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NEOPLASIA
From the Division of Hematology and Bone Marrow
Transplantation and the Department of Cytogenetics, City of Hope
National Medical Center, Duarte, CA; and the Division of Hematology and
Oncology, University of California at Los Angeles, CA.
Imatinib mesylate (STI571) is a promising new treatment for chronic
myelogenous leukemia (CML). The effect of imatinib mesylate on
primitive malignant progenitors in CML has not been evaluated, and it
is not clear whether suppression of progenitor growth represents inhibition of increased proliferation, induction of apoptosis, or both.
We demonstrated here that in vitro exposure to concentrations of
imatinib mesylate usually achieved in patients (1-2 µM) for 96 hours
inhibited BCR/ABL-positive primitive progenitors (6-week long-term culture-initiating cells [LTCICs]) as well as committed progenitors (colony-forming cells [CFCs]). No suppression of
normal LTCICs and significantly less suppression of normal CFCs were observed. A higher concentration of imatinib mesylate (5 µM) did not
significantly increase suppression of CML or normal LTCICs but did
increase suppression of CML CFCs, and to a lesser extent, normal CFCs.
Analysis of cell division using the fluorescent dye carboxyfluorescein
diacetate succinimidyl ester indicated that imatinib mesylate
(1-2 µM) inhibits cycling of CML primitive
(CD34+CD38 Chronic myelogenous leukemia (CML) is a
hematopoietic stem cell malignant disease1 that arises from
a reciprocal translocation between chromosomes 9 and 22.2
It is characterized by a massive expansion of myeloid progenitors as
well as more differentiated cells originating from the malignant clone.
The disease progresses from an initial chronic phase through an
accelerated phase followed by an inevitable acute leukemia or blast
crisis as the malignant cells lose their ability to differentiate. The
translocation (9;22)(q34;q11) fuses a truncated BCR gene
(chromosome 9) to sequences upstream of the second exon of
ABL (chromosome 22). The resulting fusion gene encodes a
protein tyrosine kinase, Bcr-Abl, with more elevated and disregulated
activity compared with c-abl. Expression of Bcr-Abl in mice was found
to induce a CML-like myeloproliferative disorder,3-7 indicating that Bcr-Abl plays a key role in leukemic transformation. Additional studies have shown that the tyrosine kinase activity is
critical to the transforming ability of Bcr-Abl,7,8 making it an attractive target for drug development.
Imatinib mesylate (STI571; formerly CGP571418B; Gleevec; Novartis
Pharmaceuticals, East Hanover, NJ) is a 2-phenylaminopyrimidine derivative developed as a potent inhibitor of the Abl protein tyrosine
kinases (v-Abl, Bcr-Abl, and c-Abl).9,10 It also has
activity against the platelet-derived growth-factor receptor (PDGF-R),10 c-Kit,11 ARG,12 and
their fusion proteins, Tel-Abl and Tel-PDGF-R,13 but does
not affect other kinases.
Phase I and phase II clinical trials of imatinib mesylate in the
treatment of chronic-phase CML showed that it is well tolerated, with
few adverse effects.14 Complete hematologic responses and cytogenetic responses, including complete cytogenetic responses, were reported in a significant proportion of patients. Activity against more advanced accelerated and blast-crisis phases of CML was also observed.15 Imatinib mesylate is currently
approved for the treatment of patients with chronic-phase CML in whom
interferon therapy has failed and for accelerated-phase and
blast-crisis disease. However, the durability of responses to imatinib
mesylate, the agent's impact on long-term survival and normal
hematopoiesis, and its role in the treatment of CML compared with other
modalities remain unclear.
In vitro studies have shown that imatinib mesylate inhibits growth of
cell lines expressing Bcr-Abl.10,13,16,17 Additionally, the numbers of colony-forming cells (CFCs) in peripheral blood or bone marrow from patients with CML have been found to be reduced, with minimal inhibition of normal cells.10,17 One study
showed that imatinib mesylate inhibited the enhanced replating ability of granulocyte-macrophage colony-forming units (CFU-GMs) from patients
with chronic-phase CML.18 However, these studies
primarily addressed the effect of imatinib mesylate on mature or
committed hematopoietic progenitors; the effects of this agent on the
more primitive cells in which the disease arises are not well
understood. Short exposure to high concentrations of imatinib mesylate
(100 µM for 48 hours or 10 µM for 7 days) was found in one study to suppress growth of long-term culture-initiating cells
(LTCICs).19 However, this study did not address whether
clinically relevant doses of imatinib mesylate suppress primitive progenitors.
