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PRECLINICAL STUDIES
Vandetanib mediates anti-leukemia activity by multiplemechanisms
and interacts synergistically with DNAdamaging agents
Margaret E. Macy & Deborah DeRyckere & Lia Gore
Received: 3 August 2010 /Accepted: 20 October 2010# Springer
Science+Business Media, LLC 2010
Summary Vandetanib is an orally active small moleculetyrosine
kinase inhibitor (TKI) with activity against severalpathways
implicated in malignancy including the vascularendothelial growth
factor receptor pathway, the epidermalgrowth factor receptor
pathway, the platelet derived growthfactor receptor pathway, and
REarranged during Trans-fection pathway. To determine if
vandetanib-mediatedinhibition of receptor tyrosine kinases is a
potentialtherapeutic strategy for pediatric acute leukemia,
thesestudies aimed to characterize the activity of
vandetanibagainst acute leukemia in vitro. Treatment of leukemia
celllines with vandetanib resulted in a dose-dependent decreasein
proliferation and survival. Vandetanibs anti-leukemic
activity appeared mediated by multiple mechanisms includ-ing
accumulation in G1 phase at lower concentrations andapoptosis at
higher concentrations. Alterations in cell surfacemarkers also
occurred with vandetanib treatment, suggestinginduction of
differentiation. In combination with DNAdamaging agents (etoposide
and doxorubicin) vandetanibdemonstrated synergistic induction of
cell death. However incombination with the anti-metabolite
methotrexate, vandetanibhad an antagonistic effect on cell death.
Although severaltargets of vandetanib are expressed on acute
leukemia celllines, expression of vandetanib targets did not
predictvandetanib sensitivity and alone are therefore not
likelycandidate biomarkers in patients with acute leukemia.
Inter-actions between vandetanib and standard chemotherapy agentsin
vitro may help guide choice of combination regimens forfurther
evaluation in the clinical setting for patients
withrelapsed/refractory acute leukemia. Taken together,
thesepreclinical data support clinical evaluation of vandetanib,
incombination with cytotoxic chemotherapy, for
pediatricleukemia.
Keywords Acute leukemia . Vandetanib . Vascularendothelial
growth factor . Tyrosine kinase inhibitor .
Combination therapy
AbbreviationALL acute lymphoblastic leukemiaAML acute
myelogenous leukemiaDMSO dimethyl sulfoxideEGFR epidermal growth
factor receptorFBS fetal Bovine SerumFlt-3 fms-like tyrosine kinase
3KDR kinase-insert domain containing regionMLL mixed lineage
leukemiaPDGFR platelet derived growth factor
This work was supported by grants from the For Julie Foundation
andNIH K12 CA086913-05, CA086913-08 (MM, LG). MM wassupported by
the University of Colorado William M. ThorkildsenResearch
Fellowship
Electronic supplementary material The online version of this
article(doi:10.1007/s10637-010-9572-6) contains supplementary
material,which is available to authorized users.
D. DeRyckere : L. GoreDepartment of Pediatrics, Section of
Hematology,Oncology, and Bone Marrow Transplantation,University of
Colorado Denver,Aurora, CO 80045, USA
L. GoreDivision of Medical Oncology, University of Colorado
Denver,Aurora, CO 80045, USA
M. E. Macy (*)Department of Pediatrics, Section of Hematology,
Oncology,and Bone Marrow Transplantation, University of Colorado
Denver,13123 East 16th Avenue B-115,Aurora, CO 80045, USAe-mail:
[email protected]
Invest New DrugsDOI 10.1007/s10637-010-9572-6
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RET Rearranged during transfectionVEGF vascular endothelial
growth factorVEGFR vascular endothelial growth factor receptor
Introduction
Acute leukemia accounts for approximately 5% of allmalignancies
with an event free survival of 40% for acutelymphoblastic leukemia
(ALL) and approximately 20% foracute myeloid leukemia (AML) [1, 2].
Despite advances intherapy the overall cure rate for either type of
acuteleukemia in adults remains relatively poor. While
acuteleukemia is a relatively uncommon disease in adults, it isthe
most common pediatric malignancy, with over 3,000new cases
diagnosed in U.S. children each year. Whilemore than 75% of
pediatric patients will be cured withcurrent intensive therapy,
patients with acute lymphoblasticleukemia (ALL) with early relapse
or acute myeloidleukemia (AML) with any relapse, or those with
specificcytogenetic or molecular abnormalities including
mixed-lineage leukemia (MLL) gene rearrangements have adismal
outcome [39]. These pediatric patients and theadult population
still desperately need new therapies.
With the recent advent of biologically-based approachesto the
treatment of human malignancies, new agents againsta wide variety
of molecular targets are currently in clinicaldevelopment. These
agents target specific genes or proteinsthat are mutated or
dysregulated in cancers. As standardchemotherapeutic agents have
significant toxicity, agentsthat are tailored to cancer-specific
abnormalities areparticularly appealing. The role of vascular
endothelialgrowth factor (VEGF) in tumor angiogenesis is an
activearea of cancer research. Neoangiogenesis is required
foradequate oxygen and nutrition to support a growing tumormass.
