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Blood, Vol. 93 No. 11 (June 1), 1999:
pp. 3973-3982
Effects of the Tyrosine Kinase Inhibitor AG957 and an Anti-Fas Receptor
Antibody on CD34+ Chronic Myelogenous Leukemia Progenitor
Cells
By
Carmelo Carlo-Stella,
Ester Regazzi,
Gabriella Sammarelli,
Simona Colla,
Daniela Garau,
Aviv Gazit,
Barbara Savoldo,
Daniela Cilloni,
Antonio Tabilio,
Alexander Levitzki, and
Vittorio Rizzoli
From the Department of Hematology, University of Parma, Parma, Italy;
the Department of Biological Chemistry, The Hebrew University of
Jerusalem, Jerusalem, Israel; and the Department of Hematology,
University of Perugia, Perugia, Italy.
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ABSTRACT |
The hallmark of chronic myelogenous leukemia (CML) is the
Philadelphia (Ph) chromosome that fuses genetic sequences of the BCR
gene on chromosome 22 with c-ABL sequences translocated from chromosome
9. BCR/ABL fusion proteins have a dysregulated protein tyrosine kinase
(PTK) activity exerting a key role in malignant transformation.
Targeting the tyrosine kinase activity of BCR/ABL or using agents
capable of triggering apoptosis might represent attractive therapeutic
approaches for ex vivo purging. AG957, a member of the tyrphostin
compounds, exerts a selective inhibition of p210BCR/ABL
tyrosine phosphorylation. We report here that preincubation of CML or
normal CD34+ cells with graded concentration of AG957 (1 to 100 µmol/L) resulted in a statistically significant,
dose-dependent suppression of colony growth from multipotent,
erythroid, and granulocyte-macrophage progenitors as well as the more
primitive long-term culture-initiating cells (LTC-IC).
However, AG957 doses causing 50% inhibition (ID50) of CML
and normal progenitors were significantly different for multilineage
colony-forming units (CFU-Mix; 12 v 64 µmol/L; P = .008), burst-forming unit-erythroid (BFU-E; 29 v 89 µmol/L; P = .004), colony-forming unit-granulocyte-macrophage
(CFU-GM; 34 v 85 µmol/L; P = .004),
and LTC-IC (43 v 181 µmol/L; P = .004). In 5 of 10 patients, analysis of BCR/ABL mRNA on single progenitors by
reverse transcription-polymerase chain reaction showed that AG957 at 50 µmol/L significantly reduced the mean (±SD) percentage of
BCR/ABL-positive progenitors (92% ± 10% v 33 ± 5%;
P = .001). Because AG957 treatment resulted in
significantly higher percentages of apoptotic cells (30% v
9%) in the BCR/ABL-transfected 32DLG7 cells as compared with 32D-T2/93
cells (BCR/ABL-negative), we investigated the combined effects of AG957
with the anti-Fas receptor (Fas-R) monoclonal antibody CH11 that
triggers apoptosis. As compared with AG957 alone, the sequential
treatment of CML CD34+ cells with AG957 (1 µmol/L) and
CH11 (1 µg/mL) increased CFU-Mix, BFU-E, and CFU-GM growth inhibition
by 1.6-fold, 3-fold, and 4-fold, respectively. In contrast, the
treatment of normal CD34+ cells with AG957 and CH11
failed to enhance AG957-induced colony growth inhibition. We conclude
that (1) AG957 inhibits in a dose-dependent manner CML CD34-derived
colony formation by both primitive LTC-IC as well as committed CFU-Mix,
BFU-E, and CFU-GM; (2) this growth inhibition is associated with the
selection of a substantial amount of BCR/ABL-negative progenitors; and
(3) the antiproliferative effect of AG957 is dramatically increased by
combining this compound with the anti-Fas-R antibody CH11. These data
may have significant therapeutic applications.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
CHRONIC MYELOGENOUS leukemia (CML) is
associated with a specific chromosomal abnormality known as the
Philadelphia (Ph) chromosome that results from a reciprocal
translocation between chromosomes 9 and 22 and fuses genetic sequences
of the BCR gene on chromosome 22 with c-ABL sequences translocated from
chromosome 9.1,2 Depending on the breakpoint in BCR, the
BCR/ABL chimeric gene generates one of three types of fusion proteins:
p210BCR/ABL is detectable in 95% of patients with CML and
also occurs in approximately one third of BCR/ABL-positive acute
lymphoblastic leukemia (ALL); p190BCR/ABL is detectable in
two thirds of BCR/ABL-positive ALL; and p230BCR/ABL is
associated with the rare Ph-positive chronic neutrophilic leukemia.3,4 As compared with the native ABL protein
(p145ABL), BCR/ABL fusion proteins have dysregulated
protein tyrosine kinase (PTK) activity,5 transforming
activity for hematopoietic cells,6 and the ability to cause
CML-like myelopoiesis in mice.7 Dysregulated PTK activity
of BCR/ABL fusion proteins and the subsequent changes in the
phosphorylation pattern of regulatory proteins play a key pathogenetic
role in CML.8,9 Several signal transduction substrates,
including p21RAS,10
p120GAP,11 and
p62DOK,12 are directly involved in
BCR/ABL-dependent leukemogenesis. The Src homology 2 (SH2) and Src
homology 3 (SH3) domain-containing Grb-2 protein links tyrosine kinases
