Content uploaded by Nadine Darwiche
Author content
All content in this area was uploaded by Nadine Darwiche on Jun 21, 2019
Content may be subject to copyright.
ST1926, an orally active synthetic retinoid, induces apoptosis
in chronic myeloid leukemia cells and prolongs survival
in a murine model
Rihab R. Nasr
1*
, Raed A. Hmadi
2*
, Rabab M. El-Eit
1
, Ahmad N. Iskandarani
1
, Mark N. Jabbour
3
, Ghazi S. Zaatari
3
,
Francois-Xavier Mahon
4
, Claudio C.P. Pisano
5
and Nadine D. Darwiche
2
1
Department of Anatomy, Cell Biology, and Physiological Sciences, American University of Beirut, Beirut, Lebanon
2
Department of Biochemistry and Molecular Genetics, American University of Beirut, Beirut, Lebanon
3
Department of Pathology and Laboratory Medicine, American University of Beirut, Beirut, Lebanon
4
Laboratoire D’h
ematologie Et Service Des Maladies Du Sang, CHU De Bordeaux, Universit
e Victor S
egalen Bordeaux 2, INSERM U876, Bordeaux, France
5
BIOGEM, Medicinal Investigational Research, Ariano Irpino (AV), Italy
The tyrosine kinase inhibitor, imatinib, is the first line of treatment for chronic myeloid leukemia (CML) patients. Unfortu-
nately, patients develop resistance and relapse due to bcr-abl point mutations and the persistence of leukemia initiating cells
(LIC). Retinoids regulate vital biological processes such as cellular proliferation, apoptosis, and differentiation, in particular of
hematopoietic progenitor cells. The clinical usage of natural retinoids is hindered by acquired resistance and undesirable side
effects. However, bioavailable and less toxic synthetic retinoids, such as the atypical adamantyl retinoid ST1926, have been
developed and tested in cancer clinical trials. We investigated the preclinical efficacy of the synthetic retinoid ST1926 using
human CML cell lines and the murine bone marrow transduction/transplantation CML model. In vitro, ST1926 induced irrevers-
ible growth inhibition, cell cycle arrest and apoptosis through the dissipation of the mitochondrial membrane potential and
caspase activation. Furthermore, ST1926 induced DNA damage and downregulated BCR-ABL. Most importantly, oral treatment
with ST1926 significantly prolonged the longevity of primary CML mice, and reduced tumor burden. However, ST1926 did not
eradicate LIC, evident by the ability of splenocytes isolated from treated primary mice to develop CML in untreated secondary
recipients. These results support a potential therapeutic use of ST1926 in CML targeted therapy.
Chronic myeloid leukemia (CML) is a clonal hematopoietic
stem cell myeloproliferative disorder caused by the formation
of the Philadelphia chromosome, due to the reciprocal trans-
location t(9;22)(q34;q11). This reciprocal translocation fuses
the Abelson kinase gene (abl) on chromosome 9 with the
breakpoint cluster region (bcr) gene on chromosome 22.
1
The new bcr-abl oncogene codes for a constitutively active
210-kD BCR-ABL tyrosine kinase implicated in the activation
of several signal transduction pathways.
1–3
CML progression
is triphasic, where the patients progress from the indolent
chronic phase into an accelerated phase, and finally into a
blast phase.
4
Imatinib mesylate (imatinib, Gleevec), the first generation
tyrosine kinase inhibitor (TKI) revolutionized the treatment
of CML.
5
Imatinib significantly decreases CML mortality by
stopping the disease progression in chronic phase
6
through
complete hematological and cytogenetic responses,
7
thus
transforming the disease from a fatal to a chronic one.
6–10
Unfortunately, resistance to imatinib due to bcr-abl gene
amplification and point mutations in its kinase domain as
well as deregulation of ABC transporters was a setback. Sec-
ond generation TKIs, such as dasatinib and nilotinib seemed
to be promising alternatives. However, patients developed
resistance to dasatinib and nilotinib as well.
7,11,12
It was
shown later that these TKIs are not curative since most
Key words: chronic myeloid leukemia, retinoids, ST1926, apoptosis,
BCR-ABL, leukemia initiating cells
Additional Supporting Information may be found in the online
version of this article.
Authors’ Contributions: R.H., R.E.E., and A.I. performed the experi-
ments; R.N., F.X.M., C.P., and N.D. designed the study; R.H., R.N.,
and N.D. wrote the manuscript; R.H., R.N., M.J., G.Z., C.P., and
N.D. analyzed the data; R.H., R.N., F.X.M., C.P. and N.D revised the
manuscript; all authors approved the final version of the manuscript.
Conflict of Interest: The authors reported no potential conflicts of
interest.
*R.R.N. and R.A.H. are primary co-authors.
Grant sponsors: American University of Beirut Medical Practice
Plan and the American University of Beirut University Research
Board
DOI: 10.1002/ijc.29407
History: Received 12 Aug 2014; Accepted 5 Dec 2014; Online 29
Dec 2014
Correspondence to: Nadine Darwiche, Department of Biochemistry
and Molecular Genetics, American University of Beirut, PO Box 11-
0236, Beirut, Lebanon, Tel.: 1961-3-860548, Fax: 1961-1-343450,
E-mail: nd03@aub.edu.lb or Rihab Nasr, Department of Anatomy,
Cell Biology, and Physiological Sciences, American University of Bei-
rut, PO Box 11-0236, Beirut, Lebanon, Tel.: 1961-70-235118,
Fax: 1961-1-343450, E-mail: rn03@aub.edu.lb
Cancer Therapy
Int. J. Cancer: 137, 698–709 (2015) V
C2014 UICC
International Journal of Cancer
IJC
patients who discontinue therapy will rapidly progress, due
to the failure of eradicating the CML leukemia initiating cells
(LIC).
3,13,14
Retinoids are natural vitamin A derivatives or synthetic
compounds with vitamin A activities that play a role in the
regulation of cell proliferation, apoptosis and differentia-
tion,
15,16
particularly in hematopoietic progenitor cells.
17
Nat-
ural retinoids, such as all-trans retinoic acid (ATRA), are
used as anticancer agents as they block proliferation and
induce differentiation in several types of cancers.
16
Neverthe-
less, the clinical usage of natural retinoids is hindered by
undesirable side effects and drug resistance.
18,19
Conse-
quently, synthetic retinoids with increased specificity and
decreased toxicity were developed. 4-hydroxy(phenyl)retina-
mide (HPR or fenritinide)
20
and (2E)-3-[30-(1-adamantyl)-40-
hydroxy[1,10-biphenyl]-4-yl]-2-propenoic acid (ST1926)
21
represent the most promising synthetic retinoids that are cur-
rently being tested in cancer clinical trials.
