)
U266 cells were cultured in medium containing 0.05% or
10% FBS for 48 hours, followed by coadministration of
1.5␮M AZD6244 for 24 hours ⫾ 300nM AZD7762 for an
additional 48 hours. Western blot analysis was performed
to monitor BimEL expression and PARP degradation. CF
indicates the cleavage fragment. (D) shBim (clones W12
and W4) and shCtrl U226 cells were treated as described
in panel C, after which time the percentage of dead
(7-AAD⫹) cells was determined by flow cytometry and
was significantly less (**P ⬍ .01 and ***P ⬍ .002) than
values for shCtrl cells treated identically in 0.05% FBS
medium. Inset shows Western blots demonstrating knockdown of Bim in shBim cell, compared with shCtrl cells. For
panels A through D (inset), each lane was loaded with
20 ␮g of protein; blots were stripped and reprobed with
anti-actin Ab to ensure equal loading and transfer. Two
additional studies yielded equivalent results.
exhibited essentially no growth compared with those cultured in
10% FBS medium (supplemental Figure 2A), with ⬎ 80% of cells
accumulating in G0/G1 (Figure 4B), but there were modest
differences in cell viability (Figure 4C; P ⬎ .05). Interestingly,
G0/G1–enriched cells were significantly more sensitive to Chk1
inhibitors (eg, AZD7762 or UCN-01) compared with controls
cultured in 10% FBS medium (Figure 4C; P ⬍ .01). However,
these cells were relatively more resistant to Taxol or VP-16 (Figure
4C; P ⬍ .02 in each case), arguing against the possibility that
serum-deprived cells were generically more sensitive to cytotoxic
agents. Effects on caspase-3 and PARP cleavage were concordant
(supplemental Figure 2B). Flow cytometric analysis also demonstrated a clear increase in the subdiploid (sub-G1) fraction in
G0/G1–enriched cells after treatment with the Chk1 inhibitors
compared with 10% FBS controls (supplemental Figure 2C).
Moreover, G0/G1 enrichment also significantly sensitized cells to
Chk1 shRNA knock-down (Figure 4D and supplemental Figure
2D), which was accompanied by increased ␥H2A.X expression
(supplemental Figure 2E), arguing for a specific role for Chk1
inhibition in Chk1 inhibitor lethality in quiescent MM cells.
G0/G1–enriched cells displayed greater susceptibility to AZD6244/
AZD7762 even when these agents were administered for shorter
intervals (ie, 24 hours after AZD7762 addition) than those cultured
under full-serum conditions (Figure 4E; P ⬍ .005 vs 10% FBS
controls). AZD6244 exposure resulted in BimEL up-regulation
under both conditions, which was associated with inhibition of
basal and AZD7762-stimulated ERK1/2 phosphorylation. However, G0/G1–enriched cells exposed to AZD7762/AZD6244 displayed more pronounced ␥H2A.X expression and caspase-3 cleavage compared with 10% FBS controls (Figure 4F). Similar results
were obtained in other MM cell lines (eg, U266 cells) and in cells
exposed to an alternative Chk1/MEK inhibitor regimen
(eg, CEP3891/PD184352; data not shown).
Bim plays a functional role in the pronounced susceptibility of
G0/G1–enriched MM cells to Chk1/MEK1/2 inhibition
As shown in Figure 5, the MEK1/2 inhibitor AZD6244 prevented
Bim phosphorylation (particularly the BimEL isoform) in H929
cells exposed to AZD7762, leading to Bim accumulation
(Figure 5A), Bax translocation, and the release of mitochondrial
apoptotic proteins (ie, cytochrome c, smac/DIABLO, and AIF;
Figure 5B). Similar results were observed in other MM cell lines
(eg, RPMI8226 and U266; data not shown). To determine whether
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PEI et al
Bim up-regulation also contributed to the susceptibility of cytokinetically quiescent MM cells to the Chk1/MEK1/2 inhibitor
regimen, U266 cells stably transfected with Bim shRNA were used.