to Ras signaling.13,14 Binding of Grb-2 to BCR/ABL is
mediated by the direct interaction of the Grb-2 SH2 domain with a
phosphorylated tyrosine within the BCR first exon.15
BCR/ABL tyrosine kinases also phosphorylate Shc proteins on tyrosine,
inducing the formation of an Shc-Grb-2 complex that also has the
potential to stimulate Ras.16 Another important substrate
is the SH2/SH3 domain-containing CRKL protein, which is
phosphorylated by and also forms specific complexes with p210BCR/ABL.17
Treatment options in CML are still limited, because only a minority of
patients are eligible for allogeneic bone marrow transplantation, which
represents the only curative treatment for CML.18 Based on
experimental and clinical findings, autologous stem cell
transplantation (ASCT) is currently considered as a strategy to be
included in the therapeutic management of CML.19-21 ASCT
leads to 5% long-term cytogenetic remission, with leukemic relapse
being the main cause of treatment failure.21 Although cure
of disease has not yet been achieved using autografting, long-term
survival is possible and indeed approaches the survival rate for
allogeneic related donor transplant recipients.20 To
improve the therapeutic index of ASCT, both purging of the leukemic
stem cells or selecting for the nonleukemic stem cells have been
explored.22 These strategies have focused on either in vivo
purging with single or double autograft23,24 or in vitro
purging with chemical, biological, or immunological techniques.25-32 Targeting the PTK activity of BCR/ABL has
been proposed as an attractive therapeutic strategy due to the
potential for malignant transformation of this kinase
activity.33
Inhibition of the BCR/ABL PTK activity has been obtained with
nonselective compounds, such as genistein, as well as with selective compounds, such as herbimycin A.34,35 More recently, a
selective inhibition of the BCR/ABL tyrosine kinase activity has been
demonstrated with the 2-phenylaminopyrimidine derivative
CGP57148B.36,37 Tyrphostins represent an additional family
of PTK inhibitors acting as competitive inhibitors of protein substrate
and/or ATP binding.38 Because of their chemical design,
tyrphostins are slightly hydrophobic, low molecular weight, nonpeptidic
compounds with high biological stability and cell
permeability.38 In K562 cells, a selective inhibition of
p210BCR/ABL tyrosine phosphorylation without inhibition of
total protein phosphorylation has been obtained with the tyrphostin
AG95739 that resulted in 13-fold more potent blocking of
the kinase activity of p210BCR/ABL than blocking of the
kinase activity of p140ABL.40
Recently, AG957 has been shown to restore integrin-mediated adhesion in
CML progenitors.41
The use of agents capable of triggering apoptosis can also be
considered in the context of in vitro purging strategies aimed at
counteracting the inhibition of the apoptotic machinery induced by
BCR/ABL gene product.42 Fas receptor (Fas-R), also termed CD95 or Apo-1, is a transmembrane glycoprotein belonging to the tumor
necrosis factor receptor family expressed in a variety of tissues,
including thymus, heart, lung, and liver, as well as hematopoietic
cells, such as CD34+ cells, T cells, B cells, and
monocytes.43,44 The characterized function of Fas-R on
normal CD34+ cells involves triggering of apoptosis upon
specific binding with the natural Fas ligand (Fas-L) or with agonistic
monoclonal antibodies (eg, CH11) against CD95.45 CML
CD34+ marrow cells constitutively express Fas-R at
significantly higher levels than do normal CD34+ marrow
cells.46 Despite a partial resistance of CML cells to apoptosis, priming with interferon- (IFN-) before Fas-R
triggering is associated with apoptosis induction and decreased BCR/ABL
protein level.47
Therefore, it was the aim of the present study to investigate the
differential effects of graded concentration of AG957 on the in vitro
growth of CML and normal hematopoietic progenitors. The capability of
AG957 to select in vitro for BCR/ABL-negative progenitors was analyzed
by detecting the BCR/ABL mRNA on single progenitors by reverse
transcription-polymerase chain reaction (RT-PCR). In addition, the
combined effects of AG957 and the anti-Fas-R monoclonal antibody CH11
on CML and normal progenitor cells were investigated.
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MATERIALS AND METHODS |
Patients.
Ten patients (5 men and 5 women) with median age of 51 years (range, 40 to 64 years) and a diagnosis of Ph-positive CML in chronic phase were
included in this study. The main clinical characteristics of the
patients are summarized in Table 1. Seven
patients were studied at diagnosis and before any treatment, whereas 3 patients had received hydroxyurea and/or IFN- therapy for 4 to 16 months before being studied. All patients were 100% Ph-positive at
direct cytogenetic analysis. Eight patients showed a b3a2 BCR/ABL
junction, and 2 cases showed a b2a2 junction when analyzed by RT-PCR.