ST1926, also known as Adarotene, is a novel adamantyl reti-
noid. It is a 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphtha-
lene carboxylic acid (CD437) analog
21
that is very active
against several hematological and solid malignancies and in in
vivo cancer models.
22–25
Due to its remarkable antineoplastic
activities, ST1926 has entered phase I clinical trials in ovarian
cancer.
26
ST1926 has been shown to be stable and highly bioa-
vailable.
21
Orally administered micromolar (lM) concentra-
tions are pharmacologically attainable.
22
In vitro, it was shown
that prior to apoptosis induction, ST1926 induces a G
1
/S
27
or
G
2
/M cell cycle arrest,
28
and blocks mitochondrial Ca
11
uptake ultimately leading to an increase in cytosolic Ca
11
lev-
els.
22
In addition, ST1926 exerts its anti-tumor activities
through retinoid receptor-independent and p53-independent
pathways.
21,23
Interestingly, ST1926 was shown to be a potent
inducer of genotoxic stress and DNA damage.
26
In a recent
study, Fratelli et al. demonstrated the involvement of the pro-
teasome in ST1926-induced cell death.
29
Importantly, orally administered ST1926 has been shown
to induce a significant growth inhibition in human mela-
noma, ovarian carcinoma and neuroblastoma growing in
nude mice,
21,23,30
and to be effective in acute promyelocytic
leukemia (APL), acute myeloid leukemia (AML) and adult T
cell leukemia/lymphoma (ATL) SCID mouse models.
22,25
In this report, we show that ST1926 inhibited the growth
of human CML cell lines and induced apoptosis through elic-
iting DNA damage and downregulating the BCR-ABL signal-
ing pathway and bcr-abl oncogene transcripts. Importantly,
oral uptake of ST1926 prolonged survival, decreased leukemic
cell infiltration in liver and lungs and reduced blood cell
counts in CML mice. However, ST1926 did not eradicate
CML LIC in untreated secondary recipient mice. These
results highlight the potential of ST1926 in CML targeted
therapy, in combination treatment with drugs that target
CML LIC.
Materials and Methods
Compounds
ATRA and HPR were purchased from Sigma, and reconsti-
tuted in 0.1% dimethyl sulfoxide (DMSO) under yellow light
(k>500 nm) at a concentration of 10
22
M and stored at
280C. ST1926 was kindly provided by Biogem (Ariano
Irpino, Italy) and reconstituted in 0.1% DMSO at a concen-
tration of 10
22
M and stored at 280C. Imatinib was pur-
chased from LC Laboratories (MA) and reconstituted in 0.1%
DMSO at a concentration of 25 mM and stored at 220C.
Cell lines
Human BCR-ABL positive cell lines were used: AR230, K562
and LAMA84 (LAMA); (Kindly provided by Franc¸ois-Xavier
Mahon, Lab INSERM, Bordeaux, France) and authenticated
by DNA fingerprinting using STR analysis (DSMZ, Ger-
many). Cells were maintained in RPMI 1640 medium (Lonza,
Switzerland) supplemented with 10% heat inactivated fetal
bovine serum (FBS; Gibco, CA), 2 mM L-glutamine, 25 mM
Hepes buffer, 1 mM sodium pyruvate, 20 lg/ml kanamycin
solution (Amresco, OH) and 50 U/ml penicillin-streptomycin
(Lonza), at 37C in a humidified incubator (5% CO
2
).
Cell growth and apoptosis
Cell growth was assayed using Thiazolyl Blue Tetrazolium
Bromide (MTT) dye (Sigma) uptake and measurement of
optical density at 595 nm using an ELISA microplate reader.
Cell viability was confirmed with trypan blue dye exclusion
assay. Apoptosis was assessed by terminal deoxynucleotidyl
transferase-mediated deoxyuridine triphosphate nick end-
labeling (TUNEL) assay (Roche Diagnostics, Germany)
according to manufacturer’s instructions. 10,000 cells were
collected and analyzed using FACScan flow cytometer (Bec-
ton Dickinson, CA) and CellQuest software (Becton-
Dickinson).
What’s new?
Imatinib is the first line of treatment for chronic myeloid leukemia (CML). Unfortunately, patients develop resistance and
relapse due to bcr-abl point mutations and the persistence of leukemia initiating cells (LIC). Here, the authors investigate for
the first time the antitumor properties of synthetic retinoids, namely ST1926, in CML therapy. They show that ST1926 inhibits
growth, induces apoptosis, and downregulates BCR-ABL signaling in CML cells. Oral treatment reduces tumor burden and pro-
longs survival of CML mice. However, ST1926 does not affect LIC activity. These results highlight the potential of ST1926 in
CML targeted therapy, in combination with CML LIC-targeting drugs.
Cancer Therapy
Nasr et al.699
Int. J. Cancer: 137, 698–709 (2015) V
C2014 UICC
Cell cycle analysis
Control and treated cells were collected, washed with
phosphate-buffered saline (PBS) and fixed in 80% ethanol.
Fixed cells were stained with propidium-iodide (PI; 50 lg/
ml) (Sigma), after incubation with 50 units RNAse A (Roche
Diagnostics). 10,000 cells were collected and analyzed using
flow cytometry as described.
31
Mitochondrial membrane potential
Quantification of mitochondrial membrane potential was
determined by Rhodamine (R123) retention (Sigma). Control
and treated cells were collected, washed with PBS and loaded
with 5 lM R123 for 30 min at 37C. Labeled cells were
washed and analyzed using flow cytometry.
Caspase activity
Involvement of caspases in ST1926-induced cell death was
assessed using z-VAD-fmk, a pan-caspase inhibitor. Cells
were pre-treated with 100 lM z-VAD-fmk (MP Biomedicals,
CA) for 2 hr, and then treated with ST1926 up to 48 hr. Cell
growth was determined after 24 and 48 hr of treatment.
Reactive oxygen species measurement
Generation of reactive oxygen species (ROS) was measured by
a fluorometric method that allows detection of intracellular
peroxides by reaction with the membrane-permeant
H
2
DCFDA (Invitrogen, CA), which will be converted inside
the cell to the fluorescent 2,7-dichlorofluorescein after oxida-
tion. Control and treated cells were collected, washed with PBS
and loaded with 50 lMH
2
DCFDA for 20 min at 37C. Labeled
cells were washed and analyzed using flow cytometry.