As shown in Figure 5C, coadministration of AZD6244 with
AZD7762 markedly increased BimEL expression in G0/G1–
enriched U266 cells transfected with negative control shRNA
(shCtrl), whereas this event was clearly attenuated in G0/G1–
enriched Bim shRNA cells. shRNA knock-down of Bim (2 clones,
W12 and W4) significantly diminished the ability of AZD6244 to
potentiate AZD7762 lethality compared with shCtrl cells (P ⬍ .002
in each case; Figure 5C-D). Similar results were obtained in other
MM cell lines (eg, RPMI8226) transfected with Bim shRNA (data
not shown). These results indicate that Bim up-regulation plays an
important functional role in the susceptibility of G0/G1–enriched
MM cells to the Chk1/MEK1/2 inhibitor regimen.
Chk1/MEK1/2 inhibition induces apoptosis in MM cell lines and
primary CD138ⴙ cells expressing the G0 marker statin
Statin is a 57-kDa nuclear envelope protein primarily expressed in
cytokinetically quiescent G0 cells and rapidly down-regulated
during progression to the G1 phase.27,33 Western blot analysis
revealed that G0/G1–enriched H929 cells exhibited a marked
increase in statin expression (supplemental Figure 3A), and
immunofluorescent staining demonstrated a marked increase in
statin-positive cells after G0/G1 enrichment by culturing in 0.1%
FBS (supplemental Figure 3B). Primary CD138⫹ MM cells clearly
expressed statin (supplemental Figure 3C), which is consistent with
their quiescent status.1-3
Untreated G0/G1–enriched H929 cells displayed robust red fluorescence (statin) but little green fluorescence (activated caspase-3; Figure
6A). AZD7762/AZD6244-treated cells displayed clear colocalization of
statin and activated caspase-3. Parallel studies in primary CD138⫹ MM
cells revealed that AZD7762/AZD6244 exposure resulted in pronounced caspase-3 activation in statin-positive cells compared with
untreated cells (Figure 6B). These findings were supported by parallel
studies involving alternative Chk1/MEK1/2 inhibitors (eg, CEP3891
and PD184352; supplemental Figure 3D-E). Colocalization of statin/
activated caspase-3 in G0/G1–enriched H929 cells was further confirmed by dual-parameter flow cytometric analysis, which indicated
increased caspase-3 activation in statin-positive cells after brief exposure (12 hours after addition of Chk1 inhibitors) to AZD6244/
AZAD7762 and to CEP3891/PD184352 (Figure 6C). Finally, dualimmunofluorescent staining revealed that ␥H2A.X expression (green
fluorescence) colocalized with statin expression (red fluorescence) in
AZD6244/AZD7762–treated G0/G1–enriched H929 cells at an early
interval (6 hours after AZD7762) (Figure 6D). Parallel results were also
obtained with CEP3891/PD184352 (supplemental Figure 4). These
findings support the notion that cytokinetically quiescent MM cells are
sensitive to DNA damage and apoptosis induced by this regimen.