Cell separation procedures.
CML marrow cells were obtained by aspiration from the posterior iliac
crest. Normal cells were obtained from healthy donors undergoing
peripheral blood progenitor cell mobilization. All patients and normal
individuals provided informed consent for these studies. Mononuclear
cells (MNCs) were separated by centrifugation on a Ficoll-Hypaque
gradient (density = 1.077 g/mL).28 MNCs were washed and
suspended in RPMI-1640 (GIBCO, Grand Island, NY) supplemented with 10%
fetal bovine serum (FBS; Stem Cell Technologies, Vancouver, British
Columbia, Canada). CD34+ cells were enriched according to a
magnetic cell sorting methodology (MACS; Miltenyi Biotec, Bergish
Gladbach, Germany).48 Briefly, MNCs were labeled with a
haptenized CD34 antibody (QBEND/10) that was then magnetically labeled
in a second-step reaction with an antihapten antibody coupled to
super-paramagnetic microbeads. Labeled cells were then separated using
a high gradient magnetic separation column placed in a strong magnetic
field. The magnetically stained cells were retained in the column while
unstained cells passed through. When the column was removed from the
magnetic field, the magnetically retained cells were eluted. Purity of CML and normal CD34+ cell fractions ranged from 63% to
97% and 75% to 86%, respectively.
Cell lines.
32D-T2/93 (BCR/ABL-negative) and 32DLG7 (BCR/ABL-positive) cell
lines14 (kindly provided by Dr A. Santucci, Hematology
Department, Bologna, Italy) were used to investigate the
apoptotic-inducing effect of AG957. Both cell lines were cultured in
RPMI-1640 supplemented with FBS (10% vol/vol) and L-glutamine (2 mmol/L). Culture medium for 32D-T2/93 cells was also supplemented (10%
vol/vol) with a conditioned medium of the WEHI-3 cell line as a source
of murine interleukin-3 (IL-3).
Multilineage colony-forming units (CFU-Mix), burst-forming
unit-erythroid (BFU-E), and colony-forming unit-granulocyte-macrophage
(CFU-GM) assay.
The assay for CFU-Mix, BFU-E, and CFU-GM was performed as described
elsewhere.28 Briefly, 1 × 103
CD34+ cells were plated in 35-mm Petri dishes in 1-mL
aliquots of Iscove's modified Dulbecco's medium (IMDM; Seromed,
Berlin, Germany) containing 30% FBS (Stem Cell Technologies),
10 4 mol/L 2-mercaptoethanol (GIBCO), and 1.1%
(wt/vol) methylcellulose. Cultures were stimulated with stem cell
factor (SCF; 50 ng/mL; Amgen Inc, Thousand Oaks, CA), IL-3 (10 ng/mL;
Sandoz, Basel, Switzerland), granulocyte colony-stimulating factor
(G-CSF; 10 ng/mL; Amgen Inc), granulocyte-macrophage colony-stimulating
factor (GM-CSF; 10 ng/mL; Sandoz), and erythropoietin (Epo; 3 U/mL;
Amgen Inc). After incubation (37°C, 5% CO2) for 14 to
18 days in a humidified atmosphere, progenitor cell growth was
evaluated according to previously published criteria.28
Long-term culture-initiating cell (LTC-IC) assay.
The LTC-IC assay was performed according to Sutherland et
al.49 Briefly, test cell suspensions (1 × 104 CD34+ cells) were resuspended in complete
medium consisting of -medium (GIBCO) supplemented with FBS (12.5%),
horse serum (12.5%; Stem Cell Technologies), L-glutamine (2 mmol/L), 2-mercaptoethanol (104 mol/L), inositol
(0.2 mmol/L), folic acid (20 µmol/L), and freshly dissolved
hydrocortisone (106 mol/L). Test cells were seeded
into cultures containing a feeder layer of irradiated (8,000 cGy)
murine M2-10B4 cells (3 × 104/cm2; kindly
provided by Dr C. Eaves, Terry Fox Laboratory, Vancouver, British
Columbia, Canada) engineered by retroviral gene transfer to produce
human IL-3 and human G-CSF.50 Cultures were fed weekly by
replacement of half of the growth medium containing half of the
nonadherent cells with fresh complete medium. After 5 weeks in culture,
nonadherent and adherent cells harvested by trypsinization were pooled,
washed, and assayed together for clonogenic cells in standard
methylcellulose cultures stimulated with IL-6 (10 ng/mL; Sandoz), SCF,
IL-3, G-CSF, GM-CSF, and Epo. The total number of clonogenic cells (ie,
CFU-Mix plus BFU-E plus CFU-GM) present in 5-week-old LTC provides a
relative measure of the number of LTC-IC originally present in the test
suspension.19 Absolute LTC-IC values were calculated by
dividing the total number of clonogenic cells by 4, which is the
average output of clonogenic cells per LTC-IC, according to limiting
dilution analysis studies reported by others.19
Cytogenetic analysis.