It is important to determine the mechanism by which imatinib mesylate
acts to restore normal hematopoiesis in patients with chronic-phase
CML, since this may help in predicting the depth of response to
imatinib mesylate, aid in designing improved therapeutic interventions,
and suggest mechanisms of resistance. The mechanism by which the
initial BCR/ABL-positive clone is able to overtake hematopoiesis is thought to be enhanced
proliferation.20,21 Inhibition of apoptosis was also
reported to play a role in expansion and preservation of the leukemic
clone,22,23 but this was not confirmed in other
studies24,25 and its role remains unclear. In vitro studies
in cell lines as well as primary cells have shown that imatinib
mesylate inhibits proliferation.10,13,16,17 Other studies
suggested that imatinib mesylate induces apoptosis in
Bcr-Abl-dependent cell lines.26,27 However, the effect of exposure to imatinib mesylate on proliferation or apoptosis in primary
cells has not been described. Because cell lines are inherently different from primary cells, this issue must be addressed directly in
primary cells from samples from patients with CML. It also needs to be
clarified whether these effects extend to primitive progenitors or are
limited to more committed progenitor cells because of their different
growth characteristics. If the primary effect of Bcr-Abl kinase
inhibition by imatinib mesylate in primary primitive progenitors is to
reduce abnormally increased proliferation, this will imply that
imatinib mesylate exposure may not by itself completely eliminate
primitive malignant progenitors.
Here, we examined the effect of imatinib mesylate on primitive and
committed progenitor cells from patients with CML and healthy donors.
Cells were exposed to imatinib mesylate for 1 to 4 days and the effects
on LTCICs and CFCs were measured. In addition to examining the effect
of imatinib mesylate treatment on CML cells relative the effect in
untreated controls, a comparison with normal treated cells addresses
the possibility of non-Bcr-Abl-specific effects through inhibition of
c-Kit, PDGF-R, or other unidentified targets. To assess how imatinib
mesylate restores normal hematopoiesis, we investigated whether a
reduction in primitive progenitor growth induced by exposure to the
agent reflects inhibition of proliferation, induction of apoptosis, or both.
Patients
Selection of CD34+ progenitors
Drug treatment of cells CD34+ cells were cultured in multiwell tissue-culture plates (Falcon) in serum-free medium (SFM; StemPro; Gibco BRL, Gaithersburg, MD) supplemented with growth factors at concentrations similar to that found in serum-conditioned medium (granulocyte-macrophage colony-stimulating factor [GM-CSF], 200 pg/mL; granulocyte colony-stimulating factor [G-CSF], 1 ng/mL; stem cell factor [SCF], 200 pg/mL; leukemia inhibitory factor [LIF], 50 pg/mL; macrophage inflammatory protein [MIP-1], 200 pg/mL; and
interleukin 6 [IL-6], 1 ng/mL)28-30 and containing 0 to
10 µM imatinib mesylate. Cells were cultured for 24 to 96 hours at
37°C in a humidified atmosphere with 5% carbon dioxide, after which
they were harvested, washed with Iscoves modified Dulbecco medium
(IMDM; Gibco, Grand Island, NY), and assayed for progenitor numbers,
apoptosis, or proliferation.
