Disparate In Vivo Efficacy of FTY720 in Xenograft Models of Philadelphia Positive and Negative B-lineage Acute Lymphoblastic Leukemia
et al. (2012) Disparate In Vivo Efficacy of FTY720 in Xenograft Models of Philadelphia
Positive and Negative B-lineage Acute Lymphoblastic Leukemia. PLoS ONE 7(5): e36429. doi:10.1371/journal.pone.0036429
Disparate In Vivo Efficacy of FTY720 in Xenograft Models of Philadelphia Positive and Negative B-lineage Acute Lymphoblastic Leukemia
Craig T. Wallington-Beddoe 0
Anthony S. Don 0
John Hewson 0
Qiao Qiao 0
Rachael A. Papa 0
Richard B. Lock 0
Kenneth F. Bradstock 0
Linda J. Bendall 0
Venugopalan Cheriyath, Texas A&M University, United States of America
0 1 Westmead Institute for Cancer Research, Westmead Millennium Institute, The University of Sydney , Sydney , Australia , 2 Lowy Cancer Research Centre, Prince of Wales Clinical School, Faculty of Medicine, University of New South Wales , Sydney , Australia , 3 Children's Cancer Institute Australia for Medical Research, Lowy Cancer Research Centre, University of New South Wales , Sydney , Australia , 4 Hematology Department, Westmead Hospital , Westmead NSW , Australia
Most patients with acute lymphoblastic leukemia (ALL) respond well to standard chemotherapy-based treatments. However a significant proportion of patients, particularly adult patients, relapse with the majority dying of leukemia. FTY720 is an immunosuppressive drug that was recently approved for the treatment of multiple sclerosis and is currently under preclinical investigation as a therapy for a number of hematological malignancies. Using human ALL xenografts in NOD/ SCIDcc2/2 mice, we show for the first time that three Ph+ human ALL xenografts responded to FTY720 with an 80612% (p = 0.048) reduction in overall disease when treatment was commenced early. In contrast, treatment of mice with FTY720 did not result in reduced leukemia compared to controls using four separate human Ph2 ALL xenografts. Although FTY720 reactivated PP2A in vitro, this reactivation was not required for death of Ph2 ALL cells. The plasma levels of FTY720 achieved in the mice were in the high nanomolar range. However, the response seen in the Ph+ ALL xenografts when treatment was initiated early implies that in vivo efficacy may be obtained with substantially lower drug concentrations than those required in vitro. Our data suggest that while FTY720 may have potential as a treatment for Ph+ ALL it will not be a useful agent for the treatment of Ph2 B-ALL.
Funding: This work was funded by the following grants: Leukemia Foundation of Australia PhD Scholarship (Clinical) (www.leukaemia.org.au), Cancer Institute
NSW Research Scholar Award No. 10/RSA/1-34, Cancer Institute NSW Early Career Fellowship 08/ECF/1-03 (www.cancerinstitute.org.au), Royal College of
Pathologists of Australasia Research Award (www.rcpa.edu.au), NHMRC CDA2 No. 511965 and NHMRC Senior Research Fellowship No. 568703 (www.nhmrc.gov.
au). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Acute lymphoblastic leukemia (ALL) represents approximately a
quarter of all childhood cancers, and a similar proportion of cases
of acute leukemia in younger adults. Although the treatment of
childhood ALL is one of the success stories of modern oncology,
treatment protocols remain imperfect . Approximately 15% of
children and the majority of adults diagnosed with ALL relapse
following treatment. The overall survival of the 60% of adults who
relapse following treatment is only 7% at 5 years [2,3]. Overall,
once relapse occurs, the success of any further treatment, including
hematopoietic stem cell transplantation, is poor.