Cytogenetic analysis and standard GTG- or QFQ-banding techniques were
performed according to standard methods.51 To exclude that
AG957 could induce false-negative results by blocking BCR/ABL gene
expression from otherwise Ph-positive colonies, in two experiments individual colonies were aspirated, divided into two aliquots, and
analyzed both at the cytogenetic and molecular level.48
Detection of BCR/ABL mRNA in individual progenitors.
Colonies were individually removed under an inverted microscope and
transferred into microcentrifuge tubes containing 40 µL phosphate-buffered saline (PBS).48 After adding guanidinium thiocyanate (40 µL), colonies were frozen at  70°C until
nested RT-PCR was performed. Briefly, after adding 4 µg of MS2 phage RNA (Boehringer Mannheim, Mannheim, Germany) as a carrier, RNA was
extracted using TRIzol (GIBCO), precipitated with isopropanol, washed
with ethanol, dried, and redissolved in RNAse-free water.52 Total RNA from each colony was reverse transcribed into cDNA in a 20 µL reaction primed with random hexamers. Total cDNA from each colony
was divided into two aliquots for detection of the BCR/ABL
rearrangement and the ABL sequence, respectively. PCR amplifications
were performed in 45 µL reactions containing 10 µL of cDNA, 0.25 mmol/L dNTP, 0.57 mmol/L of each primer (for BCR/ABL: sense
5'-GAA GAA GTG TTT CAG AAG CTT CTC CC-3' and antisense 5'-GAC CCG GAG CTT TTC ACC TTT AGT T-3'; for ABL: sense
5'-TTC AGC GGC CAG TAG CAT CTG ACT T-3' and antisense
5'-GAC CCG GAG CTT TTC ACC TTT AGT T-3'), and 2 U Taq
polymerase (GIBCO) in PCR buffer (20 mmol/L Tris-HCl and 1.5 mmol/L
MgCl2).53 Forty-five cycles, each consisting of
denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds, were performed using
a Perkin-Elmer Cetus DNA Thermal Cycler (Perkin-Elmer Cetus, Norwalk,
CT). One microliter of the first PCR product for BCR/ABL
was reamplified with internal nested primers (sense 5'-GTG AAA
CTC CAG ACT GCT CAC AGC A-3' and antisense 5'-TCC ACT GGC
CAC AAA ATC ATC ATA CAG T-3') under slightly modified conditions
(35 cycles, each consisting of denaturation at 94°C for 30 seconds,
annealing at 55°C for 30 seconds, and extension at 72°C for 30 seconds). Twenty microliters of each PCR product was electrophoresed
through a 2% agarose gel stained with ethidium bromide and
photographed under UV light. The sizes of the BCR/ABL fragments
obtained from nested PCR were 272 bp and 197 bp, depending on the
position of junction point within M-BCR. The expected product generated
by PCR for ABL was 185 bp. To adequately control that RNA was correctly
reverse transcribed and that the PCR product was generated by
amplifying cDNA and not genomic DNA eventually contaminating an RNA
sample, ABL sequence amplification was performed with two primers
located on exon 2 and exon 3 of the cDNA sequence, respectively. Only colonies positive for ABL amplification
were considered evaluable. To eliminate the possibility of
false-positive results, a blank control for RNA extraction and a
negative BCR/ABL control consisting of RNA isolated from normal
marrow-derived CFU-GM were used.54 Each PCR amplification
also included a control reaction without template cDNA (H2O
blank) and, as a positive control, RNA extracted from K562 colonies.
DNA fragmentation.
To investigate the capability of AG957 to trigger apoptosis, nuclear
DNA fragmentation was detected by terminal deoxynucleotidyl transferase
(TdT) assay.34,55 Briefly, cells were fixed in PBS
containing 4% paraformaldehyde, washed, and permeabilized with 0.1%
Triton X-100. Cells were then resuspended in 50 µL of a solution
containing 0.1 mol/L sodium cacodylate, 1 mmol/L CoCl2, 0.1 mmol/L dithiothreitol, 0.05 mg/mL bovine serum albumine, 10 U TdT, and
0.5 nmol fluorescein isothiocyanate (FITC)-conjugated biotin-16-deoxyuridine triphosphate. All chemicals and nucleotides were
purchased from Boehringer Mannheim. The cells were incubated for 60 minutes at 37°C and analyzed by flow cytometry on a FACSort (Becton
Dickinson, Mountain View, CA).
AG957 treatment.