Hst ⴙ/PYⴚ G0 MM cells are susceptible to the Chk1/MEK1/2
inhibitor regimen
To confirm that Chk1/MEK1/2 inhibition kills cytokinetically
quiescent (G0) MM cells, a multiparameter flow cytometric analysis was used. This method is specifically designed to identify G0
cells displaying Hoechst positivity (Hst⫹, corresponding to 2N
diploid cells) but low pyronin Y uptake (PY⫺, because of lack of
RNA synthesis) from other populations, particularly G1 (Hst⫹/
PY⫹).28,29 As shown in Figure 7A panels i and ii, RPMI8226 cells
cultured in 0.2% FBS medium displayed an enriched Hst⫹/PY⫺
(G0) population (gate R17), compared with 10% FBS controls
BLOOD, 10 NOVEMBER 2011 䡠 VOLUME 118, NUMBER 19
(19.14% vs 8.18% of the bulk population). 7-AAD staining was
then incorporated to monitor cell death (R16) in the Hst⫹/PY⫺
(R17 gated-in) population. After 12 hours of treatment with
AZD7762/AZD6244, the Hst⫹/PY⫺ population exhibited a clear
increase in 7-AAD uptake over untreated controls (Figure 7A
panels iii and iv; R16, 40.2% vs 26.5%; P ⬍ .01). Significant
increases in cell death of Hst⫹/PY⫺ cells (51.3%) also occurred
after CEP3891/PD184352 coexposure (Figure 7A panel v; P ⬍ .005
vs. untreated controls), providing further evidence that quiescent
(G0), noncycling MM cells are susceptible to Chk1/MEK1/2
inhibitor–mediated lethality.
Quiescent SSP MM cells are sensitive to Chk1/MEK1/2 inhibitor
lethality
Finally, the effects of Chk1/MEK1/2 inhibition were investigated
prospectively in SSP MM cells, representing quiescent cells in
which the presumed stem cell compartment resides.30,31 SP cells
were sorted from H929 cells cultured in 10% serum (Figure 7B), as
described previously.30 SP cells represented a small fraction of cells
(0.5% of PI⫺ living cells) excluding Hst (Figure 7B panels i and ii),
which was further sorted for SSP cells (Figure 7B panel iii), which
have been shown to represent a quiescent population.34 As a
control, coincubation of verapamil substantially reduced the SP
population (Figure 7B panel iv).31 Approximately 90% of sorted
SSP cells resided in G0/G1 (Figure 7B panel v). Fourteen hours of
treatment with AZD7762/AZD6244 resulted in a marked increase
in caspase-3 activation in the G0/G1 population of the sorted SSP
cells compared with untreated controls (Figure 7C). Equivalent
results were obtained with the sorted SSP population of RPMI8226
cells (data not shown). These findings provide further evidence that
Chk1/MEK1/2 inhibition is active against quiescent MM cells.
Discussion
The preceding findings demonstrate that cytokinetically quiescent
(G0/G1) MM cell lines, as well as primary CD138⫹ myeloma cells
expressing quiescent (G0/G1) markers, are fully susceptible to
strategies simultaneously targeting Chk1 and MEK1/2. It should be
emphasized that G0/G1 MM cells are not selectively killed by
Chk1/MEK1/2 inhibition, because cells in other phases of the cell
cycle are also vulnerable. However, cytokinetically quiescent cells
are generally resistant to standard cytotoxic agents, particularly
those targeting specific phases (eg, S and M phases) of the cell
cycle.1,4,5 The finding that Chk1/MEK1/2–inhibitory regimens
(eg, AZD7762/AZD6244 and CEP3891/PD184352) are active
against quiescent cells may have particular significance for MM,
generally a low-proliferative neoplasm.
Normally, cells respond to genotoxic insults through the coordinated actions of various cell-cycle checkpoints and DNA-repair
pathways. The former include biochemical signaling pathways that
monitor DNA damage and trigger cell-cycle arrest and DNA repair
if the damage is repairable, or induce apoptosis or senescence if it is
not.6,7 Transformed cells in general are susceptible to DNA damage
and oncogene-mediated DNA-replication stress.35,36 Because checkpoints are characteristically defective in neoplastic cells,12,22,36
checkpoint abrogators have become the focus of intense interest.