Progenitor assays CFCs. CD34+ cells were plated in semisolid methylcellulose progenitor culture for 14 to 18 days and assessed for the presence of colonies of CFU-GMs and erythroid burst-forming units as described previously.31 Limiting-dilution assay of primitive progenitors (LTCICs). Limiting-dilution assays of CD34+ cells were performed by plating cells on M2-10B4 feeders in 96-well plates as described previously.32 At the end of the 6-week culture period, 50% of the medium was removed and wells were overlaid with CFC-growth-supporting medium. After 14 days, wells were scored as positive or negative for the presence of CFCs. The frequency of LTCICs was calculated with L-Calc software (StemCell Technologies, Vancouver, BC) on the basis of the reciprocal of the concentration of test cells that yielded 37% negative wells. Apoptosis assay Cells treated with imatinib mesylate for 24 to 96 hours were labeled with annexin V conjugated with fluorescein isothiocyanate (FITC; BD-PharMingen, San Diego, CA) and Via-Probe 7-aminoactinomycin D (7-AAD; BD-PharMingen). Briefly, cells were washed once in phosphate-buffered saline and once in 1 × binding buffer (BD-PharMingen), and then 5 µL each of annexin V-FITC and 7-AAD was added to the cells. Cells were incubated at room temperature for 15 minutes, after which 300 µL 1 × binding buffer was added and cells were analyzed by flow cytometry. Apoptotic cells were defined as annexin V-FITC positive and 7-AAD negative.Proliferation assay CD34+-selected cells were suspended in 10 mL IMDM, and 5- (and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes, Eugene, OR) was added to obtain a concentration of 1.25 µM. Cells were incubated at 37°C in the dark for 10 minutes, with occasional mixing. Further dye uptake was stopped by the addition of 2 mL cold fetal-calf serum followed by centrifugation at 500g for 4 minutes. The cell pellet was resuspended to obtain a concentration of 250 × 103 cells/mL in SFM supplemented with growth factors (GM-CSF, 200 pg/mL; G-CSF, 1 ng/mL; SCF, 200 pg/mL; LIF, 50 pg/mL; MIP-1, 200 pg/mL; and IL-6, 1 ng/mL);
transferred to 6-well tissue-culture plates; and incubated at 37°C
overnight to allow excess unbound dye to leak out of the cells. Cells
were labeled with CD34-allophycocyanin and CD38-phycoerythrin
antibodies (Becton Dickinson, San Jose, CA).
CD34+CD38dim and
CD34+CD38+ subsets were obtained by
fluorescence-activated cell-sorter scanning (FACS). These subsets were
further selected for CFSE fluorescence by using a narrow gate
(40-channel width on a 1024-channel log amplifier; MoFlo; Cytomation,
Fort Collins, CO). Cell subsets selected by FACS were cultured for 96 hours as described above in the presence of 0 to 5 µM imatinib
mesylate. At the end of the culture period, proliferation was analyzed
by flow cytometry (FACSCalibur; Becton Dickinson). The percentage of
cells in each generation was determined by using ModFit software
(Verity, Topsham, ME), with the position of the parent generation set
on the basis of the fluorescence profile of an aliquot of cells treated
with Colcemid (0.1 µg/mL) immediately after sorting.
Fluorescent in situ hybridization analysis Cells were labeled and analyzed for the presence of the BCR/ABL translocation by using the LSI dual-labeled BCR/ABL DNA probe (Vysis, Downers Grove, IL) as described previously.32Statistical analysis Data obtained from multiple experiments were reported as the mean ± 1 SEM. Significance levels were determined by Student t test analysis. Values for the concentration that inhibits 50% (IC50) were determined by fitting a 1-phase exponential decay curve to the CFC and LTCIC data by using GraphPad Prism software (GraphPad, San Diego, CA).
Treatment of CML cells with imatinib mesylate is associated with a dose-dependent decrease in the number of primitive progenitors The effect of imatinib mesylate on progenitor growth was evaluated by in vitro exposure of CML CD34+ cells to imatinib mesylate for 24 to 96 hours under physiologic growth-factor conditions followed by enumeration of both primitive (LTCICs) and committed (CFCs) progenitors (Figure 1). Following exposure to imatinib mesylate, cells were washed to remove the drug, and progenitor assays were performed in the absence of further drug exposure. The use of this preincubation procedure allowed us to evaluate the effects of imatinib mesylate directly on progenitor cells in the absence of effects on differentiating cells generated during the progenitor assay and also to later correlate the effects of identical exposures to imatinib mesylate on proliferation and apoptosis. The number of progenitors at the end of the preincubation period would be expected to reflect both increases due to self-renewing cell divisions and decreases associated with differentiation and apoptosis.
The median frequency of CFCs in the absence of imatinib mesylate was higher for CML CD34+ cells (median, 219/1000 cells) than for normal CD34+ cells (122/1000 cells) after 24 hours of incubation and increased to 288/1000 cells after 96 hours of incubation. Normal CFC numbers did not increase during the same period (going from 122 to 111/1000 cells). In contrast, the frequency of CML LTCICs in the absence of imatinib mesylate decreased during the incubation period from 10.3 to 5.4 CD34+ cells/1000. Normal LTCICs also decreased during the incubation period (from 11.0 to 4.6/10 000). The effect of imatinib mesylate on primitive progenitors is shown in
Figure 2A. Exposure to 1 µM imatinib
mesylate for 96 hours suppressed CML LTCICs to a level 57% ± 12%
(n = 5) of that measured for cells not exposed to the drug. At a
higher concentration of imatinib mesylate (5 µM), CML LTCICs showed
an increase in suppression (35% ± 12%; n = 5). In contrast,
normal LTCICs were not suppressed after exposure to 1 µM
(93% ± 13%; n = 4) or 5 µM imatinib mesylate (132% ± 36%;
n = 4). For CML LTCICs, the IC50 was 1.7 µM imatinib
mesylate.