AG957 was diluted in dimethyl sulfoxide (DMSO) to prepare 1,000-fold
concentrated solutions. To evaluate the effect of AG957 as a single
agent, CD34+ cells (1 × 105/mL) were
exposed (30 minutes at 37°C in 5% CO2) to either
control medium (IMDM, 10% FBS) or medium containing increasing doses
of AG957 (1 to 100 µmol/L). At the end of the incubation period, the
cells were washed three times and cultured to quantitate CFU-Mix, BFU-E, CFU-GM, and LTC-IC. For each experiment, appropriate controls with vehicle alone (DMSO at 1 µL/dish) were set up. To evaluate the
combined effects of AG957 and anti-Fas-R monoclonal antibody, untreated and AG957-treated (1 to 50 µmol/L for 30 minutes)
CD34+ cells were resuspended in serum-free medium
(StemPro-34 SFM; GIBCO) and exposed (2 hours at 37°C) to the
anti-Fas-R antibody CH11 (1 µg/mL; Immunotech, Marseilles, France).
At the end of the incubation, CD34+ cells were incorporated
in a standard methylcellulose assay to quantitate hematopoietic
progenitors. For each experiment, appropriate controls with cells
exposed to either CH11 alone or an irrelevant monoclonal antibody was
set up.
Statistical analysis.
Four plates were scored for each data point per experiment and the
results were expressed as the mean ± 1 standard error of the mean
(SEM). Statistical analysis was performed with the statistical package
Statview (BrainPower Inc, Calabasas, CA) run on a Macintosh 6300 personal computer (Apple Computer Inc, Cupertino, CA). The Student's
t-test for paired or unpaired data (two-tail) or the Wilcoxon
signed-rank test were used where appropriate to test the probability of
significant differences between samples. AG957 concentrations resulting
in 50% inhibition (ID50) of colony formation were
calculated for each experiment by extrapolating from a least square
linear regression line relating AG957 concentration to the percentage
of colony growth inhibition.
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RESULTS |
Effect of AG957 ± CH11 on CML and normal progenitors.
In preliminary experiments, the time-dependent effect of AG957 was
investigated. Preincubation of CML cells with a single dose of AG957
(10 µmol/L) resulted in a maximal inhibition of colony formation
after 30 minutes of exposure (data not shown). This length of time was
therefore used in subsequent experiments aimed at exploring the
dose-dependent effect of AG957.
As shown in Fig 1, preincubation of CML
CD34+ cells (n = 10) with AG957 (1 to 100 µmol/L)
followed by extensive washings resulted in a statistically significant
(CFU-Mix: P = .04 at 1 µmol/L; BFU-E: P = .01 at 1 µmol/L; CFU-GM: P = .04 at 5 µmol/L), dose-dependent suppression of colony growth from multipotent (Fig 1A), erythroid (Fig
1B), and granulocyte-macrophage (Fig 1C) progenitors. Regression analysis showed that inhibition was linearly related (CFU-Mix: r = .73, P = .04; BFU-E: r = .87, P = .01; CFU-GM: r = .91, P = .01) to AG957 concentration
over the range tested. Although individual CML samples exhibited
variable sensitivity to the inhibitory effects of AG957, no one failed
to respond to this agent. The degree of colony suppression was not
related to colony number in control cultures.
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| Fig 1.
Effect of AG957 on CML ( ) and normal ( )
CD34+-derived CFU-Mix (A), BFU-E (B), and CFU-GM (C).
Each data point represents the mean (±SEM) percentage of inhibition
from separate experiments using 10 CML and 6 normal samples. For CML,
control colonies per 1 × 103 CD34+ cells
ranged from 2 to 7 for CFU-Mix, 39 to 80 for BFU-E, and 31 to 180 for
CFU-GM. For normal samples, control colonies 1 × 103
CD34+ cells ranged from 4 to 8 for CFU-Mix, 24 to 96 for
BFU-E, and 40 to 64 for CFU-GM. When compared with control cultures
(Wilcoxon signed-rank test), the inhibitory effect of AG957 on CML
progenitors was statistically significant at the dose of 1 µmol/L for
CFU-Mix (P = .04) and BFU-E (P = .01) and of 5 µmol/L for CFU-GM (P = .04). The inhibitory effect of AG957
on normal progenitors was statistically significant at the dose of 5 µmol/L for CFU-Mix (P = .02), and BFU-E (P = .04)
and 10 µmol/L for CFU-GM (P = .03).
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Colony formation by normal CD34+ cells (n = 6) exposed to
AG957 was also inhibited in a dose-dependent manner (Fig 1A through C).
However, normal cells were suppressed at a significantly lower extent
than CML cells, with a relevant toxic effect being evident only when
AG957 doses in excess of 50 µmol/L were used. As shown in
Fig 2, AG957 doses causing 50% inhibition
(ID50) of CML and normal progenitor cell growth were
significantly different for CFU-Mix (12 v 64 µmol/L;
P = .008), BFU-E (29 v 89 µmol/L; P = .004),
and CFU-GM (34 v 85 µmol/L; P = .004).
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| Fig 2.
Mean (±SEM) concentrations of AG957 inducing 50%
inhibition (ID50) of CML () and normal ( ) colony
formation. ID50 values were calculated for each experiment
by extrapolating from a least square linear regression line relating
AG957 concentration to the percentage of colony growth inhibition.
*Statistically significant (P = .004, at least) when compared
with normal samples (Student's t-test for unpaired data).