FTY720 is an immunosuppressive drug recently approved for
the treatment of multiple sclerosis . Once phosphorylated by
sphingosine kinases, phosphorylated FTY720 (FTY720-P) down
regulates four of the five sphingosine 1-phosphate (S1P) receptors,
trapping lymphocytes in secondary lymphoid organs . More
recently, FTY720 has been investigated for the treatment of
malignancies and has documented in vitro and/or pre-clinical
activity against a number of hematological disorders including
Tcell acute lymphoblastic leukemia (T-ALL), multiple myeloma,
chronic lymphocytic leukemia (CLL), mantle cell lymphoma
(MCL), acute myeloid leukemia (AML) with c-kit mutations,
mouse models of chronic myeloid leukemia (CML) and Ph+
(Philadelphia chromosome positive) ALL, Ph2 ALL and NK cell
The anti-leukemic efficacy of FTY720 is thought to be due to
reactivation of the protein phosphatase type 2A (PP2A), an essential
protein serine/threonine phosphatase, the activity of which is
reduced in certain malignancies . The involvement of PP2A
reactivation in Ph+ disease has been well documented with interplay
between PP2A and Bcr/Abl being clearly demonstrated .
Indeed PP2A activation and caspase-dependent cell death were
required for its cytotoxic effect in AML, CML and Ph+ ALL [10,11]
whilst caspase-dependence without PP2A activation was recently
reported for NK cell leukemia . However we, and others, have
reported caspase-independent death mechanisms of FTY720,
suggesting that the mechanism of action of FTY720 in malignant
cell killing is varied and still unclear [8,9,12]. Regardless of the
mechanism of cell death, the IC50 values have been similar between
studies, ranging from 2.4 to 12 mM (Table 1). Study of the in vivo
efficacy of FTY720 for the treatment of CLL, MCL, AML, CML,
Ph+ ALL and NK cell leukemia demonstrated increased survival of
mice and/or reduced leukemic cell burden [8,9,10,11,13].
Wallington-Beddoe et al 2011. Ph+ & Ph2 ALL
Table 1. Studies of FTY720 in hematological malignancies.
ND - Not determined. In vitro IC50 values were assessed at 24 hours unless otherwise indicated. FTY720 was administered by the intra-peritoneal route in all in vivo
studies. Cell lines used for in vivo studies are indicated.
We have previously reported the in vitro efficacy of FTY720 in
Ph2 ALL . Here, consistent with reports by others in mouse
models of Ph+ ALL , we show that FTY720 was effective in vivo
in a human xenograft model of Ph+ ALL. On the other hand, we
found that FTY720 had no therapeutic effect in vivo against Ph2
ALL. This disparity in the in vivo response occurred despite Ph+
and Ph2 ALL cells demonstrating similar in vitro sensitivities to
FTY720. In some Ph2 ALL xenografts FTY720 appeared to
exacerbate the disease, suggesting that clinical trials of FTY720 in
Ph2 ALL are unlikely to succeed.
Materials and Methods
Cells and Reagents
Leukemic blasts were obtained from 4 ALL patients with
written informed consent, or in the case of minors from the parents
of patients, and institutional ethics committee approval from the
Sydney West Area Health Service Human Ethics Committee
(Approval No. HREC/2009/8/4.1 3028), while xenografts
ALL3, ALL-55 and ALL-56 were previously established. The clinical
details of some patient samples have been previously published but
information on all patient samples are summarized in Table S1
[14,15,16,17]. Xenografts were established in NOD/SCID mice
from mononuclear cells as described previously . The
phosphatase inhibitor okadaic acid was purchased from
SigmaAldrich (St Louis, MO) and FTY720 from Selleck Chemicals
Assessment of In Vivo FTY720 Efficacy
This study was carried out in strict accordance with the
recommendations in the National Health and Medical Research
Council Guidelines and Policies to Promote the Wellbeing of
Animals Used for Scientific Purposes and the Australian Code of
Practice for the care and use of animals for scientific purposes.
Protocols were approved by the Sydney West Area Health Service
Animal Ethics Committee (Approval No. 5049.08-09) and The
University of New South Wales Animal Care and Ethics
Committee (Approval No. 09/130A).
Groups of NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NOD/
SCIDcc2/2) mice were engrafted with 256106 ALL cells by
tail vein injection. Peripheral blood was collected weekly from the
tail vein of all mice for the monitoring of ALL. FTY720, prepared
in 2% ethanol (experiments using ALL-3) or saline (all other
experiments), was administered by intra-peritoneal (IP) injection,
except where gavage was indicated. For the early disease model,
treatment commenced within a week of cell transfer and for the
advanced disease model, when 1% ALL was detected in the blood.