Similarly, the number of committed progenitors (CFCs) decreased in CML cells in a dose-dependent manner following treatment with imatinib mesylate (Figure 2B). Exposure to 1 µM imatinib mesylate for 96 hours suppressed CML CFCs to a level 48% ± 5% (n = 5) of that for untreated cells, whereas normal CFCs showed minor suppression (87% ± 7%; n = 4). CML CFCs were further suppressed when the imatinib mesylate concentration was increased to 5 µM (22% ± 6%; n = 5), resulting in an IC50 of 0.6 µM. However, normal CFCs were also suppressed by 5 µM imatinib mesylate, though to a lesser extent (63% ± 13%; n = 4); the IC50 was 7.6 µM, suggesting that non-Bcr-Abl-dependent mechanisms may contribute to the increased CFC suppression at higher concentrations of imatinib mesylate. Varying the time that CD34+ cells were exposed to imatinib
mesylate (24 to 96 hours) showed that the effect of imatinib mesylate was enhanced by prolonged exposure (Figure
3). The addition of imatinib mesylate led
to a decrease in CFC frequency for CML CD34+ cells,
reversing the increase observed in the absence of imatinib mesylate. At
all concentrations analyzed, there was significantly more suppression
of CML CFCs after a 96-hour exposure to imatinib mesylate than after a
24-hour exposure (P = .0005 for 1 µM,
P = .0003 for 2 µM, and P = 0.0032 for
5 µM).
There was considerable variability in CFC and LTCIC inhibition by
imatinib mesylate among samples from different patients with CML
(Figure 4). Because this variability
could reflect intersample differences in the number of
BCR/ABL-negative progenitors, which would not be expected to
be suppressed, cells were harvested following colony-formation assays
and analyzed for the presence of BCR/ABL by FISH. With the
exception of one sample (discussed below), in the absence of imatinib
mesylate, LTCICs and CFCs were highly BCR/ABL positive after
96 hours of incubation (42%-89%; mean, 78.4%).
The percentage of BCR/ABL-positive cells did not decrease
significantly on treatment with imatinib mesylate (Figure
5). Although there was a suppression of
BCR/ABL-positive progenitor numbers (calculated as the
progenitor number times the percentage of cells that were
BCR/ABL positive on FISH analysis), the failure to increase the percentage of BCR/ABL-negative cells might indicate that
additional stimuli may be required to allow efficient outgrowth of
normal progenitors. The variability among samples did not correlate
with differences in the stage of the disease or prior treatment.
Imatinib mesylate treatment is associated with decreased proliferation of CML primitive and committed progenitors We next investigated whether inhibition of primitive progenitor growth by imatinib mesylate reflected inhibition of proliferation or induction of apoptosis. Cell division was analyzed by using the fluorescent tracking dye CFSE. Cells labeled with CFSE were sorted to provide a narrow band of CFSE fluorescence to enhance definition of generations and then further subdivided into populations of primitive progenitors (CD34+CD38dim) or committed progenitors (CD34+CD38+; Figure 6A). The sorted cells were grown with various concentrations of imatinib mesylate (0 to 5 µM) under physiologic growth-factor conditions for 96 hours and then analyzed for cell division. Representative data are shown in Figure 6B, and the percentages of cells in each generation, as determined with ModFit software, are summarized in Figure 6C. Exposure to imatinib mesylate resulted in an increase in the fraction of undivided cells and a reduction in the number of cell divisions undergone by CML primitive progenitors during 4 days of culture. There was a significant increase in undivided cells with exposure to 5 µM imatinib mesylate (P = .047). In addition, the number of CML primitive progenitor cells undergoing only a single cell division increased significantly in response to imatinib mesylate (P = .002 at 1 µM and P = .003 at 5 µM).