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The effect of AG957 was also assayed by plating CML and normal
CD34+ cells on irradiated murine M2-10B4 cells engineered
by retroviral gene transfer to produce human IL-3 and human G-CSF.
After 5 weeks, cultures were evaluated for the production of secondary
clonogenic cells in the nonadherent supernatant and the adherent
stromal layers. After exposure to increasing doses of AG957, a
statistically significant and dose-dependent suppression of CML
(r = .94, P = .005) and normal (r = .89, P = .01) LTC-IC growth was observed (Table 2). Again, normal LTC-IC were
significantly less inhibited than CML LTC-IC, as demonstrated by
statistically different ID50 values (181 ± 8 v
43 ± 5 µmol/L; P = .004).
Fas-R is constitutively expressed at significantly higher levels on CML
than on normal CD34+ cells.46,56 Fas-R
triggering by the natural Fas-L or agonistic monoclonal antibodies
results in apoptosis, reduced colony growth, and a decrease in the
BCR/ABL protein level.46,47,56 To investigate whether Fas-R
triggering could have enhanced AG957-induced colony suppression, the
effect of a sequential treatment with AG957 (0 to 50 µmol/L for 30 minutes) and CH11 (1 µg/mL for 120 minutes) was investigated. In
agreement with already reported data,45-47 treatment of
CD34+ cells with CH11 alone suppressed the growth CFU-Mix,
BFU-E, and CFU-GM by both CML (59%, 54%, and 42%, respectively) and
normal (28%, 19%, and 33%, respectively) CD34+ cells.
The sequential treatment of CML CD34+ cells with AG957 and
CH11 significantly increased growth suppression
(Fig 3A through C). When incubation with
AG957 concentrations as low as 1 µmol/L was followed by CH11
exposure, CFU-Mix, BFU-E, and CFU-GM growth inhibition was increased by
1.6-fold, 3-fold, and 4-fold, respectively (Fig 3A through C). In
contrast, the sequential treatment of normal CD34+ cells
with AG957 and CH11 failed to enhance AG957-induced colony growth
inhibition (Fig 3D through F).
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| Fig 3.
Effect of AG957 alone () or AG957 plus CH11 () on
CML (upper panels) and normal (lower panels) progenitors. The effects
on CFU-Mix (A and D), BFU-E (B and E), and CFU-GM (C and F) are shown.
To evaluate the combined effects of AG957 and CH11, untreated and
AG957-treated (1 to 50 µmol/L for 30 minutes) CD34+
cells (1 × 105/mL) were resuspended in serum-free medium
and exposed (2 hours at 37°C) to CH11 (1 µg/mL). At the end of
the incubation, CD34+ cells were incorporated in a
standard methylcellulose assay to quantitate hematopoietic progenitors.
Each histogram represents the mean (±SEM) percentage of inhibition
from separate experiments using CML (n = 4) and normal (n
= 2) CD34+ cells. For CML, control colonies per 1 × 103 CD34+ cells ranged from 2 to 7 for
CFU-Mix, 39 to 80 for BFU-E, and 31 to 180 for CFU-GM. For normal
samples, control colonies 1 × 103 CD34+
cells ranged from 2 to 6 for CFU-Mix, 53 to 96 for BFU-E, and 97 to 154 for CFU-GM. When compared with cultures treated with AG957 alone
(Wilcoxon signed-rank test), the inhibitory effect of AG957 plus Fas-L
on CML progenitors was significantly different at the dose of 1 µmol/L for CFU-Mix (P = .04), BFU-E (P = .01),
and CFU-GM (P = .04). For normal samples, no statistically
significant difference was detected by comparing the inhibitory effects
of AG957 or AG957 plus Fas-L. *Statistically significant when compared
with samples treated with AG957 alone (Student's t-test for
unpaired data).
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DNA fragmentation.
Data obtained by AG957 preincubation suggest that the effect of the
drug does involve toxicity. To examine whether apoptosis is involved in
AG957-induced inhibition of progenitor cell growth, two cell lines,
namely 32D-T2/93 and 32DLG7, were treated with AG957 (0 to 100 µmol/L
for 12 hours) and apoptosis was analyzed by the TdT assay. Upon AG957
exposure, 9% of 32D-T2/93 cells (BCR/ABL-negative) were in a
progressive stage of apoptosis, whereas virtually no apoptotic cells
were detected in the control (Fig 4, left
panels). AG957 treatment of the BCR/ABL-transfected 32DLG7 cells
resulted in significantly higher percentages of apoptotic cells (Fig 4, right panels).
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| Fig 4.
Detection of apoptotic cells by means of TdT assay
in untreated and AG957-treated 32D-T2/93 (BCR/ABL-negative) and
32DLG7 (BCR/ABL-positive) cell lines. Mean (±SEM) percentages of
apoptotic cells in the live gated cell populations are indicated for
32DT2/93 (left panels) and 32DLG7 (right panels) cells exposed to
graded concentrations of AG957 for 12 hours.
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BCR/ABL mRNA expression on single colonies.