Immunoblot analysis
Total cellular protein lysates [0.25 mM Tris-HCL (pH 57.4),
20% b-mercaptoethanol and 5% sodium dodecyl sulfate
(SDS)] were prepared, quantified and separated by SDS-
PAGE electrophoresis. Subsequently, separated proteins were
transferred into nitrocellulose membranes. After blocking
with 5% skimmed milk in TBS (50 mM Tris-HCL and
150 mM NaCl), membranes were incubated overnight with
primary antibodies at 4C. Secondary antibodies were added
for 2 hr at room temperature, and the antibody-protein com-
plex was visualized by enhanced chemiluminescence (ECL)
using the ECL system (Santa Cruz, TX). Antibodies: p-BCR-
ABL (2865), BCR-ABL (2862), p-STAT-5 (9359), STAT-5
(9358), p-Crk-L (3181), Crk-L (3182), PARP (9542) and c-
H2AX (2577) were purchased from Cell Signaling, MA.
GAPDH (MAB5476) was purchased from Abnova, (Heidel-
berg, Germany).
Real-time PCR
Bcr-abl transcript levels were assessed by real-time PCR in
control and treated cells. Total cellular RNA was extracted
using RNeasy Mini Kit (Qiagen, Netherlands) and reverse
transcribed into cDNA using superscript II reverse transcrip-
tase (Invitrogen). Real-time PCR was performed using CFX96
Real-Time PCR machine (Bio-Rad, CA). We used the
following DNA primers for human abl (forward: 50-
AGGACAGCTCTTGATTTG-30; reverse: 50-GACAGATGG
AAAGGACATG-30). Abl results were normalized to actin
transcript levels. Bcr-abl DNA in splenocytes was quantified
by real-time PCR for human abl using the above mentioned
primers and the following probe (probe: 50-AAACAGG
GTGCTAAAGCCAAC-30). Murine abl gene was used as
internal control (forward: 50-CTGCACTTGAAACTTCTC-30;
reverse: 50-TACCGTCATTGAGCTATTC-30; probe: 50-
CACAGCCAGTCTCAGTTCAGG-30). Analysis was per-
formed using the 2-DCT method.
Murine bone marrow retroviral transduction/
transplantation model of CML
Experiments were approved by the Institutional Animal Care
and use Committee of the American University of Beirut. The
murine bone marrow retroviral transduction/transplantation
model was used as described.
32
Briefly, Plat-E cells were trans-
fected by the retroviral expressing vector MIGp210
BCR-ABL
.
Then, retroviral supernatants were used to infect murine bone
marrow cells collected from Balb/c donor mice (6- to 8-weeks
old) that were given intravenous injections of 5-fluorouracil 4
days prior to bone marrow isolation. Spinoculation of cells was
done twice in the presence of IL-3, IL-6 and stem cell factor
(Abcys SA, France), and transduced cells were subsequently
injected intravenously into lethally irradiated syngeneic recipi-
ent mice (female Balb/c 6- to 8-weeks old). Seven days post-
transplantation, mice were randomly assigned into vehicle,
imatinib, or ST1926 groups. Imatinib was administered orally
at 50 mg/kg every AM and 100 mg/kg every PM as previously
described.
33
This treatment regimen assures continuous inhibi-
tion of BCR-ABL kinase activity. ST1926 was administered
orally at 15 mg/kg every AM. In secondary transplantation
experiments, 10 310
6
spleen cells isolated from control and
treated primary mice were injected intravenously into suble-
thaly irradiated syngeneic mice. Secondary recipients were left
untreated and monitored for survival.
Spleens, livers and lungs obtained from control and
treated mice were fixed in 10% neutral buffered formalin,
paraffin embedded, sectioned, stained with hematoxylin and
eosin, and examined by light microscopy. Scoring of leukemic
cell infiltrate was done accordingly: Score 1 represents 1–
25%, Score 2 represents 26–50%, Score 3 represents 51–75%
and Score 4 represents 76–100%. White blood cell (WBC)
and neutrophil counts were determined using routine clinical
laboratory techniques.
Statistical analysis
All experiments were performed at least three times.
Reported data represent mean 6standard error (SE), unless
mentioned otherwise. Statistical analyses used were Student’s
ttest and one-way analysis of variance (ANOVA) test.
Cancer Therapy
700 ST1926 induces apoptosis in CML cells and prolongs survival in mice
Int. J. Cancer: 137, 698–709 (2015) V
C2014 UICC
Kaplan–Meier method with statistical significance assessed
using the log rank test was used for survival analysis; differ-
ences were considered significant only when p<0.05.
Results
ST1926 induces growth inhibition in CML cell lines at
pharmacologically achievable concentrations
Three well-characterized human blast crisis CML cell lines,
AR230, K562 and LAMA, were used to test for the effects of
the natural retinoid ATRA, and the synthetic retinoids, HPR
and ST1926. Achievable pharmacological concentrations of
ATRA (1 lM) had a minimal effect on the growth of all
CML cells (Fig. 1a). Similarly, 1 lM HPR did not have any
effect on AR230 and K562 and had only minimal effect on
LAMA cells after 3 days of treatment (Fig. 1b). It has been
shown previously that HPR concentrations up to 3 lM can
be maintained in the plasma of patients for prolonged time
with minimal side effects; while 10 lM levels did not seem to
be pharmacologically attainable.
34
Interestingly, pharmacolog-
ically achievable 1 lM concentrations of ST1926 resulted in
time-dependent growth inhibition of all tested CML cells, evi-
dent after 1 day of treatment and completed after 3 days
(Fig. 1c). AR230 and LAMA cells were sensitive to ST1926,
where 1 lM ST1926 resulted in approximate 50% growth
inhibition (IC50) after 1 and 2 days of treatment, respec-
tively. However, K562 cells were relatively less sensitive to
ST1926, where IC50 values were achieved at 1 lM concentra-
tions after 3 days of treatment (Fig. 1c). Similar trends were
observed when cell viability was assessed through trypan blue
dye exclusion assay (data not shown). Furthermore, ST1926-
induced growth inhibition in 24 hr treated CML cells per-
sisted even after 72 hr of drug removal (Supporting Informa-
tion Fig. 1) indicating an irreversible mode of growth
suppression. To decipher the mechanism of ST1926-induced
growth inhibition and cell death of CML cells, we selected 1
lM ST1926 concentrations for AR230 cells, and 5 lM
Figure 1. CML cells are more sensitive to ST1926 than to ATRA and HPR. Effects of ATRA (a), HPR (b) and ST1926 (c) on the growth of three
human CML cell lines, AR230, K562 and LAMA. All CML cells were seeded at a concentration of 2 310
5
cell/ml in 96-well plates and
treated with 0.1% DMSO or the indicated concentrations of ATRA, HPR and ST1926 up to 3 days. Cell growth was assayed in quadruplicate
wells using the MTT cell proliferation assay. Results are expressed as percentage of control (0.1% DMSO) and represent an average of at
least three independent experiments 6SE.