A proliferation index (PI), which represents the sum of the cells in all generations divided by the computed number of original parent cells theoretically present at the start of the experiment, can be generated by ModFit software. This single index simplifies comparison between samples and conditions. The results indicated that imatinib mesylate inhibits division of CML primitive and committed progenitor cells to a much greater extent than in normal cells. Data on proliferation of primitive progenitors exposed to imatinib mesylate are summarized in Figure 6D. Untreated CML primitive progenitors were significantly more proliferative than untreated normal primitive progenitors (P = .006), reflecting the presence of fewer quiescent cells and an increased number of cell divisions. Exposure to 1 µM imatinib mesylate significantly reduced the PI for CML primitive progenitors (P = .006). At 2 µM imatinib mesylate, the PI of CML primitive progenitors was reduced further, such that they no longer showed significantly enhanced proliferation compared with normal primitive progenitors (P = .227). When the data were normalized by comparison with the frequency of progenitors in samples not exposed to imatinib mesylate, the PI was significantly reduced for CML progenitors at all concentrations of imatinib mesylate assessed (P < .0001 at 1 µM, P < .0015 at 2 µM, and P < .0024 at 5 µM). This inhibition was significantly greater for CML than for normal primitive progenitors (P < .0001 at 1 µM, P < .0011 at 2 µM, and P < .0008 at 5 µM). The normalized PI for committed progenitors also indicated that there was a significant inhibition of proliferation in CML progenitors exposed to imatinib mesylate (P = .0052 at 1 µM, P < .0019 at 2 µM, and P < .0004 at 5 µM). These results suggest that the inhibition of cell division is an important mechanism in the suppression of both primitive and committed CML progenitors, suppressing the proliferative advantage present in these cells. One CML patient sample that showed almost no suppression of LTCICs and
only minimal suppression of CFCs (Figure
7A) was found on FISH analysis to have a
very low percentage of BCR/ABL-positive cells. In this
sample, the fraction of BCR/ABL-positive CFCs decreased with
exposure to imatinib mesylate. CFCs without such exposure were 27.5%
BCR/ABL positive, 23.5% at 1 µM imatinib mesylate and 15% at 5 µM imatinib mesylate. However, this decrease was
not observed for LTCICs. The minimal progenitor suppression might represent the presence of a large proportion of nonresponsive normal
cells. A mixed cell population was also apparent in the results of the
CFSE-labeling experiments (Figure 7B). In the absence of imatinib
mesylate, there were 2 distinct populations of cells. A large peak of
undivided cells (67% undergoing 0-1 cell division) was readily
apparent, as was a second population of highly proliferative cells that
had undergone 2 to 5 cell divisions (33%). On exposure to 1 µM
imatinib mesylate, proliferation was decreased and the cells
were more uniformly quiescent (90% undergoing 0-1 cell division).
Effects of imatinib mesylate treatment on apoptosis of CML progenitors To assess the effect of imatinib mesylate on apoptosis, CD34+ cells were grown with various concentrations of imatinib mesylate (0 to 5 µM) under physiologic growth-factor conditions for 96 hours, labeled with annexin V-FITC and 7-AAD, and then analyzed by flow cytometry for cells positively labeled by annexin V but negative for 7-AAD. Results are summarized in Figure 8. Treatment with 1 µM imatinib mesylate did not significantly increase apoptosis in CML CD34+ cells compared with no such treatment (P = .12). When 5 µM imatinib mesylate was used, a significant increase in apoptosis of CML cells was observed (from 16.1% ± 3.8% to 29.8% ± 4.1%; n = 6; P = .007). However, apoptosis was also significantly increased in normal samples at this concentration (from 10.9% ± 1.6% to 16.4% ± 2.6%; n = 3; P = .046). Under no conditions was apoptosis of CML cells significantly greater than that of normal cells, thus suggesting that non-Bcr-Abl-dependent mechanisms may contribute to the increased apoptosis at higher concentrations of imatinib mesylate. For 2 patient samples, there was an increase in apoptosis at 1 µM imatinib mesylate. This may correlate with increased sensitivity to imatinib mesylate, since these samples also showed a high degree of suppression of CFCs and LTCICs with exposure to the agent.