Stimulation of VEGF receptors results in activation ofsignaling
pathways that promote endothelial cell survival,migration,
differentiation, and increase vascular permeability[10, 11]. VEGF
is over-expressed in multiple solid tumorsand is associated with
poor outcome [1215]. As a result,VEGFR signaling has been targeted
as an anti-angiogenicstrategy in cancer.
Vandetanib (ZD6474) is an orally active small moleculetyrosine
kinase inhibitor with activity against the VEGFreceptor 2 (VEGFR2),
also known as kinase insert domaincontaining receptor (KDR) [16].
It possesses inhibitoryactivity at submicromolar concentrations
against VEGFreceptor3 (VEGFR3), the epidermal growth factor
receptor(EGFR), REarranged during Transfection (RET)
tyrosinekinase, and at micromolar concentrations activity againVEGF
receptor 1 (VEGFR1) and platelet-derived growthfactor receptor beta
(PDGFR) [1719]. In Phase I clinical
trials in patients with solid malignancies vandetanib waswell
tolerated at doses up to 300 mg once daily [20, 21].Common dose
related side effects included rash, diarrhea,hypertension and
asymptomatic QTc prolongation. Phar-macokinetic analyses
demonstrate a long half-life ofapproximately 120 h [20]. Vandetanib
is currently beingstudied in Phase II and III trials. Phase II
studies in patientswith non-small cell lung cancer, breast cancer,
or medullarythyroid cancer have shown stable disease or prolonged
timeto progression in patients treated with vandetanib [2226].Newer
studies with vandetanib plus gemcitabine andcapcitabine in biliary
cancers have shown significantprolongation of survival and clinical
responses [27].Vandetanib was granted orphan drug status by the
FDAfor treatment of medullary thyroid carcinoma where itsactivity
is mediated in part by inhibition of RET [28, 29].
While the role of vandetanib as an angiogenesis inhibitoris
being further elucidated in multiple clinical studies, itsactivity
against hematopoietic tumors is less well defined.Hematopoietic
malignancies express VEGF and VEGFsignaling plays an important role
in hematopoiesis, medi-ating hematopoietic stem cell survival and
repopulation viaan autocrine loop and regulating angiogenesis via a
para-crine loop [30]. Paracrine stimulation of other cells in
thetumor microenvironment can also result in production ofgrowth
factors that stimulate leukemia proliferation and/orsurvival [30].
All three VEGFRs are expressed on ALL andAML patient cells and cell
lines [30]. Activation ofVEGFR2 in leukemia cells promotes survival
through thenuclear factor B (NF-B), mitogen-activated proteinkinase
(MAPK)/Extracellular signal-regulated kinase(ERK), and
phosphatidylinositol-3 kinase/Akt pathways[31]. VEGFR1 and VEGFR3
signaling have been shownto promote leukemia cell migration,
survival, proliferationand chemoresistance [30]. VEGF expression in
leukemiasis associated with poorer prognosis and decreased
relapse-free survival [32, 33]. In addition, pediatric patients
withleukemia have increased bone marrow microvessel
densitysuggesting that leukemia may induce and be dependentupon
angiogenesis within the bone marrow environmentitself [30, 34].
These observations indicate that VEGF andangiogenesis may be
desirable targets in leukemia. Addi-tionally, other vandetanib
targets may also play importantroles in leukemia. The RET
proto-oncoogene is expressedon normal human CD34+ progenitor cells
and leukemicblasts from AML patients and RET expression is
increasedin more differentiated leukemia subtypes [35, 36]. Of
note,EGFR is not felt to have a significant role in
leukemiasurvival or proliferation as its expression is rarely
detectedin patient samples, and activating EGFR mutations are
notobserved in leukemia [37, 38].
In a previous preclinical report, vandetanib inducedgrowth
arrest and apoptosis in 3 of 14 leukemia cell lines
Invest New Drugs
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and 10 of 13 patient samples with AML [39]. The goal ofthe
studies presented here was to characterize the activityand
mechanisms of vandetanib against acute leukemia invitro, thereby
investigating the role of autocrine signalingthrough the pathways
inhibited. Additionally, we evaluatedthe interactions between
vandetanib and selected chemo-therapeutic agents. This is
particularly important in pediatriconcology where preclinical
studies can facilitate rationaldesign of clinical trials and
thereby maximize the amountand relevance of information obtained
from the limitednumber of patients available for clinical
studies.
Methods and materials
Cell culture The Molt-4, REH, RCH-AcV, RS4;11, Nalm-6, and
HAL-01 cell lines were obtained from Dr. StephenHunger (University
of Colorado Denver) in 2001. Jurkat,Molt-3, CCRF-HSB-2, and HL-60
were obtained from Dr.Douglas Graham (University of Colorado
Denver) in 2004.HEL, Eol-1, Molm-13, and Molm-14 were obtained
fromDr. Robert Arceci (Johns Hopkins University) in 2005.THP-1 was
obtained from Dr. Terzah Horton (TexasChildrens Hospital) in 2005.