Chk1 is a distal signal transducer that is activated by ATM/ATR
after DNA damage8 and that has been implicated in the G2/M,
intra-S, and mitotic spindle checkpoints.37 More recently, Chk1 has
been shown to play a more direct role in cell survival- and DNA
repair–related processes.10,38,39 In this context, it has been reported
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BLOOD, 10 NOVEMBER 2011 䡠 VOLUME 118, NUMBER 19
Chk1/MEK INHIBITOR KILLS QUIESCENT MYELOMA CELLS
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Figure 6. Chk1/MEK1/2 inhibition induces caspase-3
activation and ␥H2A.X expression in statin-positive
(G0/G1) MM cells. (A) H929 cells were incubated in 0.1%
FBS medium for 64 hours, and then exposed to 1.5␮M
AZD6244 for 24 hours, followed by 300nM AZD7762 for
an additional 18 hours. (B) Primary CD138⫹ MM cells
isolated from the BM sample of a patient with MM were
exposed to 5␮M AZD6244 and 250nM AZD7762 for
24 hours. For panels A and B, cells were immunofluorescently stained with Abs against statin (red fluorescence)
and cleaved (activated) caspase-3 (green fluorescence)
and counterstained with DAPI (blue) after treatment.
Images were captured microscopically at 60⫻/1.40 under oil and then merged as indicated. Arrows indicate
cells exhibiting colocalization of statin and activated
caspase-3 expression. Results are representative of
3 separate experiments. (C) H929 cells enriched for
G0/G1 as described in panel A were exposed to MEK1/2
inhibitors (1.5␮M AZD6244 or 1.5␮M PD184352) for
24 hours, followed by Chk1 inhibitors (300nM AZD7762
or 300nM CEP3891 for an additional 12 hours. Flow
cytometric analysis was used to quantify the percentage
of cells coexpressing statin and activated caspase-3
(upper right quadrant). In parallel, untreated cells were
incubated with IgG instead of statin or cleaved caspase-3
Abs as a negative control to demonstrate the specificity
of the immunostaining. Numbers indicate the percentage
of activated caspase-3–negative (blue) or –positive (red)
in the statin⫹ population. Two additional studies yielded
equivalent results. (D) Alternatively, G0/G1–enriched H929
cells were treated 1.5␮M AZD6244 for 24 hours, followed by 300nM AZD7762 for an additional 6 hours,
immunofluorescently stained with Abs against statin
(red fluorescence) and ␥H2A.X (green fluorescence),
and then counterstained with DAPI (blue). Images were
captured microscopically at 60⫻/1.40 under oil and then
merged as indicated. Arrows indicate cells coexhibiting
statin and ␥H2A.X expression/foci formation.
that exposure to Chk1 inhibitors (eg, UCN-01 and CEP3891) or
Chk1 shRNA knock-down is sufficient to induce DNA damage,
even in the absence of exogenous genotoxic insults.23 The initial
development of Chk1 inhibitors focused on the multikinase inhibitor UCN-01, but because of its unfavorable pharmacokinetic and
toxicity profiles,40 interest has shifted to more specific secondgeneration Chk1 inhibitors such as AZD7762, CEP3891, XL-844,
and PF-477736, among many others.37 Chk1 inhibitors dramatically enhance genotoxic agent lethality by disrupting the G2/M
checkpoint (in the case of DNA-damaging agents such as camptothecin and SN-38) or the intra-S phase checkpoints (in the case of
DNA synthesis inhibitors such as gemcitabine).12,39,41 In these
settings, cells with DNA damage progress inappropriately through
the G2/M or S phase, triggering cell death. Implicit in these
mechanistic models is the presumption that cell-cycle progression
must occur for such disruptions to be lethal. Consequently, few
attempts have been made to apply this approach to disorders
involving low-proliferative malignancies, such as MM, or to
cytokinetically quiescent (G0/G1) tumor cells, a characteristic of
cancer stem (initiating) cells. Studies by our group have shown that
interruption of the MEK1/2/ERK1/2 pathway strikingly increases
UCN-01 lethality in MM cells.19-21 However, the cell-cycle relatedness of this interaction, in contrast to strategies combining Chk1
inhibitors with DNA-damaging agents, has not yet been defined nor
have these findings been extended to include newer-generation,