The major mechanism of suppression of CML progenitors by imatinib mesylate is decreased proliferation The loss of both committed and primitive progenitors (Figure 2) appears to primarily reflect a decrease in proliferation. Table 1 shows a summary of the data from each assay for CML cells exposed to 1 µM imatinib mesylate compared with those not exposed to imatinib mesylate. The first row shows the mean percentage decrease in progenitor frequency for primitive progenitors (LTCICs) or committed progenitors (CFCs). The second row indicates the percentage decrease in cell division measured by the proliferation assay based on CFSE labeling. Results for primitive progenitors reflect data from the CD34+CD38dim population, whereas the committed progenitor data are from the CD34+CD38+ population. The percentage decrease in viability (bottom row) is based on data for all CD34+ cells and is therefore the same in each column. These data show that the major mechanism associated with the decrease in both primitive and committed progenitors is a decrease in proliferation, not an increase in apoptosis.
We evaluated the effect of imatinib mesylate on primitive progenitor cells from patients with CML. Previous studies have shown that imatinib mesylate effectively suppresses BCR/ABL-positive committed progenitor cells.10,17 Here, we showed that this effect extends to primitive progenitors as well. Importantly, suppression was shown to occur under physiologic growth-factor conditions and at clinically relevant concentrations of imatinib mesylate. The amount of suppression observed was dose-dependent, with an average IC50 of 0.6 µM for CML CFCs and 1.7 µM for CML LTCICs exposed to the drug for 96 hours. Therefore, LTCICs appear to be somewhat less responsive to imatinib mesylate than are CFCs. Although suppression was observed with an exposure to imatinib mesylate as short as 24 hours, the amount of suppression increased with longer exposure to the drug. Under the most stringent conditions (5 µM imatinib mesylate for 96 hours), CML LTCICs decreased by 63% and CML CFCs by 78%. However, exposure for this length of time did not completely eliminate malignant CFCs or LTCICs. Under these same conditions, progenitors from healthy donors were not suppressed, except at the highest concentrations, and then only at the level of CFCs and not LTCICs. We analyzed the mechanisms underlying the growth suppression. CFSE
assays confirmed that CML primitive and committed progenitors had
increased proliferation compared with their normal counterparts, including both increased cell division and a reduced number of quiescent cells, particularly for primitive progenitor cells. Although
the increased proliferation was quite apparent in the CFSE assay, it
did not translate into an increased frequency of LTCICs. This can be
attributed to the observation that CML progenitors have increased
differentiation and decreased self-renewal compared with normal
progenitors.32-34 Imatinib mesylate suppressed
proliferation of primitive and committed CML progenitors. It largely
reversed the proliferative advantage of primitive progenitors,
decreasing cell divisions and increasing the proportion of undivided
cells. Because cell division may be associated with loss of primitive progenitor capacity, the decrease in proliferation by imatinib mesylate
may in fact reduce the loss of primitive progenitors through this
mechanism and contribute to the trend toward the increased number of
normal LTCICs observed following exposure to imatinib mesylate. This
could also explain why the suppression of LTCIC frequency was less than
the suppression of CD34+CD38 Although there is evidence from studies of BCR/ABL-positive cell lines that apoptosis is induced by treatment with imatinib mesylate,26,27 we did not consistently observe an increase in apoptosis in primary CD34+ cells at clinically achieved concentrations of the agent. Increased apoptosis was observed in 2 samples that also had the greatest sensitivity to inhibition of proliferation. However, the percentage of apoptotic cells never exceeded 32% in any sample for cells exposed to imatinib mesylate at a concentration below 5 µM, suggesting that increased apoptosis was not the major contributor to suppression of progenitor growth. Although apoptosis was induced when cells were exposed to 5 µM imatinib mesylate, increased apoptosis was also observed in normal samples, though to a lesser extent than in CML samples, suggesting that non-Bcr-Abl-dependent mechanisms may contribute to this process. Our findings are consistent with a hypothetical model in which Bcr-Abl tyrosine kinase activity results in enhanced proliferation of primitive and committed progenitors derived from the malignant clone, leading to a growth advantage over their normal counterparts, and in which exposure to imatinib mesylate, by reducing the abnormally increased proliferation of BCR/ABL-positive progenitors, removes their growth advantage. The effect of imatinib mesylate on primitive progenitor growth must reflect a balance of its effects on proliferation, survival, and self-renewal. Here, we showed that the predominant effect of imatinib mesylate is to inhibit proliferation. These results are consistent with those of a study by Marley et al18 in which a colony-replating assay was used to show that imatinib mesylate suppresses progenitor cell proliferation. In addition, Marley