CCRF-CEM, Kasumi-1 andMV4-11 were obtained from the American Type
CultureCollection (ATCC; Manassas, VA) in 2008. NOMO-1 wasobtained
from the German Collection of Microorganismsand Cell Cultures
(Braunschweig, Germany) in 2007. Allcell lines were received
frozen, and passaged to confluentgrowth. Experiments were performed
on established pas-sages less than 3 months of age. The identity
and purity ofthe THP-1, REH, CCRF-CEM, MV4-11, RS4;11, CCRF-HSB-2,
Eol-1, and Nalm-6 cell lines, the purity of the REHcell line and
the purity and common source of the Molm-13and Molm-14 cell lines
were confirmed by multiplexpolymerase chain reaction DNA profiling
using the ABIIdentifiler kit (Applied Biosystems, Carlsbad, CA)
followedby comparison with the ATCC database. Continuouscultures
were verified by DNA profiling and used forexperiments within 3
months. Cell lines were maintained at37C in 5% CO2 in RPMI medium
(InVitrogen, Carlsbad,CA) supplemented with 10% fetal bovine serum
(FBS) andpenicillin/streptomycin.
Treatment with vandetanib and other chemotherapeuticagents
Vandetanib was kindly provided by AstraZeneca(Macclesfield, UK).
Vandetanib and methotrexate (SigmaAldrich, St Louis MO) stocks were
prepared at 10 mM indimethylsulfoxide (DMSO). Etoposide (EMD
Biosciences,Gibbstown, NJ) stocks were prepared at 20 mM in DMSOand
doxorubicin (Sigma Aldrich, St Louis, MO) stockswere prepared at 5
mM in distilled, deionized water.Preliminary experiments were
performed to determine the
maximum density for each cell line such that nutrientswould not
be limiting for proliferation. Cells were platedand cultured
overnight prior to addition of therapeutic agent(s) or vehicle for
an additional 48 h.
Assessment of anti-tumor activity
For determination of relative number of metabolicallyactive
cells, cells were cultured in 96-well dishes andtreated in
triplicate with a therapeutic agent(s) and/orvehicle.
3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide
(MTT, Sigma Aldrich, St. Louis,MO) in PBS was added to a final
concentration of0.65 mg/ml after 48 h of treatment with vandetanib
and/or chemotherapeutic agents and cells were cultured for
anadditional 4 h. Solubilization solution (2, 10% SDS in0.01 M HCl)
was added and plates were incubated at 37Covernight. Optical
density was determined at 562 nm with areference wavelength of 650
nm. Relative numbers ofmetabolically active cells were calculated
by subtraction ofbackground absorbance and normalization to
untreatedcontrols. IC50 values were determined by
non-linearregression using Graphpad Prism v4.0 software
(GraphpadSoftware, La Jolla, CA).
Determination of cell death and cell cycle distribution
Cellswere cultured in 24-well dishes and collected by
centrifu-gation at 240 g for 5 min after treatment with
therapeuticagent(s) for 48 h. For assessment of cell death, cell
pelletswere resuspended in PBS containing 1 M YO-PRO-1(InVitrogen,
Carlsbad, CA) and 1.5 M propidium iodide(InVitrogen, Carlsbad, CA)
and incubated on ice for 2030 min. Fluorescence was detected and
analyzed using anFC500 flow cytometer and CXP data analysis
software(Beckman Coulter, Miami, FL). For assessment of cellcycle
distribution, cell pellets were resuspended in PBS andethanol was
gradually added with vortexing to a finalconcentration of 70% to
permeabilize membranes. Cellswere incubated at 4C overnight,
collected by centrifugation,resuspended in PBS containing 20 g/ml
propidium iodideand 2 g/ml RNase A, and incubated again at 4C
overnightprior to analysis by flow cytometry as above.
Determination of Differentiation Marker Expression Cellswere
cultured in 6-well dishes or 10 cm tissue culture platesand
collected by centrifugation at 240 g for 5 min aftertreatment with
vandetanib for 48 h. For assessment of cellsurface marker
expression, aliquots of 105 cells wereresuspended directly in 5%
FBS/PBS + 40 g/ml humanIgG (Sigma Aldrich, St. Louis, MO). Cells
were incubatedon ice for 30 min to block non-specific antibody
binding.An equal volume of fluorochrome-linked or biotin-linked
Invest New Drugs
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antibody in 5% FBS/PBS + 40 g/ml human IgG wasadded and samples
were incubated on ice for an additional60 min. Where indicated,
cells were washed with 0.75 ml5% FBS/PBS and resuspended in 50 L of
fluorochrome-linked streptavidin in 5% FBS/PBS + human IgG for
anadditional 30 min on ice. After staining, all samples werewashed
with and resuspended in 5% FBS/PBS, andfluorescence was quantitated
by flow cytometry as above.Aliquots of cells were stained with
fluorochrome-linkedisotype control antibodies to define background
fluores-cence and positive-staining cells. Antibodies were
obtainedfrom BD Biosciences (San Jose, CA). 2X solutions
wereprepared at the following dilutions: IgG1-FITC (#556028)1:5;
CD15-FITC (#555401); IgG1-PE (#559320) 1:5;CD69-PE (#555531);
IgG1-Biotin (#555747) 1:5;Streptavidin-FITC (#554060) 1:100;
CD28-Biotin(#555727) 1:5; CD2-FITC (#347593);CD11b-PE(#347557) 1:5;
CD4-FITC (#340133) 1:5; CD25-FITC(#347643) 1:5.