more specific Chk1 and MEK1/2 inhibitors currently under clinical
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BLOOD, 10 NOVEMBER 2011 䡠 VOLUME 118, NUMBER 19
Figure 7. Chk1/MEK1/2 inhibitor regimens induce cell death in G0 MM cells characterized by the Hstⴙ/PYⴚ phenotype. (A) RPMI8226 cells were incubated in 0.2% or
10% FBS medium for 72 hours, after which time flow cytometry was performed to assess G0 populations by double staining DNA with Hst and RNA with PY. The G0 population
(2N DNA/low levels of RNA, Hst⫹/PY⫺) was discriminated from the G1 population (2N DNA/high levels of RNA, Hst⫹/PY⫹), whereas the S and G2/M populations
displayed ⬎ 2N DNA. G0 cells (R17) were gated-in for further analysis. Compared with those cultured in 10% FBS (i), flow cytometric profiles indicate enrichment of G0
population in cells cultured in 0.2% FBS (ii). In parallel, PI staining and flow cytometry were performed to monitor cell-cycle distribution (inset). Values indicate the percentage of
cells in each phase. G0/G1–enriched 8226 cells were then exposed to MEK1/2 inhibitors (1.5␮M AZD6244 or 1.5␮M PD184352) for 24 hours, followed by Chk1 inhibitors
(400nM AZD7762 or 500nM CEP3891) for an additional 12 hours. Flow cytometric analysis was performed to assess cell death (7-AAD positivity, R16) in the G0 (Hst⫹/PY⫺,
R17 gate-in) population (iii-v). To ensure specificity of this assay, control experiments were performed in parallel, including negative controls (without fluorescent dye),
individual staining with each dye, and double staining with each pair of dyes. Results are representative of 3 separate sets of experiments. (B) H929 cells cultured in 10% FBS
medium reached a density of 1 ⫻ 106 cells/mL, after which time viable SSP cells were sorted by exclusion of Hst staining of MM cells, as described in supplemental Methods. In
brief, cells were incubated with Hst and then stained with PI. For flow cytometric analysis, after gating out PI⫹ dead cells (i), gates were set for both the SP (ii) and low FSC
(small size) population (iii). In parallel, cells were coincubated with 50␮M verapamil (iv), which blocks Hst efflux, as a control. The cell-cycle profile was determined by PI
staining immediately after sorting (v top, unsorted cells; v bottom, sorted SSP cells). (C) Sorted SSP cells in 5% FBS medium were then treated with the AZD6244/AZD7762
regimen for 14 hours and stained with activated caspase-3 plus PI to determine the percentage of apoptotic cells within the G0/G1 population. Two additional experiments yield
roughly identical results.
evaluation. To minimize—although not completely exclude—the
possible contribution of off-target effects, parallel studies were
performed using 2 unrelated specific Chk1 inhibitors (AZD7762
and CEP3891) in combination with 2 MEK1/2 inhibitors (AZD6244
and PD184352).
The bulk of evidence indicates that lethality stemming from
simultaneous interruption of Chk1 and MEK1/2 occurs independently of cell-cycle progression in MM cells, which, at least at
early stages of the disease, display low proliferative indices and
largely reside (⬎ 95%) in G0/G1.1 This evidence includes the
findings that: (1) Chk1/MEK1/2 inhibitor regimens
(eg, AZD7762/AZD6244 and CEP3891/PD184352) were highly
active against primary CD138⫹ MM cells, which have a low
proliferative capacity1-3; (2) in contrast to regimens incorporating Chk1 inhibitors and genotoxic agents, which lead to
inappropriate progression through G2/M,12 Chk1/MEK1/2 inhibition arrested asynchronized MM cells cultured in 10% FBS
medium in G0/G1 (notably, the G0/G1 population was as or more
sensitive to these regimens than their cycling S or G2/M
counterparts); (3) Chk1/MEK1/2 inhibition induced pronounced
cell death in G0/G1–enriched MM cells, whereas these cells were
relatively less sensitive to VP-16 or Taxol; (4) the Chk1/
MEK1/2 inhibitor regimens induced cell death in cultured and
primary MM cells characterized by quiescent G0 phase phenotypes (eg, statin⫹, Hst⫹/PYlow)29,33; and (5) these regimens were
active against the sorted quiescent SSP population of MM cells.