Quantitative differences in CML and normal colony growth suppression
argue for an antiproliferative effect of AG957 that is specifically
related to BCR/ABL inhibition. To investigate whether AG957 could
affect CML colony formation not only quantitatively but also
qualitatively, CML colonies were individually harvested and analyzed by
RT-PCR for the expression of hybrid BCR/ABL mRNA. At the time of the
study, all patients were 100% Ph-positive by standard cytogenetics.
Table 3 shows that preincubation with 1 to
10 µmol/L of AG957 failed to show any antileukemic effect. However,
AG957 at 50 µmol/L significantly reduced the mean (±SD) percentage of BCR/ABL-positive progenitors (92% ± 10% v
33% ± 5%; P = .001).
Because the combined treatment with AG957 and CH11 was associated with
a significant increase of the antiproliferative effect of the
tyrphostin, in four experiments colonies generated by CD34+
cells sequentially exposed to low doses of AG957 (1 and 5 µmol/L) and
CH11 were analyzed for BCR/ABL expression. Indeed, the combined AG957/CH11 treatment failed to improve the selection of nonleukemic progenitors (data not shown).
To rule out the possibility that AG957 could affect BCR/ABL
transcription, thus inducing the growth of Ph-positive progenitors with
nonfunctional BCR/ABL gene (BCR/ABL negative), in two experiments (cases no. 2 and 5) colonies were individually harvested and split into
two aliquots, one for cytogenetics and the other for RT-PCR. In both
cases, no difference was observed in the percentages of Ph-positive and
BCR/ABL-positive colonies generated by AG957-treated cultures (data not
shown), thus confirming that AG957-induced increase in the percentage
of nonleukemic progenitors was not related to suppression of BCR/ABL transcription.
| |
DISCUSSION |
The increasing knowledge of transmembrane and intracellular signal
transduction phenomena now allows us to manipulate cell growth by
altering signaling pathways.9,33 Because PTKs participate in the establishment and progression of several malignant diseases, inhibitors of PTKs represent attractive antiproliferative
agents.33-37 AG957 prevents the phosphorylation of
p210BCR/ABL,39,40 thus downregulating the
activation of regulatory proteins that stimulate Ras and play a crucial
role in the pathogenesis of CML.9,10,14-16 In addition,
AG957 restores integrin-mediated adhesion and proliferation inhibition
in CML progenitors.41
In the present study, we demonstrate that AG957 inhibits in a
dose-dependent manner the growth of CML-derived LTC-IC, CFU-Mix, BFU-E,
and CFU-GM. The inhibitory effect of AG957 on highly purified CD34+ cells indicates that the antiproliferative action of
AG957 does not involve accessory cells. A significant growth inhibition
is seen after exposure to AG957 concentrations as low as 1 µmol/L and
a nearly complete inhibition (>80%) is detected at 50 µmol/L.
Inhibition of CML progenitor cell growth to 50% of control values is
achieved with doses of AG957 that are 2.5-fold to 5-fold lower than
those required for normal progenitors, suggesting a differential effect
of AG957 on normal and CML cells. However, exposure of normal
CD34+ cells to AG957 concentrations  50 µmol/L is
associated with a relevant toxicity on committed (CFU-Mix, BFU-E, and
CFU-GM) but not primitive (LTC-IC) progenitors. The toxicity of AG957
on Ph-negative cells is also demonstrated by detection of nuclear DNA
fragmentation of the BCR/ABL-negative 32D-T2/93 cell line. However,
once again, the quantitative evaluation of apoptotic cells by the TdT
assay suggests a more effective action of AG957 on Ph-positive rather than Ph-negative cells. The loss of selectivity of AG957 may be intrinsic to the mechanism of action of tyrphostins that are
competitive inhibitors not only of protein substrate but also of ATP
binding.33 In fact, the ATP antagonism is likely to mediate
inhibition of tyrosine kinases other than BCR/ABL, including normal
c-ABL, that are essential for the proliferation of normal
colony-forming cells.
Analysis of BCR/ABL mRNA expression in individual progenitors
demonstrates that exposure of CML cells to 10 µmol/L of AG957 fails
to enrich BCR/ABL-negative progenitors, whereas a substantial depletion
of leukemic colonies is observed with AG957 at 50 µmol/L, a dose
suppressing the growth of CML and normal LTC-IC by 76% and 19%,
respectively. The limited, nonspecific toxic effect on normal LTC-IC
associated with a selective antileukemic action supports the use of
AG957 for purging malignant progenitors from CML marrow. Indeed, when
CD34+ cells were treated with 50 µmol/L AG957, only half
of the patients generated sufficient numbers of colonies to be analyzed
at the molecular level. BCR/ABL-negative progenitors were detected in 4 patients studied at diagnosis and 1 patient who had received hydroxyurea for a short period, whereas we were unable to detect significant levels of nonleukemic progenitors in 2 patients analyzed after 12 months of therapy as well as in 3 additional patients analyzed at diagnosis. These results are in keeping with previous studies demonstrating a progressive reduction of the BCR/ABL-negative compartment in chronic-phase CML.57,58 The failure of AG957 to select nonleukemic colonies may be explained by the rarity of
BCR/ABL-negative progenitors that could therefore be underscored when a
limited number of colonies (on average, 20 to 30) is analyzed. Alternatively, the presence of additional mutations in a subset of
progenitors would allow cell proliferation independently of BCR/ABL.