Cancer Therapy
Nasr et al.701
Int. J. Cancer: 137, 698–709 (2015) V
C2014 UICC
ST1926 concentrations for K562 and LAMA cells, as they are
less sensitive at the selected time points compared to AR230.
ST1926 treatment causes cell cycle arrest and apoptosis
in CML cells
The effects of ST1926 on cell cycle progression were investi-
gated using flow cytometric analysis of DNA content. ST1926
treatment induced pronounced changes in the cell cycle dis-
tribution of all treated CML cells (Fig. 2a). Treatment of
AR230 cells with 1 lM ST1926 resulted in preG
1
accumula-
tion of cells, with levels reaching 56% of control after 48 hr.
Treatment of K562 and LAMA cells with 5 lM ST1926
resulted in 12% and 33% preG
1
accumulation after 48 hr of
treatment respectively, demonstrating again that K562 and
LAMA cells are less sensitive to ST1926. Furthermore,
ST1926 induced a G
1
cell cycle arrest after 24 hr of treatment
Figure 2. Cell cycle arrest and apoptosis induction by ST1926 in CML cells. AR230 cells were treated with 1 lM ST1926, whereas K562
and LAMA cells were treated with 5 lM ST1926 at the indicated time points. Control cells were treated with 0.1% DMSO. (a) cell cycle anal-
ysis of CML cells treated with 0.1% DMSO or ST1926. The preG
1
population presumably represents apoptotic cells. Cycling cells, the sum
of S1G
2
/M, are a percentage of nonapoptotic cells. Results represent the mean of three independent experiments (6SE). (b) TUNEL analy-
sis of 1 lM treated-AR230 cells and 5 lM treated-K562 and -LAMA cells up to 72 hr. Results represent the mean of three independent
experiments (6SE). Significance from ST1926 treatment is indicated by ** (p<0.01) and *** (p<0.001).
Cancer Therapy
702 ST1926 induces apoptosis in CML cells and prolongs survival in mice
Int. J. Cancer: 137, 698–709 (2015) V
C2014 UICC
Figure 3. ST1926 treatment of CML cells results in loss of mitochondrial membrane potential and caspase activation. AR230 cells were
treated with 1 lM ST1926, whereas K562 and LAMA were treated with 5 lM ST1926. Control cells were treated with 0.1% DMSO. (a) Effect
of ST1926 on mitochondrial membrane potential. CML cells were treated with 0.1% DMSO or ST1926 up to 48 hr and stained with
Rhodamine-123 as described in materials and methods. Histograms represent analysis of Rhodamine-123 fluorescence (X-axis) over cell
counts (Y-axis), and panels represent overlay of ST1296-treated cells (gray) over control cells (black). Results are representative of three
independent experiments. (b) ST1926 treatment of CML cells causes PARP cleavage. AR230, K562 and LAMA cells were treated with 0.1%
DMSO or ST1926 (ST) up to 48 hr. Total SDS protein lysates (50 lg/lane) were prepared and immunoblotted against PARP. Arrow indicates
cleaved PARP. Results are representative of three independent experiments. (c) Effects of the pan-caspase inhibitor z-VAD on ST1926-
induced growth inhibition. AR230 and LAMA cells were pretreated with z-VAD for 2 hr followed by treatment with ST1926 at the indicated
concentrations up to 48 hr. Cell growth was assayed in quadruplicate wells using the MTT cell proliferation assay. Results are expressed as
percentage of control (6SD) and are representative of three independent experiments. Significance from ST1926 treatment is indicated by
*(p<0.05). (d) effects of the pan-caspase inhibitor z-VAD on PARP cleavage induced by ST1926. AR230 and LAMA cells were pretreated
with z-VAD for 2 hr followed by treatment with ST1926 (ST) up to 24 hr. Total SDS protein lysates (50 lg/lane) were prepared and immuno-
blotted against PARP. Arrow indicates cleaved PARP.
Cancer Therapy
Nasr et al.703
Int. J. Cancer: 137, 698–709 (2015) V
C2014 UICC
in all tested CML cells. In AR230 cells, 1 lM ST1926 resulted
in a decrease in the percentage of cycling cells, S1G
2
/M,
from 53% in untreated cells to 27% after 24 hr of treatment.
In K562 and LAMA cells, 5 lM ST1926 resulted in a
decrease of cycling cells, S1G
2
/M, from 60% to 46% and
from 42% to 23%, respectively.
ST1926 treatment induced a pronounced increase in the
presumably apoptotic preG
1
region which was further con-
firmed using the TUNEL assay (Fig. 2b). Treatment of
AR230 cells with 1 lM ST1926 resulted in an increase in the
percentage of TUNEL-positive cells from 6% to 71% after 72
hr of treatment. This increase was observed as early as 24 hr
post-treatment (data not shown). Apoptosis induction by
ST1926 was also detected, but to a lower extent, in 5 lM-
treated K562 and LAMA cells, resulting in an increase in the
percentage of TUNEL-positive cells up to 16 and 43%,
respectively, 72 hr post-treatment (Fig. 2b).
ST1926-induced apoptosis involves dissipation of
mitochondrial membrane potential and caspase activation
ST1926-induced apoptosis in CML cells was associated with a
time-dependent dissipation of the mitochondrial membrane
potential, evident by the decrease in Rhodamine-123 fluores-
cence, and correlated with the sensitivity to ST1926 (Fig. 3a).
In fact, the loss of mitochondrial membrane potential was
detected in AR230 and LAMA cells after 24 and 48 hr of treat-
ment, respectively. However, K562 cells did not show any loss
of mitochondrial membrane potential up to 48 hr of treatment
(Fig. 3a). The effect of the dissipation of the mitochondrial
membrane potential in ST1926-treated CML cells on the cas-
pase cascade was studied. ST1926-induced apoptosis was asso-
ciated with the activation of caspases as evident by PARP
cleavage (113 kDa) into its death-associated fragment (89
kDa). This was detected as early as 24 hr post-treatment in
AR230 and LAMA cells, but to a lesser extent in K562 cells
(Fig. 3b). ST1926-induced growth inhibition of AR230 cells
was partially but significantly reversed by the pan-caspase
inhibitor z-VAD after 24 and 48 hr of treatment (Fig. 3c). Con-
versely, no significant change of ST1926-induced growth inhi-
bition by z-VAD was detected in LAMA cells (Fig. 3c) despite
the complete reversal of PARP cleavage (Fig. 3d).