Interaction models and statistical analysis Interactionsbetween
vandetanib and cytoxic chemotherapies wereassessed by the Bliss
independence model [40]. Thefrequency of affected (Fa) expected for
an additiveinteraction between 2 agents was calculated based on
the
Bliss independence model using the following formula:
Fa1 2 Fa1 1 Fa1 Fa2where Fa1 and Fa2 are the effects for the
individual drugs(1 and 2) when used as single agents at the
concentrationsof interest.
Statistically significant differences (p
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determined to be sensitive if the respective IC50 was at orbelow
the clinically-achievable serum level of 2.5 M.Based on this
criterium, 6 cell lines were sensitive and 13cell lines were
resistant. The 6 sensitive lines included theALL line HSB-2, the
AML lines Kasumi-1 and Eol-1, MLLlines with predominate myeloid
features (Molm-13 andMolm-14), and an MLL line with predominant
lymphoidfeatures (MV4-11).
Vandetanib mediates anti-tumor activity by
multiplemechanisms
Cytoxicity assays To elucidate the mechanism(s) by
whichvandetanib exerts its anti-leukemia effect, we evaluated
thecytotoxic potential of vandetanib. Six
vandetanib-sensitiveleukemia cell lines were treated with various
concentrationsof vandetanib for 48 h and then stained with
propidiumiodide and YoPro-1-iodide in order to quantitate the
fraction of viable, apoptotic and dead cells using
flowcytometry. Vandetanib induced dose dependent cell death(Fig.
2a). However, induction of significant cell deathrequired treatment
with relatively high concentrations ofvandetanib (IC75
concentrations or higher). In all cases,reduction in cell number
was observed at lower concen-trations than those required to induce
apoptosis (Figs. 1and 2a).
Cell Cycle Analysis To investigate other potential mecha-nisms
of vandetanib-mediated anti-leukemia activity, wecharacterized the
cell cycle profile of vandetanib-sensitiveleukemia cell lines. When
treated with vandetanib, thevandetanib-sensitive cell lines
exhibited alterations in cellcycle distribution, with all lines but
Eol-1, demonstratingsignificant accumulation in the G1 phase (Fig.
2b). Thesedata demonstrate that vandetanib mediates alterations
incell cycle progression.
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100110
G2/M S G1
*
*
****
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** **
HSB-2 Eol-1 Molm-13 Molm-14 MV4-11 Kasumi-1
HSB-2 Eol-1 Molm-13 Molm-14 MV4-11 Kasumi-1
0102030405060708090
100110
% c
ells
% c
ells
ApoptoticDead
*
**
**
** **
*
*
* **
****
**
*
*
a.
b.
Fig. 2 Vandetanib inducesdose-dependent cell death
andaccumulation in G1 phase inleukemia cell lines.
Vandetanib-sensitive acute leukemia celllines were treated with
theindicated concentrations ofvandetanib for 48 h. a. Dead
andapoptotic cells were quantitatedby flow cytometric analysis
ofcells stained with YoPro-1iodide and propidium iodide.Mean values
+/ SEM derivedfrom 3 independent experimentsare shown.
Statistically-significant differences in thefraction of dead and
apoptoticcells in vandetanib-treatedcultures relative to
controlcultures were determined usingthe students paired t-test.b.
Cell cycle profiles weredetermined by flow cytometricanalysis of
permeabilized cellsstained with propidium iodide.Mean values +/ SEM
werederived from 3 independentexperiments.
Statistically-significant differences in thefraction of cells in G1
phase invandetanib-treated culturesrelative to control cultures
weredetermined using the studentspaired t-test. * p
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Selectivity of tyrosine kinase inhibition We
characterizedexpression of known vandetanib targets in a subset of
bothsensitive and resistant leukemia cell lines to investigate
thepossible biochemical mechanism of
vandetanib-mediatedanti-leukemic activity. All of the cell lines
expressed one ormore of the known targets of vandetanib
(VEGFR1,VEGFR3, PDGFR and/or RET) (Supplementary Figure 1).Only one
vandetanib-resistant line (THP-1) expressedVEGFR2 or EGFR. No
correlation was observed betweenexpression of receptors and
sensitivity to vandetanib.