All mice were treated for 3 weeks unless otherwise indicated, with
mice engrafted with xenograft ALL-3 receiving drug 6 days a
week, while all others received treatment daily. Animals from
xenografts 1345, 2070, 1999, 0398, ALL-55 and ALL-56 were
sacrificed after 21 days of treatment and disease assessed in the
peripheral blood, bone marrow and spleen by flow cytometry and
in the liver by histology. Single cell suspensions of blood, bone
marrow and spleen were prepared and red cells removed by lysis
where required. Total leukemia burden was calculated by totaling
the number of ALL cells in the bone marrow, blood and spleen.
The calculation of total ALL cells in the bone marrow was based
on the accepted standard that the marrow from one femur
represents 5.8% of the total bone marrow. Total ALL cells in the
blood was based on the total blood volume being 80 mL/g of
Cell viability was measured as previously described  after a
16-hour exposure to FTY720. Cells were labelled with propidium
iodide and annexin V-FITC (BD Biosciences, San Jose CA)
according to the manufacturers instructions, with negative cells
Cells from mice were stained with anti-human CD19PE or
antihuman CD45APC and anti-murine CD45FITC (BD Biosciences
and Invitrogen, Carlsbad CA) for 10 or 30 minutes according to
the manufacturers instructions, and as previously described
[14,17]. All cells were analysed using a FACSCanto flow
cytometer (BD Biosciences).
PP2A Activity Assay
Cells were lysed in a low-detergent lysis buffer (1% Nonidet
P40, 10 mM HEPES, 150 mM NaCl, 10% glycerol, 1 mM PMSF,
5 mM benzamidine and 10 mg/mL leupeptin). PP2A phosphatase
activity was determined using the malachite green-phosphate
complex assay as described by the manufacturer (Millipore,
Billerica MA) using a PP2A-specific reaction buffer and 750 mM
phosphopeptide substrate. After 10 minutes incubation at 30uC,
malachite dye was added and free phosphate measured by optical
density at 620 nm using a Wallac 1420 Multilabel Counter
(PerkinElmer, Turku Finland).
Measurement of FTY720 in plasma
Blood was drawn by tail vein bleeding and immediately
transferred to ice. A 50 mL aliquot was centrifuged at 1500 rpm
for 10 min to pellet cells, and 20 mL of plasma was added to
380 mL ice-cold methanol. Extracts were spiked with 20 pmoles
C17 sphingosine and C17 S1P (Avanti Polar Lipids, Alabaster,
AL), which act as the internal standards for FTY720 and
FTY720phosphate (FTY720-P), respectively. Extracts were then vortexed,
sonicated for 30 seconds in an ice bath, and centrifuged for
20 min at 14,000 rpm, at 4uC. Supernatants were transferred to
5 mL glass tubes, and the pellets were re-extracted as above with
600 mL ice-cold 80% methanol/20% water (v/v). The
supernatants from both extraction steps were combined, dried in a
SpeediVac, resuspended in 200 mL 80% methanol/0.1% formic
acid (v/v), and stored at 220uC for quantification of FTY720 and
FTY720-P using liquid chromatography-tandem mass
spectrometry (LC-MS/MS), as described previously .
Comparisons between two groups were performed using the
Students t test and between multiple groups using two-way
ANOVA with Bonferroni post-test. Comparison of the response of
Ph2 and Ph+ ALL xenografts was done using a Fischers exact test.
FTY720 Did Not Reduce the Progression of a Ph2 ALL in
Vivo Using Multiple Treatment Dosing Schedules
FTY720 has been reported to induce cell death in ALL cells in
vitro  and inhibit the development of ALL in the murine cell
line BaF3 transduced with Bcr/Abl in immuno-compromised mice
. Here we examined the effect of FTY720 on Ph2 human
ALL xenografts in NOD/SCID mice. In an initial experiment
using xenograft ALL-3, we compared an advanced disease model,
where treatment was not commenced until disease was clearly
detectable in the blood, with an early disease model where
treatment commenced only 4 days post engraftment. Mice were
administered vehicle control or FTY720 by IP injection at 5 mg/
kg/day in the early disease arm or 10 mg/kg/day in the late
disease arm. Mice were treated for a total of 18 days and the level
of ALL in the blood assessed weekly. Although a trend towards
reduced disease with FTY720 was observed, this did not achieve
statistical significance (Fig. 1A). To further escalate plasma
concentrations of FTY720, this same xenograft was examined
using orally administered FTY720 at 25 mg/kg/day, with
treatment commencing when human leukemia cells were first
detected in peripheral blood (advanced disease model). Again
there was no reduction in disease observed in the FTY720-treated
animals (Fig. 1B). Assessment of FTY720 plasma levels revealed
mice receiving the higher dose of drug by gavage had higher
FTY720 levels (427.1658 nM) than those receiving FTY720 by
the IP route (205658 nM) (Fig. 1C). Consistent with previous
reports, FTY720-P was present at higher levels than the parental
drug in the plasma of treated mice [18,19].