ST1926 induces DNA damage and downregulates
BCR-ABL signaling
Several recent studies reported that ST1926 treatment induces
DNA damage
26,29
and that ST1926-induced resistance results
Figure 4. ST1926 induces DNA damage and BCR-ABL downregulation in CML cells. AR230 cells were treated with 1 lM ST1926, whereas
K562 and LAMA cells were treated with 5 lM ST1926. Control cells were treated with 0.1% DMSO. (a) ST1926 causes DNA damage. CML
cells were treated with 0.1% DMSO or ST1926 up to 24 hr. Total SDS protein lysates (50 lg/lane) were prepared and immunoblotted
against c-H2AX antibody. All blots were re-probed with GAPDH antibody to ensure equal protein loading. Similar trends were observed in
three independent experiments. (b) effects of ST1926 on BCR-ABL signaling pathway. CML cells were treated with 0.1% DMSO or ST1926
up to 48 hr. Total SDS protein lysates (50 lg/lane) were prepared and immunoblotted against the specified antibodies. All blots were
reprobed with GAPDH antibody to ensure equal protein loading. Similar trends were observed in three independent experiments. (c) Effects
of ST1926 on bcr-abl transcript levels. Total cellular RNA was extracted and reverse-transcribed into cDNA. Measurements of bcr-abl tran-
scripts were relatively normalized to control cells, and were performed using the DDC
T
method. Significance from ST1926 treatment is indi-
cated by ** (p<0.01) and *** (p<0.001).
Cancer Therapy
704 ST1926 induces apoptosis in CML cells and prolongs survival in mice
Int. J. Cancer: 137, 698–709 (2015) V
C2014 UICC
in its loss.
35
Protein expression levels of gamma-H2AX (c-
H2AX), a sensitive marker for DNA damage, were measured
to investigate whether ST1926 induces DNA damage in CML
cells. Interestingly, ST1926 induced a major upregulation in
the expression levels of c-H2AX in all tested CML cells (Fig.
4a), clearly indicating that ST1926 is a potent inducer of
DNA damage. DNA damage induction in AR230 cells was
also confirmed in ST1926-treated AR230 cells by the single-
cell electrophoresis COMET assay (Supporting Information
Fig. 2). LAMA cells showed some levels of endogenous c-
H2AX (Fig. 4a), which may be due to several factors includ-
ing genetic instability and damaged telomeres.
36
To investigate whether ST1926-induced cell death of CML
cells is mediated by oxidative stress, ROS levels were meas-
ured. As has been observed in neuroblastoma cells,
30
ST1926
did not increase ROS levels in all tested CML cells following
12 and 24 hr treatment (Supporting Information Fig. 3).
The bcr-abl oncogene is the molecular signature of CML,
hence the effects of ST1926 treatment on the BCR-ABL sig-
naling pathway were studied. Western blot analysis showed a
time-dependent downregulation in the expression levels of
total BCR-ABL, and p-BCR-ABL, the active form of BCR-
ABL (Fig. 4b). This reduction was detected in all tested CML
cells, but was most pronounced in the most sensitive AR230
cells as early as 24 hr and was maximal after 48 hr of treat-
ment (Fig. 4b; densitometry analysis in Supporting Informa-
tion Fig. 4). We then tested the effect of ST1926 on
downstream mediators of BCR-ABL such as STAT5 and Crk-
L. ST1926 treatment also induced a downregulation in the
protein expression levels of STAT-5 and its phosphorylated
Figure 5. Oral treatment with ST1926 prolongs survival and reduces tumor burden in murine CML model. (a) Seven days post-transplanta-
tion, CML mice were left untreated or treated with ST1926 or imatinib. Some mice were monitored for survival while others were sacrificed
at the end of treatment. (b) Kaplan–meier analysis of survival curves of CML mice. A significant prolongation in survival was observed in
mice treated with ST1926 or imatinib (p<0.05). (c) Effect of treatment on WBC and neutrophil counts. Error bars show SD. Significance
from untreated is indicated by * (p<0.05). (d) Scores of organ infiltration and histopathology analysis of the spleen, liver and lungs from
untreated or treated CML mice. Left panel: scores of organ infiltration. Score 1 represents 1–25% leukemic cell infiltrate, Score 2: 26–50%,
Score 3: 51–75% and Score 4: 76–100%. Results represent average 6SD. Right panel: representative pictures of histopathology analysis.
Cancer Therapy
Nasr et al.705
Int. J. Cancer: 137, 698–709 (2015) V
C2014 UICC
active form, p-STAT-5 (Fig. 4b). Surprisingly in all tested
CML cells, ST1926 treatment did not affect the expression
levels of p-Crk-L, the phosphorylated active form of Crk-L
(Fig. 4b). To test whether ST1926-induced reduction in BCR-
ABL protein levels was proteasome-dependent, AR230 cells
were treated with 1 lM ST1926 in the presence or absence of
the proteasome inhibitor PS-341. The data clearly shows that
inhibition of the proteasome did not rescue cells from
ST1926-induced cell death in AR230 cells (Supporting Infor-
mation Fig. 5a). Moreover, ST1926-induced reduction of
BCR-ABL protein levels was not reversed by PS-341 (Sup-
porting Information Fig. 5b). Therefore, ST1926-induced cell
death and BCR-ABL reduction are proteasome-independent
in CML cells.
The observed downregulation in the expression levels of
the BCR-ABL oncoprotein may be partly due to a reduction
in the bcr-abl oncogene transcripts. In fact, real-time PCR
showed that ST1926 treatment significantly reduced the bcr-
abl mRNA levels after 24 hr of treatment (Fig. 4c).
Oral treatment with ST1926 prolongs survival and reduces
tumor burden in CML mice
Based on the effective anti-tumor properties of ST1926 in
CML cells, the effects of ST1926 in CML mice were investi-
gated. Toward this aim, a well-characterized murine trans-
duction/transplantation model of CML that successfully
recapitulates human CML
37
was used. BCR-ABL/GFP virally
transduced bone marrow cells from 5-fluorouracil-treated
donor Balb/c mice were injected into lethally irradiated syn-
geneic primary mice. Within few days, all primary mice
developed CML, which was characterized by splenomegaly,
hepatomegaly, leukocytosis and infiltration of the spleen, liver
and lungs by leukemic cells. All untreated CML mice died 3
to 4 weeks post-injection.