Treatment with vandetanib alters expression of differentia-tion
markers on leukemia cell lines The observed accumu-lation of
leukemia cells in G1 phase of the cell cyclefollowing treatment
with vandetanib is consistent with thepossibility that vandetanib
mediates differentiation ofleukemic blasts. To investigate this
possibility, a subset ofthe vandetanib-sensitive cell lines were
treated with IC50concentrations of vandetanib and cell surface
expression ofdifferentiation markers was determined. Both ALL
andAML cell lines demonstrated alterations in expressionof
hematopoietic differentiation markers (Fig. 3). HSB-2, a T-lineage
ALL, down-regulated expression of CD15(a myeloid marker) and CD69
(a T-cell activation markerthat is also expressed on immature
myeloid precursors).Expression of CD28, which is expressed on
mature T-cells was up-regulated in response to treatment with
vandetanib. Both AML cell lines, Eol-1 and Molm-14,exhibited
down-regulation of the lymphoid markersincluding CD25 (an activated
T-cell marker, also expressedon monocytes), CD4 (a T-cell
co-receptor) on Eol-1 cells andCD2 (T-cell marker involved in
interactions with antigenpresenting cells) on Molm-14 cells.
Molm-14 also demon-strated decreased expression of CD11b, a myeloid
marker.These cell surface alterations suggest that exposure
tovandetanib promotes altered cell surface characteristics
ofleukemic blasts.
Vandetanib interacts synergistically with topoisomerase
IIinhibitors and antagonistically with an anti-metaboliteagent As
vandetanib mediates anti-leukemia activity bymultiple mechanisms,
we investigated the interaction ofvandetanib with standard
chemotherapeutic agents used inthe treatment of leukemia. A
vandetanib-resistant cell line(Nalm6), the most
vandetanib-sensitive cell line (HSB-2),and a moderately
vandetanib-sensitive cell line (Molm-13)were treated with IC50
concentrations of vandetanib and/ora standard chemotherapeutic
agent (doxorubicin, etoposide,or methotrexate), alone and in
combination for 48 h andcell death was assessed by flow cytometric
analysisfollowing staining with YoPro-1-iodide and propidiumiodide.
Interactions between agents were assessed usingthe Bliss additivity
model [40]. Upon concurrent treatmentwith vandetanib and a
topoisomerase II inhibitor (doxoru-
0
20
40
60
80
100
CD25 CD4
% P
ositi
ve C
ells
0
20
40
60
80
100
CD15 CD69 CD28
% P
ositi
ve C
ells
0
20
40
60
80
100
CD11b CD2
% P
ositi
ve C
ells
*
*
***
HSB-2
Molm-14*
*
**
Eol-1
untreatedvandetanib
Fig. 3 Treatment with vandetanib alters cell surface expression
ofdifferentiation markers on leukemia cell lines.
Vandetanib-sensitiveleukemia cell lines were treated with IC50
concentrations ofvandetanib (grey bars) or with an equal volume of
media alone (whitebars) for 48 h. Cells were then stained with
fluorochrome-conjugatedantibodies against the indicated cell
surface differentiation markers
and analyzed by flow cytometry. Mean values +/ SEM were
derivedfrom 4 to 5 independent experiments.
Statistically-significant differ-ences in the fraction of cells
expressing the relevant marker invandetanib-treated cultures
relative to control cultures were deter-mined using the students
paired t-test. * p
-
bicin or etoposide), all three cell lines exhibited
statisticallysignificant increases in the fraction of dead or
apoptoticcells relative to the predicted values for additive
interac-tions indicating a synergistic interaction (Fig. 4a and b).
Incontrast, treatment with the anti-metabolite methotrexate
incombination with vandetanib resulted in significantly lesscell
death than the predicted additive values for all threecell lines,
indicating an antagonistic interaction (Fig. 5).Synergistic and
antagonistic interactions with topoisomer-ase II inhibitors and
methotrexate respectively, were also
observed when the Molm-13 cell line was treated withhigher
concentrations (IC75) of vandetanib, indicating thatthese
interactions are not concentration dependent (Fig. 6).
Discussion
In this study, we investigated the anti-tumor effect
ofvandetanib on acute leukemia cell lines in vitro. Wedescribe the
anti-leukemic activity of this agent andelucidate mechanisms by
which it has its effect, both alone
0102030405060708090
Rel %
Dea
d Ce
lls
Vandetanib + Etoposide
***
*
b.
0102030405060708090
Rel
% D
ead
Cells
Vandetanib + Doxorubicin
*
***
a.
Nalm-6 Molm-13 HSB-2
Nalm-6 Molm-13 HSB-2
Fig. 4 Concurrent treatment with vandetanib and topoisomerase
IIinhibitors results in synergistic induction of leukemia cell
death.Cultures of the indicated leukemia cell lines were treated
with IC50concentrations (determined by MTT assay) of vandetanib
(Vand)alone, chemotherapy alone or both drugs concurrently for 48 h
asindicated. Apoptotic and dead cells were quantified by
flowcytometric analysis of cells stained with YoPro-1 iodide
andpropidium iodide dyes and normalized to untreated.