FTY720 Is Effective in Ph+ but Not Ph2 ALL Xenografts
Using an Early Disease Model
To further examine whether FTY720 may be effective when
used earlier in the development of the disease, and in light of the
possibility that the ALL-3 xenograft may have been resistant to
FTY720, we engrafted NOD/SCIDcc2/2 mice with three
additional human Ph2 ALL xenografts (1345, 0398 and 1999).
Treatment was commenced on day 3 (xenograft 1345) or 7
(xenografts 0398 and 1999) post engraftment, when ALL cells
were not yet detectable in the peripheral blood, and continued for
3 weeks. No significant reduction in disease was observed in any
tissue examined at the end of 3 weeks of treatment with FTY720.
Indeed, the amount of ALL increased in the bone marrow from
2.762.3 to 9.863.36106 cells/femur (3.7 fold, p = 0.004) and
blood from 3.162.1 to 8.062.56106 cells/mL (2.5 fold, p = 0.01)
of mice with xenograft 1345 treated with FTY720 and in the
spleens, from 0.0360.01 to 0.3060.246106 cells (11.5 fold,
p = 0.02) of mice receiving xenograft 0398 (Fig. 2A). In the
experiment using xenograft 1345 there were also two deaths in the
Figure 1. FTY720 does not reduce disease burden in a xenograft model of advanced human ALL. (A) Groups of 78 NOD/SCIDcc2/2
mice were engrafted with xenograft ALL-3 and treated with vehicle control or FTY720 by IP injection 6 days a week for 3 weeks. The early disease arm
received FTY720 at 5 mg/kg/day 6 days a week commencing on day 4 post engraftment, and the advanced disease arm at 10 mg/kg/day
commencing in week 5. The mean 6 SD of the percentage of ALL cells in the blood is indicated. (B) Groups of 4 mice were engrafted with ALL-3 and
treated with 25 mg/kg/day of FTY720 or vehicle control by gavage, commencing in week 6 and continuing for 2 weeks. The mean 6 SD of the
percentage of ALL cells in the blood is indicated. (C) Plasma concentrations of FTY720 and FTY720-P were determined 2 h after IP administration and
6 h after oral administration of FTY720.
FTY720 group prior to the three-week endpoint with very high
levels of leukemia in both animals (data not shown). In addition,
the livers of mice engrafted with xenografts 1345 and 1999 also
showed increased leukemic cell infiltration (Fig. 2B). The dose of
FTY720 was reduced from 10 mg/kg to 5 mg/kg in the
experiment using xenograft 1999 due to concerns regarding
toxicity of the drug, with extreme lethargy noted post injection in
To confirm the validity of this model we examined the response
of 3 Ph+ ALL samples, 2070, ALL-55 and ALL-56. Mice were
engrafted and treated as described above with treatment
commencing 7 days after the injection of cells. Consistent with reports
on murine Ph+ ALL , FTY720 produced a significant reduction
in disease burden in the Ph+ ALL xenografts using an early disease
model (Fig. 2A). This decrease was most obvious in the blood
(reduced from 0.1560.04 to 0.0360.006106/mL (p = 0.0002),
0.0160.00 to 0.0060.006106/mL (p = 0.0009), and 2.4560.76 to
1.1260.416106/mL (p = 0.005) respectively) and the bone marrow
(reduced from 1.3860.51 to 0.4160.186106/femur (p = 0.003),
1.3260.51 to 0.1060.066106/femur (p = 0.0002) and 3.6361.16
to 0.9660.516106/femur (p = 0.0004) respectively) with variable
responses being noted in the spleens (from 1.5360.87 to
1.0060.396106/spleen (p = n.s.), 0.0260.01 to 0.0460.026106/
spleen (p = n.s.) and 83.54665.04 to 11.3966.446106/spleen
(p = 0.02) respectively). Overall the Ph+ xenografts were reduced
from an estimated total disease burden of 25.4869.06 to
7.9763.406106 cells (p = 0.002), 22.8568.96 to 1.8161.086106
cells (p = 0.0002) and from 149.96674.08 to 29.92613.366106
cells (p = 0.003) respectively. The response of Ph+ xenografts was
significantly different from that observed in Ph2 xenografts
(p = 0.029).