These observations suggest that the mechanisms through which
combined Chk1/MEK1/2 inhibition induces cell death differ
from those responsible for the potentiation of genotoxic agent
lethality by Chk1 inhibitors, and that the former are operative in
both cycling and quiescent tumor cells. In this context, very
recent studies have shown that targeting the SP population of
MM cells represents an important mechanism contributing to
the anti-MM activity of lenalidomide.31 It will be interesting to
determine whether Chk1/MEK1/2 inhibitor regimens act against
this population through similar or different mechanisms.
The Ras/Raf/MEK1/2/ERK1/2 pathway plays an important role
in cell-cycle progression. For example, ERK1/2 activation is
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BLOOD, 10 NOVEMBER 2011 䡠 VOLUME 118, NUMBER 19
required for progression from the G1 to S phase or through G2/M.42
Moreover, recent findings suggest a functional role for MEK1/2/
ERK1/2 in cell-cycle checkpoints.43,44 However, the MEK1/2/
ERK1/2 pathway also mediates multiple survival functions, including down-regulation of proapoptotic proteins such as Bad and
Bim.45 The latter protein, implicated in potentiation of Chk1
inhibitor lethality by inhibitors of the Ras/MEK1/2/ERK1/2 pathway in the bulk population of MM cells,20 also plays a functional
role in the lethality of these regimens toward quiescent (G0/G1)
MM cells. In this context, the Ras pathway has been linked to
checkpoint activation in quiescent cells.32 Therefore, whereas the
lethal consequences of combined exposure to DNA-damaging
agents and Chk1 inhibitors have been attributed to inappropriate
cell-cycle progression, coadministration of MEK1/2 inhibitors may
act coordinately with Chk1 inhibitors to promote genotoxicity in
both cycling and quiescent cells. The ability of Chk1 inhibitors
(eg, UCN-01 and CEP3891) and Chk1 knockdown by shRNA to
induce DNA damage has been well documented.23 Interestingly,
results of one study suggest that nonproliferating ovarian cancer
cells are particularly sensitive to the lethal consequences of Chk1
inhibition.4 These findings raise the possibility that intact Chk1
function is required even in quiescent cells to circumvent the lethal
consequences of spontaneously occurring DNA damage. In this
context, emerging evidence indicates that, in addition to its classic
checkpoint function, Chk1 plays diverse roles, including those
related to survival and DNA repair.10,11,39 For example, Chk1 has
been implicated in the latter through interactions with the DNArepair machinery.39,46 Conversely, ERK1/2 activation is also involved in DNA repair through regulation of DNA-repair proteins.47
Interestingly, the type of DNA repair is cell-cycle phase dependent;
that is, in quiescent (G0/G1) cells, it primarily proceeds via
nonhomologous end-joining, whereas in cycling cells (S and
G2/M), homologous recombination predominates.48 Therefore, it is
possible that disruption of Chk1 function, particularly in conjunction with disruption of MEK1/2/ERK1/2–related DNA-repair
events,47,49 may increase the susceptibility of quiescent (G0/G1)
cells to DNA damage. The pronounced induction of DNA damage
(eg, ␥H2A.X expression/foci formation) by the Chk1/MEK1/2
inhibitor regimens in G0/G1–enriched cells and in the G0/G1–
labeled population supports this notion. Although a more direct role
for Chk1 in homologous recombination has been recognized
recently,50 evidence specifically relating Chk1 to nonhomologous
end-joining or other processes characteristic of G0/G1 cells is not
yet available. Further studies will be required to define the
mechanism(s), such as disruption of DNA repair, that renders
quiescent (G0/G1) cells vulnerable to this strategy.