Interactionsbetween the two drugs were assessed using the Bliss
IndependenceModel. The predicted response for an additive
interaction (BlissAdditivity) is determined based on the single
agent data. The observed% dead cells for the combination therapies
are greater than the % deadcells predicted for an additive
interaction, indicating synergy. Meanvalues and standard errors
were derived from 4 to 7 independentexperiments.
Statistically-significant differences in the fraction ofapoptotic
or dead cells observed relative to the fraction predicted foran
additive interaction were determined using the students paired
t-test. * p
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and in combination with cytotoxic chemotherapies. Whilethe
effect of vandetanib on various solid tumors is welldocumented, the
role of this multi-targeted tyrosine kinaseinhibitor with effects
on VEGFR2, RET, EGFR and to alesser extent, VEGFR1, VEGFR3, and
PDGFR has beenmuch less studied in acute leukemia. In addition, it
is likelythat there are off target effects that remain undefined at
thistime. Our results demonstrate that vandetanib exhibits
anti-leukemic activity against several leukemia cell lines invitro,
including both ALL and AMLs, three of which havenot been previously
described as vandetanib-sensitive(HSB-2, Molm-13 and Molm-14). The
anti-leukemiaactivity of vandetanib does not appear to be specific
to aparticular lineage or subtype. Of note, three of the
sensitivecell lines possess MLL translocations (Molm-13,
Molm-14,and MV4-11) [42, 43]. This is of potential clinical
interest,as MLL rearranged leukemias are typically more
refractoryand associated with poorer outcomes [6, 44, 45].
Similarlya large majority of MLL-rearranged leukemias
demonstrateover-expression of fms-like tyrosine kinase 3 (flt-3),
aknown negative risk factor [4648].
The concentrations at which vandetanib exerts its anti-tumor
effect on sensitive leukemia cell lines in this studyare within the
range of concentrations expected to affect theknown selective
targets of the drug and are clinicallyachievable. However,
vandetanib sensitivity does notappear to correlate with expression
of any one specificreceptor. While this observation does not
preclude inhibi-tion of known vandetanib targets as a mechanism
ofvandetanib-mediated anti-leukemia activity, it suggests
thatreceptor expression will not be a useful clinical marker
ofvandetanib sensitivity. These data are also consistent withthe
possibility that vandetanibs anti-tumor activity occursthrough
different signaling pathways in different cell lines,by inhibition
of multiple pathways in individual cell lines,or by off-target
effects which have not yet been elucidated.The kinase inhibitor
profile of vandetanib and the broadrange of IC50 values for
sensitive cell lines are consistentwith the possibility that
inhibition of different receptorsresults in anti-leukemia activity
in different cell lines.Notably, anti-leukemia activity against
cell lines containingMLL translocations requires higher
concentrations ofvandetanib compared to other sensitive cell lines.
Thisobservation is consistent with the possibility that
anti-leukemia activity is mediated by inhibition of FLT3 in
thesecell lines as they all express internal tandem duplications
ofFLT3 and vandetanib is known to inhibit FLT3 phosphory-lation at
concentrations greater than or equal to 1 M [39]. Incontrast,
vandetanib's anti-leukemia activity against theEol-1 and HSB-2 cell
lines occurred at much lowerconcentrations and may therefore be
mediated by morespecific inhibition of known target receptors, such
asVEGFR1 and VEGFR3. Thus, it is possible that a subset
of the sensitive cell lines are dependent on VEGFRsignaling for
proliferation and/or survival. VEGFR signal-ing may also play roles
in proliferation and survival ofleukemia cell lines that are not
sensitive to vandetanibwhere other signaling pathways function
redundantly suchthat VEGFR signaling is not absolutely
required.
We also expanded on previous studies by investigatingmultiple
cellular mechanisms involved in the anti-leukemiceffect of
vandetanib. Our data indicate that the anti-tumoractivity mediated
by vandetanib occurs through multiplemechanisms and is
concentration dependent with inductionof apoptosis and cell death
occurring at relatively highconcentrations, well above the IC50
concentrations. Atlower concentrations, little to no apoptosis and
cell deathare observed, but alterations in cell cycle progression
dooccur. The increased fraction of cells in G1 phase suggestsa
delay or arrest in cell cycle progression induced byvandetanib. An
similar increase in the fraction of tumorcells in G1 phase has been
described in response tovandetanib and other EGFR antagonists and
this accumu-lation in G1 phase in solid tumor cell lines has been
shownto be due to decreased cyclin D expression [49, 50],suggesting
a mechanism by which cell cycle effects may bemediated. However,
the relevance of this mechanism inleukemia cell lines is
questionable as none of the sensitivecell lines expressed EGFR.