CML mice were randomly distributed 7-days post-injec-
tion between three groups: untreated controls, treated with
imatinib or with ST1926 (Fig. 5a). Based on the survival
plots, ST1926 and imatinib treatments significantly prolonged
the longevity of CML mice when compared to untreated con-
trol mice (p50.013 and p50.014, respectively; Fig. 5b). Fur-
thermore, ST1926 treatment significantly reduced total WBC
and neutrophil counts (p<0.05; Fig. 5c), but did not have
any effect on spleen or liver weight (data not shown). Histo-
pathology analysis of spleen, liver and lungs in ST1926-
treated animals revealed a decrease in the leukemic cell infil-
tration mainly in the livers and lungs (Fig. 5d). In fact, leuke-
mic cell infiltration in liver decreased significantly with
imatinib treatment (score 52.4), and reached complete
Figure 6. ST1926 does not reduce LIC activity in CML mice. (a) Experimental design: serial transplantation of splenocytes (10 310
6
cells)
from untreated and treated primary CML mice into untreated syngeneic secondary recipients. (b) Percentage of human abl in transplanted
splenocytes from primary CML mice. Error bars represent SD. (c) Kaplan–meier analysis of survival curves of secondary CML mice.
Cancer Therapy
706 ST1926 induces apoptosis in CML cells and prolongs survival in mice
Int. J. Cancer: 137, 698–709 (2015) V
C2014 UICC
resolution, except for the presence of erythroid precursors, in
ST1926-treated group (score 51.9). Strikingly, on ST1926
treatment the lung infiltrate was reduced to a score of 0.6
versus a score of 3.5 in untreated controls and 2.0 in
imatinib-treated group (Fig. 5d). ST1926 treatment also
induced complete resolution of nodule formation and any
associated hemorrhage (Fig. 5d). Importantly, normal Balb/c
mice receiving 30 mg/kg ST1926 every other day for 3 weeks
did not show any signs of toxicity (data not shown).
ST1926 does not reduce CML LIC activity
To investigate the effects of ST1926 on LIC eradication, serial
transplantation experiments were performed. Spleen cells iso-
lated from untreated or treated (ST1926 or imatinib) primary
mice were injected into untreated syngeneic secondary mice
to reconstitute sublethally irradiated Balb/c recipients (Fig.
6a). Secondary recipients received similar amounts of leuke-
mic cells from untreated and treated primary mice, as evident
by the amounts of human abl in the inoculated splenocytes
(Fig. 6b). All reconstituted animals died. Some developed
CML-like myeloproliferative disease with the characteristic
splenomegaly and hepatomegaly. Others showed a lymphatic
malignancy with mild splenomegaly and enlarged lymph
nodes reminiscent of the acute lymphoblastic leukemia in
secondary transplantation in this CML mouse model.
37
As expected, secondary recipients that received spleen cells
from imatinib-treated primary mice died before the control
mice (Fig. 6c) due to the ability of imatinib to enrich CML
LIC.
32,38
Similar results were obtained with ST1926 treat-
ment, suggesting the inability of ST1926 to target CML LIC.
Therefore, even though ST1926 treatment prolongs the lon-
gevity of CML mice and reduces tumor burden, it does not
eradicate LIC, indicating the need of combination therapy
with drugs that target LIC for CML cure.
Discussion
In this study, ST1926 has been found to inhibit the prolifera-
tion of CML cell lines accompanied by induction of cell cycle
arrest and apoptosis. To our knowledge this is the first report
that investigated the antitumor properties of synthetic retinoids
in CML treatment. Interestingly, cell growth assays have shown
that ST1926, at pharmacologically achievable lM concentra-
tions, is at least five-fold more active than the synthetic retinoid
HPR in these ATRA-resistant CML cells. Furthermore, inhibi-
ting the retinoid-receptor signaling pathway using the general
RAR antagonist did not rescue the cells from ST1926-induced
cell death (data not shown). This finding is in agreement with
previously reported studies that ST1926 acts through retinoid-
receptor independent mechanisms.
21
ST1926 treatment of CML cells induced early G
1
arrest and
accumulation of apoptotic cells with varying sensitivities in the
three cell lines used. Cell cycle arrest and cell death were more
pronounced in AR230 and LAMA cells compared to K562
cells, which harbor a mutated p53. These results are in agree-
ment with previous reports demonstrating that the absence of
functional p53 might decrease the responsiveness of cancer
cells to synthetic retinoids.
30
However, other reports indicated
that ST1926 can function in a p53-independent manner.
21,23
Other factors that might explain this variability include cyto-
solic Ca
11
homeostasis and mobilization, which has been
shown to play a role in ST1926-induced cell death of AML
cells
22
and may play a role in CML cells.
Interestingly, BCR-ABL oncoprotein downregulation was
due to a reduction in the bcr-abl oncogene transcript levels,
through yet an unknown mechanism. Recently, the protea-
some was implicated in ST1926 mode of action in AML
cells.
29
Based on our results, it is unlikely that the proteasome
is involved in the observed ST1926-induced cell death of
CML cells and in the degradation of BCR-ABL protein levels.
ST1926 was previously shown to be a potent DNA dam-
aging agent in different hematological malignancies and solid
tumors.
25,26,29,35
The mechanism of genotoxic stress induced
by ST1926 is still unclear. Interestingly, our results clearly
indicate that while ST1926-induced cell death does not
involve the generation of ROS, it was accompanied by a mas-
sive induction of DNA damage. Further studies are needed to
decipher the nature and mechanism of ST1926-induced DNA
damage which seems to be irreversible.
The importance of ST1926 in CML targeted therapy is high-
lighted after showing a reduction in the BCR-ABL protein and
its downstream mediator STAT-5. Crk-L is an adapter protein
and has been shown to play a pivotal role in several signaling
pathways involved in cell survival, motility and invasion.
39,40
Although Crk-L is a known target of BCR-ABL, ST1926-
induced BCR-ABL downregulation did not drastically reduce
its phosphorylation levels in CML-treated cells. This may be
due to the fact that Crk-L may be phosphorylated by other
tyrosine kinases than BCR-ABL or to the difference in the rela-
tive pools of phosphorylated versus unphosphorylated forms of
these two substrates in CML cells.