Finally, individual progenitors expressing p190BCR/ABL at
high levels might not be efficiently suppressed by AG957.40
Analysis of individual colonies by both cytogenetics and RT-PCR showed
similar percentages of Ph-negative and BCR/ABL-negative colonies
generated by AG957-treated samples, thus ruling out that AG957 acts by
suppressing BCR/ABL transcription, as has been described for
IFN-.59 This is further supported by the evidence of
Kaur et al,39 who have shown that preincubation of K562
cells with the tyrphostin AG957 inhibits cell growth,
p210BCR/ABL tyrosine kinase activity, and DNA synthesis as
early as 2 hours, a time at which RNA and protein synthesis were not affected.
BCR/ABL signals to cause transformation through several mechanisms,
including activation of mitogenic signaling pathways,8,60 induction of anchorage-independent growth,61 and
suppression of apoptosis.42,62 Inhibition of BCR/ABL
tyrosine kinase activates a death program, as shown by apoptosis of
32DLG7 cells upon exposure to AG957. Because recent findings
demonstrate that, after priming with IFN-, Fas-R triggering on CML
CD34+ cells is associated with dose-dependent decrease of
colony formation, apoptosis, and decrease in the BCR/ABL protein level,
we hypothesized that Fas-R triggering after AG957 treatment could have
enhanced the antiproliferative action of the tyrphostin. In agreement
with previously reported results,45,46 exposure to CH11
alone results in an antiproliferative effect on both CML and normal
progenitors. Under our experimental conditions, CH11 inhibits CML
progenitors more than normal progenitors. This finding is likely to be
related either to different levels of Fas-R expression by CML and
normal CD34+ cells or the use of normal CD34+
cells that were not preincubated with inducers of Fas-R expression.
As compared with AG957 alone, the combined AG957/CH11 treatment
resulted in a dramatic increase of CML, but not normal, growth inhibition. The different behavior of CML and normal progenitors in
response to the sequential AG957/CH11 treatment might be due to
different levels of Fas-R expression. These data suggest that AG957-induced inhibition of BCR/ABL tyrosine kinase enhances the response of CML cells to Fas-L and allow us to hypothesize the existence of a link between reduced PTK activity of BCR/ABL and apoptosis triggering by the Fas-R/Fas-L system. Although we were not
able to demonstrate a better antileukemic effect by sequentially combining low doses of AG957 with CH11, the marked antiproliferative effect seen on CML progenitors in the absence of an additive toxicity on normal progenitors may be of therapeutic relevance and is currently under investigation in an animal model. In fact, the combined AG957/CH11 treatment could allow the use of the tyrphostin at low dose,
thereby reducing the risk of nonspecific toxicity on normal progenitors.
The in vitro selection for normal hematopoietic stem and progenitor
cells from within CML marrow and the potential for using these cells as
leukemia-free autografts has been the topic of increasing
discussion.19,25,28,30-32 Data reported here demonstrate the possibility to select nonclonal CML progenitors by means of a
simple incubation with the PTK inhibitor AG957, thus suggesting that it
may be feasible to select a population of benign progenitors from CML
marrow that could be used for autografting patients without suitable
allogeneic bone marrow donors. Further investigations are also required
to explore the therapeutic potential of PTK inhibitors in combination
with other agents, including biological response modifiers and
apoptosis-inducing molecules.
| |
FOOTNOTES |
Submitted August 5, 1998; accepted January 29, 1999.
Supported in part by grants from "Ministero dell'Università e
della Ricerca Scientifica e Tecnologica" (MURST-40% & 60%), "Associazione Italiana per la Ricerca sul Cancro" (AIRC), and "Associazione Italiana Leucemie (AIL)-Trenta Ore per la Vita." D.G. is supported by a grant from the Azienda Ospedaliera di Parma. E.R. is a recipient of an AIRC fellowship.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Carmelo Carlo-Stella, MD, Unità
Trapianto di Midollo, Istituto Nazionale Tumori, Via Venezian, 1, 20133 Milano, Italy; e-mail: ccs{at}ipruniv.cce.unipr.it.
| |
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M. Gianni', Y. Kalac, I. Ponzanelli, A. Rambaldi, M. Terao, and E. Garattini
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X. Sun, J. E. Layton, A. Elefanty, and G. J. Lieschke
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D. Cilloni, C. Carlo-Stella, F. Falzetti, G. Sammarelli, E. Regazzi, S. Colla, V. Rizzoli, F. Aversa, M. F. Martelli, and A. Tabilio
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TOP
ABSTRACT
INTRODUCTION