The observed accumulation of leukemia cells in G1phase is
consistent with the possibility that the cells areundergoing
differentiation in response to treatment withvandetanib. Indeed,
treatment with vandetanib led toalterations in the expression of
hematopoietic differentia-tion markers. These alterations generally
suggest a moremature phenotype and/or down-regulation of
aberrantlyexpressed markers. HSB-2 is an immature T-cell
leukemiacell line [51]. When treated with vandetanib, HSB-2
cellsdemonstrated increased expression of the mature T-cellmarker
CD28, decreased expression of CD69, an immatureT-cell marker and a
marker of mature T cell activation, anddecreased levels of CD15, an
aberrantly expressed myeloidmarker. These changes could be
consistent with differenti-ation towards a less proliferative, more
mature phenotype.Similarly, Eol-1 and Molm-14 exhibit
down-regulation ofCD25, CD4, and/or CD2, markers that are expressed
on Tcells and on immature myeloid precursors, again
suggestingdifferentiation to a more mature phenotype [52]. To
ourknowledge, this is the first description of a small
moleculeinhibitor with VEGFR inhibition properties altering
expres-sion of differentiation markers as a single agent.
Consistentwith our data, down-regulation of cellular VEGF levels
hasbeen associated with induction of leukemia cell differenti-ation
into functional leukemic dendritic cells in AMLpatient samples
[53]. Taken together, these data suggestthat inhibition of the VEGF
pathway can induce differen-
Invest New Drugs
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tiation of leukemia cells. These observations are
particularlyrelevant given that abrogation of differentiation is a
commonmechanism of leukemogenesis, particularly in
AMLs.Differentiating agents are also used to treat other
cancersthat demonstrate an undifferentiated phenotype, such
asneuroblastoma, and vandetanib may also be therapeutic inthis
context. Interestingly, vandetanib does mediate syner-gistic
anti-tumor activity in combination with retinoic acid,
adifferentiating agent, in neuroblastoma cell lines. In thiscase,
retinoic acid mediates increased activation of vandeta-nibs target
RETand may thereby render neuroblastoma cellsmore susceptible to
apoptosis in response to treatment withvandetanib [54]. Similarly,
vandetanib-mediated differenti-ation may play a role in increasing
sensitivity to apoptosisin response to normal cell stressors or
treatment withchemotherapy agents. Studies investigating changes
indifferentiation mediated by vandetanib or other VEGFinhibitors in
combination with other agents have not beendescribed.
Additionally, and perhaps more relevant clinically, weevaluated
interactions with other therapeutic agents that arecurrently in
clinical use. Vandetanib demonstrates synergywith topoisomerase II
inhibitors, even in cell lines in whichit does not demonstrate
single agent activity, and thus maypotentiate certain conventional
chemotherapy. These datasuggest combination strategies which may be
clinicallyrelevant particularly for AML, as more of the sensitive
lineswere AMLs and topoisomerase II inhibitors are commonlyused in
AML therapy. The synergy observed whenvandetanib is combined with a
DNA-damaging agent hasbeen previously described in solid tumors and
is thought tobe mediated in part by effects on pro-survival
pathways[49, 55, 56]. Interestingly, when vandetanib is
combinedwith oxaliplatin in human colon cancer cell lines, there
wasa marked synergistic decrease in the expression in
VEGF-Asecretion by the tumor cells [56]. This may be part
ofvandetanibs anti-leukemic effect, as the leukemia cell linesall
express high levels of VEGF as part of an autocrinestimulatory loop
(data not shown) [30, 5761]. With theaddition of chemotherapy, VEGF
secretion may be furtherimpeded compared with vandetanib alone.
Vandetanib canalso affect multi-drug resistance through inhibition
of thetransport functions of both the p-glycoprotein and
ABCG2proteins [62, 63], suggesting another rational reason
forclinical evaluation of the synergistic combinations. How-ever,
while this mechanism may play a role in some of thesynergistic
interactions we observed, this is not the onlyrole for vandetanib
as the Nalm-6 cell line does not expresspGP [64].
The antagonism seen with methotrexate is not surprising,as
methotrexate is an anti-metabolite that affects purinemetabolism,
and therefore DNA synthesis. As vandetanibinduces a G1 phase
arrest, it likely prevents leukemia cell
progression into S phase, where methotrexate exerts itseffect.
Similarly, we would expect antagonistic interactionsbetween
vandetanib and other cell cycle specific agents,including other
anti-metabolite agents affecting DNAsynthesis and therapies that
exert their effects in G2/Mphase, such as the vinca alkaloids and
taxanes. In contrast,vandetanib may be particularly effective in
combinationwith G1 phase specific agents such as L-asparaginase. It
isalso possible that a more favorable interaction betweenvandetanib
and methotrexate could be achieved if thetreatments were applied
sequentially. Although studies havedemonstrated that the sequence
in which receptor tyrosinekinase inhibitors and chemotherapeutic
agents are adminis-tered can affect the interaction between agents,
we evaluatedonly concurrent therapy because the long half-life
ofvandetanib (120 h in human serum), makes sequencing
lessclinically feasible [20, 41, 49, 50, 65].