41
Most importantly, using the murine bone marrow retroviral
transduction/transplantation model of CML that successfully
recapitulates CML in humans, orally administered ST1926 was
shown to prolong the survival of primary CML mice and to
significantly reduce tumor burden and leukemic cell infiltra-
tion. Several reports have indicated the promising role of
ST1926 in several solid and liquid tumor models, but this is the
first report to investigate the role of ST1926 in the eradication
of cancer stem cells, particularly LIC. However, similarly to
imatinib, ST1926 did not succeed in targeting LIC activity.
14
Indeed, stem cells in general, and cancer stem cells particularly,
demonstrate higher DNA repair activity compared to differen-
tiated cells.
42
A plausible explanation of the unresponsiveness
of LIC to ST1926 treatment might be due to their ability to
evade DNA damage and/or upregulate DNA repair pathways,
thus favoring antiapoptotic conditions. Moreover, although
CML is initiated and driven by the oncogene bcr-abl, CML ini-
tiating cells are not BCR-ABL addicted and their survival is
BCR-ABL kinase independent.
43,44
Cancer Therapy
Nasr et al.707
Int. J. Cancer: 137, 698–709 (2015) V
C2014 UICC
Novel anticancer therapies have shown the promise of
combination strategies in the successful treatment of cancer,
due to their targeting of tumor cells as well as cancer stem
cells.
45,46
In fact, we have recently shown that the combina-
tion of arsenic trioxide and interferon alpha reduces the clo-
nogenicity of primary CML cells and targets the LIC activity
in CML mice.
32
It is noteworthy to mention that the combi-
nation of ST1926 with imatinib showed additive growth inhi-
bition in CML cell lines; however, it caused severe toxicity in
CML mice (data not shown). Interestingly, recent reports
have shown the feasibility of selectively targeting LIC in CML
without affecting normal hematopoietic stem cells.
47,48
Overall, the efficacy and tolerability of the novel and
orally active ST1926 strongly highlight its promise in CML
targeted therapy. Further evaluations are needed to determine
combination treatments with ST1926 that effectively target
both tumor cells and LIC for a CML cure.
Acknowledgments
The authors would like to thank Drs. Fadia Homeidan and Samira Kaissi for
their critical editing of the manuscript. This work was supported by the
American University of Beirut Medical Practice Plan, and the American
University of Beirut University Research Board. We are grateful to the per-
sonnel of the Core Laboratory Facilities and the Medical Laboratories and
the Animal Care Facility of the American University of Beirut.
References
1. Rumpold H, Webersinke G. Molecular pathoge-
nesis of Philadelphia-positive chronic myeloid
Leukemia-is it all BCR-ABL?. Curr Cancer Drug
Targets 2011;10:3–19.
2. An X, Tiwari AK, Sun Y, et al. BCR-ABL tyrosine
kinase inhibitors in the treatment of philadelphia
chromosome positive chronic myeloid leukemia:
a review. Leuk Res 2010;10:1255–68.
3. Stuart S, Minami Y, Wang JY. The CML stem
cell. Cell Cycle 2009;9:1338–43.
4. Gaiger A, Henn T, Horth E, et al. Increase of
bcr-abl chimeric mRNA expression in tumor cells
of patients with chronic myeloid leukemia pre-
cedes disease progression. Blood 1995;6:
2371–8.
5. Melo J, Hughes T,, Apperley J. Chronic myeloid
Leukemia. Hematology Am Soc Hematol Educ
Program 2003;132–52.
6. Rea D, Rousselot P, Guilhot J, et al. Curing
chronic myeloid leukemia. Curr Hematol Malig
2012;7:103–8.
7. Vigil C, Griffiths EA, Wang ES, Wetzler M.
Interpretation of cytogenetic and molecular
results in patients treated for CML. Blood Rev
2011;5:139–46.
8. Hochhaus A. Educational session: managing
chronic myeloid leukemia as a chronic disease.
Hematology 2011;11:128–35.
9. Rousselot P, Charbonnier A, Cony-Makhoul P,
et al. Loss of major molecular response as a trig-
ger for restarting tyrosine kinase inhibitor ther-
apy in patients with chronic-phase chronic
myelogenous leukemia who have stopped imati-
nib after durable undetectable disease. J Clin
Oncol 2014;5:424–30.
10. Mahon FX, Etienne G. Deep molecular response
in chronic myeloid leukemia: the new goal of
therapy? Clin Cancer Res 2014;2:310–22.
11. Kantarjian H, Baccarani M, Jabbour E, et al. Sec-
ond-generation tyrosine kinase inhibitors: the
future of frontline CML therapy. Clin Cancer Res
2011;17:1674–83.
12. DiPersio JF, Ramirez P. Therapy options in ima-
tinib failures. Oncologist 2008;4:424–34.
13. Chen Y, Peng C, Sullivan C, et al. Critical molec-
ular pathways in cancer stem cells of chronic
myeloid leukemia. Leukemia 2010;9:
1545–54.
14. Zhang H, Li S. Molecular mechanisms for sur-
vival regulation of chronic myeloid leukemia
stem cells. Protein Cell 2013;3:186–96.
15. Lippman SM, Lotan R. Advances in the develop-
ment of retinoids as chemopreventive agents.
J Nutr 2000;130:479S
16. Tang X, Gudas L. Retinoids, retinoic acid recep-
tors, and cancer. Annu Rev Pathol 2011;6:345–64.
17. Zauli G, Visani G, Vitale M. All trans-retinoic
acid shows multiple effects on the survival, prolif-
eration and differentiation of human fetal CD41
haemopoietic progenitor cells. Br J Hematol 1995;
90:274–82.
18. de The H. Altered retinoic acid receptors. FASEB
1996;9:955–60.
19. Darwiche N, Scita G, Jones C, et al. Loss of reti-
noic acid receptors in mouse skin and skin
tumors is associated with activation of the ras
Ha
oncogene and high risk for premalignant progres-
sion. Cancer Res 1996;56:4942–59.
20. Gander R, Gurney J. All-trans retinoic acid esters
and amides. Chem Abstr 1978;88:89892
21. Cincinelli R, Dallavalle S, Merlini L, et al. A novel
atypical retinoid endowed with proapoptotic and
antitumor activity. J Med Chem 2003;46:
909–12.
22. Garattini E, Parrella E, Diomede L, et al. ST1926,
a novel and orally active retinoid-related molecule
inducing apoptosis in myeloid leukemia cells:
modulation of intracellular calcium homeostasis.
Blood 2004;103:194–207.
23. Pisano C, Vesci L, Foder
a R, et al. Antitumor
activity of the combination of synthetic retinoid
ST1926 and cisplatin in ovarian carcinoma mod-
els. Ann Oncol 2007;9:1500–5.