In summary, the present findings provide evidence that the Chk1/
MEK1/2–inhibitory strategy, in addition to killing cycling cells, is also
active against cytokinetically quiescent (G0/G1) MM cells while exerting relatively minimal toxicity toward normal cells. Our findings also
indicate that the mechanism(s) responsible for inducing cell death in
cytokinetically quiescent (noncycling) transformed cells by this regimen
may differ fundamentally from those involved in the potentiation of
Chk1/MEK INHIBITOR KILLS QUIESCENT MYELOMA CELLS
5199
genotoxic agent lethality in cycling cells by DNA-damage checkpoint
abrogation. For example, the present results suggest that cytokinetically
quiescent transformed cells, which characteristically exhibit resistance
to conventional chemotherapeutic agents,3-5 may be particularly susceptible to intrinsic DNA damage, possibly reflecting the essential function
of Chk1 in the maintenance of genomic stability.39 In this setting, Bim
could serve as a death trigger to promote the elimination of cells
containing damaged DNA. Dysregulation of the Ras/Raf/MEK/ERK
pathway, which is one of the most common aberrations in cancer,
including MM,13,14 might therefore act to down-regulate Bim, allowing
such cells to survive. Conversely, by up-regulating Bim, interruption of
MEK/ERK signaling could lower the threshold for apoptosis in transformed cells, including those in a nonproliferative, quiescent state. The
finding that Bim up-regulation is required for Chk1/MEK1/2–inhibitor
lethality in G0/G1–arrested cells supports this notion. Based on in vitro
and in vivo evidence of activity of AZD6244 in MM models,17 a phase
2 trial of AZD6244 in refractory MM has been initiated. Such a study
could provide a foundation for successor trials combiningAZD6244 and
Chk1 inhibitors such as AZD7762 in MM, which would determine the
in vivo relevance of the present findings more definitively.
Acknowledgments
The authors thank Dr Eugenia Wang (University of Louisville,
Louisville, KY) for her generous gift of the anti-statin mAb;
AstraZeneca for supplying AZD7762 and AZD6244; Cephalon for
supplying CEP3891; and Dr Daniel H. Conrad and Ms Julie
Farnsworth (Flow Cytometry Share Resource of Virginia Commonwealth University/Massey Cancer Center, Richmond, VA) for their
technical assistance in flow cytometry, which was supported in part
by Massey Cancer Center core National Institutes of Health grant
P30 CA16059.
This work was supported by the National Institutes of Health (grants
CA93738, CA100866, P50 CA130805-01, P50 CA142509-01, and
RC2 CA148431-01), the Multiple Myeloma Research Foundation, the
Leukemia & Lymphoma Society of America (grant R6181-10), the V
Foundation, and by the Department of Defense.
Authorship
Contribution: X-Y.P. designed and performed the research, analyzed the data, and wrote the manuscript; L.E.Y., S.C., W.W.B.,
Y.T., J.F., J.A.A., and L.B.K. performed the research; P.D. helped
design the research; and Y.D. and S.G. designed the research,
analyzed the data, and wrote the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Dr Steven Grant, Division of Hematology/
Oncology, Virginia Commonwealth University Health Sciences
Center, Rm 234 Goodwin Research Bldg, 401 College St, Richmond, VA 23298; e-mail: [email protected].
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From www.bloodjournal.org by guest on August 2, 2017. For personal use only.
2011 118: 5189-5200
doi:10.1182/blood-2011-02-339432 originally published
online September 12, 2011
Cytokinetically quiescent (G0/G1) human multiple myeloma cells are
susceptible to simultaneous inhibition of Chk1 and MEK1/2
Xin-Yan Pei, Yun Dai, Leena E. Youssefian, Shuang Chen, Wesley W. Bodie, Yukie Takabatake,
Jessica Felthousen, Jorge A. Almenara, Lora B. Kramer, Paul Dent and Steven Grant
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