The data presented here focus on the direct anti-tumoreffect of
vandetanib, however it is also important to notethat the bone
marrow microenvironment is not representedin our model system. It
is well known that the bone marrowstroma abundantly expresses VEGF
and its receptors. Inaddition, leukemic bone marrow has a higher
density ofmicrovessels than normal bone marrow, suggesting a
rolefor increased angiogenesis. The studies presented hereassess
autocrine inhibition but do not account for theparacrine
involvement of the endothelial cells and otherhematopoietic cells
found in the bone marrow microenvi-ronment or the contribution of
increased blood supply toleukemogenesis in this environment, both
of which can beaffected by VEGFR inhibition. As such, further in
vivo andclinical studies are warranted to investigate potential
anti-leukemic effects that are dependent upon the milieu of thebone
marrow microenvironment.
Our results demonstrate that vandetanib has direct anti-leukemic
activity in vitro. Its activity is mediated bymultiple mechanisms
and probably through different recep-tors or pathways depending on
the cell line. Furtherevaluation of the specific signaling pathways
affected byvandetanib will be necessary to elucidate the
exactbiochemical mechanisms of this drugs anti-leukemicactivity and
to facilitate development of robust pharmaco-dynamic markers for
clinical trials. This study also providespreclinical data to
facilitate rational clinical development ofa therapeutic regimen
containing vandetanib for pediatricacute leukemias, based on its
synergistic interactions withdoxorubicin and etoposide. For this
combination, thesensitivity of leukemia cells to vandetanib may not
beimportant, as treatment with vandetanib increased sensitiv-ity to
cytotoxic chemotherapies independent of whether thecell line was
sensitive or resistant to vandetanib. Takentogether, our data
support further evaluation of vandetanib,both in animal models and
in clinical trials. Further
Invest New Drugs
-
evaluation in an in vivo setting will be necessary todetermine
whether the autocrine effects we demonstratehere are clinically
relevant and to evaluate the contribution ofparacrine signaling
inhibition and anti-angiogenic effectsmediated by vandetanib, both
as a single agent and incombination with DNA damaging agents.
However, vande-tanib provides an exciting new molecularly-targeted
optionfor treatment of acute leukemia.
Acknowledgements We thank AstraZeneca Pharmaceutical for
thegenerous gift of vandetanib. We are also grateful to Lori
Gardner forlaboratory support, Gail Eckhardt for comments and
discussion, andRobert Arceci and Stephen Hunger for critical review
of thismanuscript. This work was supported by grants from the For
JulieFoundation and National Institutes of Health K12
CA086913-05,CA086913-08. MM was supported by the University of
ColoradoWilliam M. Thorkildsen Research Fellowship.
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Invest New Drugs
Vandetanib mediates anti-leukemia activity by multiple
mechanisms and interacts synergistically with DNA damaging
agentsAbstractIntroductionMethods and materialsAssessment of
anti-tumor activity
ResultsVandetanib mediates anti-tumor activity by multiple
mechanisms
DiscussionReferences
/ColorImageDict > /JPEG2000ColorACSImageDict >
/JPEG2000ColorImageDict > /AntiAliasGrayImages false
/CropGrayImages true /GrayImageMinResolution 150
/GrayImageMinResolutionPolicy /Warning /DownsampleGrayImages true
/GrayImageDownsampleType /Bicubic /GrayImageResolution 150
/GrayImageDepth -1 /GrayImageMinDownsampleDepth 2
/GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages true
/GrayImageFilter /DCTEncode /AutoFilterGrayImages true
/GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict >
/GrayImageDict > /JPEG2000GrayACSImageDict >
/JPEG2000GrayImageDict > /AntiAliasMonoImages false
/CropMonoImages true /MonoImageMinResolution 600
/MonoImageMinResolutionPolicy /Warning /DownsampleMonoImages true
/MonoImageDownsampleType /Bicubic /MonoImageResolution 600
/MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000
/EncodeMonoImages true /MonoImageFilter /CCITTFaxEncode
/MonoImageDict > /AllowPSXObjects false /CheckCompliance [ /None
] /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false
/PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000
0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true
/PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ]
/PDFXOutputIntentProfile (None) /PDFXOutputConditionIdentifier ()
/PDFXOutputCondition () /PDFXRegistryName () /PDFXTrapped
/False
/Description > /Namespace [ (Adobe) (Common) (1.0) ]
/OtherNamespaces [ > /FormElements false /GenerateStructure
false /IncludeBookmarks false /IncludeHyperlinks false
/IncludeInteractive false /IncludeLayers false /IncludeProfiles
true /MultimediaHandling /UseObjectSettings /Namespace [ (Adobe)
(CreativeSuite) (2.0) ] /PDFXOutputIntentProfileSelector /NA
/PreserveEditing false /UntaggedCMYKHandling /UseDocumentProfile
/UntaggedRGBHandling /UseDocumentProfile /UseDocumentBleed false
>> ]>> setdistillerparams> setpagedevice