24. Zuco V, Benedetti V, De Cesare M, Zunino F.
Sensitization of ovarian carcinoma cells to the
atypical retinoid ST1926 by the histone deacety-
lase inhibitor, RC307: enhanced DNA damage
response. Int J Cancer 2010;5:1246–55.
25. El Hajj H, Khalil B, Ghandour B, et al. Pre-clini-
cal efficacy of the synthetic retinoid ST1926 for
treating adult T cell leukemia/lymphoma. Blood
2014;124:2072–80.
26. Valli C, Paroni G, Di Francesco AM, et al. Atypi-
cal retinoids ST1926 and CD437 are S-phase-
specific agents causing DNA double-strand
breaks: significance for the cytotoxic and antipro-
liferative activity. Mol Cancer Ther 2008;9:2941–
54.
27. Pisano C, Merlini L, Penco S, et al. Cellular and
pharmacological bases of the antitumor activity
of a novel adamantyl retinoid, ST1926.
J Chemother 2004;4:74–6.
28. Parrella E, Giann
ı M, Fratelli M, et al. Antitumor
activity of the retinoid-related molecules (E)-3-
(40hydroxy-30- adamantylbiphenyl-4-yl)acrylic
acid (ST1926) and 6-[3-(1-adamantyl)-4-hydroxy-
phenyl] -2-naphthalene carboxylic acid (CD437)
in F9 teratocarcinoma: role of retinoic acid recep-
tor gamma and retinoid-independent pathways.
Mol Pharmacol 2006;3:909–24.
29. Fratelli M, Fisher JN, Paroni G, et al. New
insights into the molecular mechanisms underly-
ing sensitivity/resistance to the atypical retinoid
ST1926 in acute myeloid leukaemia cells: the role
of histone H2A.Z, cAMP-dependent protein
kinase a and the proteasome. Eur J Cancer 2013;
6:1491–500.
30. Di Francesco AM, Meco D, Torella AR, et al.
The novel atypical retinoid ST1926 is active in
ATRA resistant neuroblastoma cells acting by a
different mechanism. Biochem Pharmacol 2007;5:
643–55.
31. Darwiche N, Sinjab A, Abou-Lteif G, et al. Inhi-
bition of mammalian target of rapamycin signal-
ing by everolimus induces senescence in adult T-
cell leukemia/lymphoma and apoptosis in periph-
eral T-cell lymphomas. Int J Cancer 2011;4:993–
1004.
32. El Eit R, Iskandarani A, Saliba J, et al. Effective
targeting of chronic myeloid leukemia initiating
activity with the combination of arsenic trioxide
and interferon alpha. Int J Cancer 2014;4:988–96.
33. Wolff N, Randle D, Egorin M, et al. Imatinib
mesylate efficiently achieves therapeutic intratu-
mor concentrations in vivo but has limited activ-
ity in a xenograft model of small cell lung cancer.
Clin Cancer Res 2004;10:3528–34.
34. Formelli F, Clerici M, Campa T, et al. Five-year
administration of fenretinide: pharmacokinetics
and effects on plasma retinol concentrations.
J Clin Oncol 1993;11:2036–42.
35. Zuco V, Zanchi C, Lanzi C, et al. Development
of resistance to the atypical retinoid, ST1926, in
the lung carcinoma cell line H460 is associated
with reduced formation of DNA strand breaks
and a defective DNA damage response. Neoplasia
2005;7:667–77.
36. Nakamura A, Redon C, Bonner W, Sedelnikova
O. Telomere-dependent and telomere-
independent origins of endogenous
DNA damage in tumor cells. Aging 2009;
2:212–8.
37. Pear W, Miller J, Xu L. Efficient and rapid induc-
tion of a chronic myelogenous leukemia-like
myeloproliferative disease in mice receiving P210
bcr/abl-transduced bone marrow. Blood 1998;92:
3780–92.
Cancer Therapy
708 ST1926 induces apoptosis in CML cells and prolongs survival in mice
Int. J. Cancer: 137, 698–709 (2015) V
C2014 UICC
38. Helgason GV, Karvela M, Holyoake TL.
Kill one bird with two stones: potential efficacy
of BCRABL and autophagy inhibition in CML.
Blood 2011;118:2035–43.
39. Feller S. Crk family adaptors-signalling complex
formation and biological roles. Oncogene 2001;44:
6348–71.
40. Kumar S, Fajardo JE, Birge RB, Sriram G. Crk at
the quarter century mark: perspectives in signal-
ing and cancer. J Cell Biochem 2014;5:
819–25.
41. Shah NP, Kasap C, Weier C, et al. Transient
potent BCR-ABL inhibition is sufficient to com-
mit chronic myeloid leukemia cells irreversibly to
apoptosis. Cancer Cell 2008;6:485–93.
42. Maugeri-Sacc
a M, Bartucci M, De Maria R. DNA
damage repair pathways in cancer stem cells. Mol
Cancer Ther 2012;8:1627–36.
43. Graham SM1, Jørgensen HG, Allan E, et al.
Primitive, quiescent, Philadelphia-positive stem
cells from patients with chronic myeloid leukemia
are insensitive to STI571 in vitro. Blood 2002;99:
319–25.
44. Hamilton A, Helgason GV, Schemionek M, et al.
Chronic myeloid leukemia stem cells are not
dependent on Bcr-abl kinase activity for their
survival. Blood 2012;119:1501–10.
45. Deau B, Nicolini FE, Guilhot J, et al. The addi-
tion of daunorubicin to imatinib mesylate in
combination with cytarabine improves the
response rate and the survival of patients with
myeloid blast crisis chronic myelogenous leuke-
mia (AFR01 study). Leuk Res 2011; 6:777–82.
46. LoRusso PM, Canetta R, Wagner JA, et al. Accel-
erating cancer therapy development: the impor-
tance of combination strategies and collaboration.
Summary of an institute of medicine workshop.
Clin Cancer Res 2012;22:6101–9.
47. Peng C, Chen Y, Yang Z, et al. PTEN is a tumor
suppressor in CML stem cells and BCR-ABL–
induced leukemias in mice. Blood 2010;3:626–35.
48. Zhang H, Peng C, Hu Y, et al. The blk pathway
functions as a tumor suppressor in chronic mye-
loid leukemia stem cells. Nat Gen 2012;8:861–71.
Cancer Therapy
Nasr et al.709
Int. J. Cancer: 137, 698–709 (2015) V
C2014 UICC