FTY720 Kills Ph2 ALL Xenograft Cells in Vitro in a
We considered the possibility that the Ph2 patient xenografts
may have been resistant to FTY720 but in vitro culture
demonstrated that 1345, 0398, 1999, ALL-3, ALL-55 and
ALL56 were all sensitive, with comparable IC50 values to ALL cell lines
and patient samples examined previously, including the Ph+
sample 2070  (Fig. 3). FTY720 induced activation of PP2A in
all xenografts which was inhibited by 5 nM okadaic acid, a
concentration previously shown to be optimal for specific
inhibition of PP2A , However, FTY720-induced cell death
was independent of PP2A activation (Fig. 4).
The main findings of this study were that FTY720 was
ineffective in treating human Ph2 ALL xenografts whilst effective
for Ph+ ALL in a clinically relevant mouse model of human ALL,
implying that this drug is unlikely to be suitable for clinical trial
development for Ph2 disease. Overall there were no significant
reductions in ALL in Ph2 xenografts as a result of FTY720
treatment and in one xenograft we observed a clear and significant
worsening of the disease. This lack of effect was observed not only
when animals had advanced disease but also in an early disease
model. The increased splenic infiltration observed in one xenograft
could be due to FTY720-induced loss of S1P1 expression and
retention of ALL cells in the spleen as S1P1 is required for
lymphocyte egress from the splenic white pulp . The reduced
efficacy of FTY720 on inhibiting the infiltration of two of the three
Ph+ ALL into the spleens is also consistent with an effect on ALL
cell trafficking. However, the increased splenic disease in xenograft
0398 was not associated with significantly reduced disease in the
blood or bone marrow suggesting that altered trafficking was not
the only cause of increased ALL in the spleens of FTY720-treated
animals. We found FTY720 to be efficacious in a human xenograft
model of Ph+ ALL in the early treatment model, consistent with
previous reports in mouse models of Ph+ ALL .
Figure 4. FTY720 reactivates PP2A but induces PP2A-independent cell death. (A) Xenografts were treated with 10 mM FTY720 for 4 hours
with or without a 2 hour pre-incubation with 5 nM okadaic acid. Activation of PP2A was assessed as described in Materials and Methods. (B) All
xenografts were treated with the indicated concentrations of FTY720 with or without a pre-incubation with 5 nM okadaic acid. Viability was assessed
by flow cytometry using annexin V and propidium iodide staining. The mean 6 SD of duplicate determinations are shown.
It is not clear why FTY720 was not effective in vivo for Ph2 disease
but there are a couple of potential explanations. Firstly the bone
marrow microenvironment has been reported to afford protection
from the effects of a range of chemotherapeutic agents in a number
of hematological malignancies including ALL . Although the
mechanism is not fully understood, stromal cells are known to
provide factors that support ALL survival, activating pro-survival
pathways such as the PI-3K/mTOR pathway [22,23]. It is also
possible that FTY720 simply did not attain sufficient concentrations
in the animals to produce the cytotoxic effects in ALL cells that we
observed in vitro. Pharmacokinetic studies of FTY720 performed in
rats suggest that micromolar concentrations may not be achievable
[19,24,25]. In our study, plasma FTY720 levels were approximately
200 nM and 400 nM when administered at 10 mg/kg/day by IP
injection and 25 mg/kg/day oral, respectively. However, in vivo
responses have been observed in a range of hematological
malignancies where in vitro IC50 values were in the micromolar
range (Table 1). The reasons for the discrepancies in effective drug
concentrations in vitro and in vivo remain unclear. We have
previously reported a similar situation with the mTOR inhibitor
RAD001 in ALL , with IC50s in the low micromolar ranges, but
in vivo responses observed at plasma concentrations ten-fold lower,
and it is possible that effects on the microenvironment are
contributing to the observed in vivo response. Lower concentrations
of FTY720 appear to be required in vivo for Ph+ ALL than the in vitro
studies would suggest. It is also possible that the previously reported
accumulation of FTY720 in lymphoid tissues enhances its effective
concentration on leukemia cells, relative to the concentration
measured in plasma . This increases the likelihood of FTY720
being useful in the setting of malignant disease. Although this may
explain greater than expected in vivo activity, it would not explain the
discrepancy between Ph2 and Ph+ ALL. While FTY720 has
relatively low toxicity compared to standard chemotherapeutic
agents, there are suggestions that long-term exposure produces
macular degeneration  and conditions associated with increased
vascular leak , as well as increased risk of viral infections and
skin cancer . FTY720 also induces transient bradycardia, which
could explain the lethargy observed following injection .
In contrast to most previous studies, the ultimate mechanism of
cell death in vitro in Ph+ and Ph2 ALL cells was both caspase- and
PP2A-independent. Although PP2A was activated by FTY720,
inhibition of PP2A activity using okadaic acid did not impact on
cell death. This finding contrasts with many previous studies
including those in hematological malignancies [8,10,11]. The
major exception was a study in natural killer cell leukemia .
Despite reporting in vitro and in vivo efficacy, FTY720 was not
found to activate PP2A and the PP2A inhibitor okadaic acid did
not reverse FTY720-induced cell death. While our data is similar,
in that cell death was PP2A independent, we did observe a
significant activation of PP2A following FTY720 exposure. Our
data also differs in that the mechanism of cell death was not
Our previous in vitro studies also demonstrated the production of
reactive oxygen species in response to FTY720, a finding that is in
keeping with previous reports [9,30]. In these studies blockade of
ROS production by antioxidants partially reversed the cytotoxic
effects of FTY720. In the study by Liao et al  it was suggested
that ROS production down regulated the anti-apoptotic protein
Mcl-1, the loss of which was required for cell death . Similarly,
we have shown that FTY720 reduced expression of Mcl-1, however
we did not detect apoptosis as a result of FTY720 exposure .
Mcl-1 also inhibits the development of autophagy through
inhibition of beclin-1 [31,32]. Although we did not detect
induction of beclin-1 expression with FTY720, reduced inhibition
would be expected due to lower Mcl-1 protein levels thereby
permitting the development of autophagy. Furthermore,
autophagy induced by a sub-cytotoxic concentration of FTY720 partially
counteracted the cytotoxic effects of agents such as vincristine
supporting its cell protective role . FTY720-induced
autophagy is at least partly due to the induction of S1P signaling by
FTY720-P [12,19]. Brinkmann et al noted that FTY720-P peaked
at more than triple the peak levels of the cytotoxic
unphosphorylated FTY720 . In our study, the non-cytotoxic
phosphorylated derivative was found to be approximately 1.5
and 6 fold higher than FTY720 in animals receiving FTY720 by
the IP and oral route respectively. It therefore appears that the
induction of autophagy may offer protection, which may have
played a role in our animal experiments, potentially explaining the
outcomes observed for Ph2 disease.
Our observation that a drug with excellent in vitro efficacy did not
universally translate into positive effects in the in vivo setting was
unexpected. The disparity between the in vivo responses of Ph2 ALL
samples to FTY720, despite good in vitro efficacy currently lacks an
adequate explanation, but highlights the importance of conducting
well-designed pre-clinical studies prior to initiating clinical trials. In
conclusion, we present here the first report of FTY720, in a relevant
human xenograft model, demonstrating differential efficacy in Ph+
versus Ph2 B-lineage ALL, a finding which questions the utility of
further testing of FTY720 in the latter form of the disease.
We would like to thank the staff of the Westmead Animal Facility and Ms
Virginia James who prepared the histological sections presented herein. We
would also like to thank Dr Vivek Bhadri from the Childrens Cancer
Institute Australia for Medical Research for the provision of ALL
xenografts ALL-55 and ALL-56. Childrens Cancer Institute Australia
for Medical Research is affiliated with Sydney Childrens Hospital and the
University of New South Wales.
Conceived and designed the experiments: LB CW KB AD RL. Performed
the experiments: CW AD JH QQ RP. Analyzed the data: CW LB AD.
Wrote the paper: LB CW KB AD.
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