et al were unable to detect an increase in apoptosis associated with imatinib mesylate treatment. Although we did not observe a decrease in proliferation to a level below that of normal progenitors under the conditions of our assay, an increase in the time of exposure to imatinib mesylate beyond the 96 hours we examined would be expected to increase suppression of CML progenitors further. Consistent with this idea, we observed increased progenitor suppression after 96 hours of exposure to imatinib mesylate compared with 24 or 48 hours. Another factor leading to preferential loss of CML progenitors may be their decreased self-renewal capacity.32-34 Inhibition of progenitor proliferation in combination with decreased self-renewal may be sufficient to preferentially inhibit growth of malignant compared with normal progenitors during prolonged exposure to imatinib mesylate. This would also be consistent with the observation that more than 3 months of treatment is usually required for cytogenetic responses to imatinib mesylate treatment in patients with CML. An important implication of our studies is that in the absence of a significant apoptotic effect, malignant primitive progenitors may persist and not be completely eliminated by treatment with imatinib mesylate. Studies to determine the effects of long-term exposure to imatinib mesylate on residual malignant progenitors are under way. It should be noted that although we did not observe a consistent induction of apoptosis in our assays, it is possible that apoptosis may play a role in the elimination of progenitors in some patients. In the future, it will be important to explore the possibility that, in addition to the direct effects on progenitor growth described here, imatinib mesylate may indirectly affect progenitor growth by correcting abnormalities in responsiveness to microenvironmental influences such as ligands for adhesion30,35-37 and chemokine receptors.38-40 Our studies suggest that short-term exposure to imatinib mesylate ex vivo may not be sufficient to eliminate malignant progenitors. This indicates that use of imatinib mesylate alone may not adequately deplete malignant stem cells from CML autografts. Although apoptosis was not induced at low concentrations of imatinib mesylate, there was an increase in apoptosis when the agent was used at a concentration of 5 µM. The use of additional apoptosis-inducing agents may increase the elimination of BCR/ABL-positive cells and enhance the efficiency of imatinib mesylate as a purging agent. The range of responsiveness under the conditions studied was quite broad (residual CFC values ranged from 2% to 52% and LTCIC values from 0% to 114%). Our data do not indicate a strong association between response or lack of response to imatinib mesylate and stage of disease, time since diagnosis, or prior treatment. Only in a single sample (Figure 7) did the presence of a low percentage of BCR/ABL-positive cells (< 30%) correlate with decreased inhibition of progenitor growth. This sample contained a mixture of cells that were sensitive to imatinib mesylate and cells that were not responsive to the agent. Correlative studies to determine whether differences in the responsiveness of CML progenitors to imatinib mesylate in vitro correspond to in vivo responses are under way. In addition, correlation of the effect of imatinib mesylate with Bcr-Abl kinase activity and downstream signaling pathways may provide information about why progenitors from some samples are more responsive than others. To understand further the effect of imatinib mesylate on primary CML progenitors, it will be important to examine downstream signaling pathways affected by inhibition of Bcr-Abl tyrosine kinase activity. Decreased quiescence of CML progenitors may be related to activation of Ras, Raf, and mitogen-activated protein kinase, leading to activation of c-Myc and c-Fos and subsequent increases in transcription.41 Enhanced cell-cycle progression from G1 to S may be related to increased activated cyclin D complexes42 or decreases in p27KIP.43-46 The role of these signaling pathways in the response to imatinib mesylate will be the focus of future studies. In summary, we found that imatinib mesylate suppresses growth of CML but not normal primitive progenitors. At clinically relevant concentrations, imatinib mesylate reversed the abnormally increased proliferation that characterizes CML primitive progenitors but did not significantly increase apoptosis. These results suggest that inhibition of Bcr-Abl tyrosine kinase activity by imatinib mesylate restores normal hematopoiesis in patients with CML by removing the proliferative advantage of CML progenitors but that it may not eliminate all CML primitive progenitors. In future studies, we will investigate the signaling mechanisms underlying the effect of imatinib mesylate on primary primitive progenitor cells and correlate the results with clinical response.
We thank Novartis Pharmaceuticals, East Hanover, NJ, for providing imatinib mesylate for these studies. We acknowledge the excellent technical support of Lucy Brown and Claudio Spalla, Analytical Cytometry Core, who performed all the cell sorting; and we thank Kewei Cui, Allen Lin, Paula Goldfarb, Alison Ahlers, Jeanine Stevenson, and the physicians and staff in the Division of Hematology and Bone Marrow Transplantation for assistance with patient samples.
Submitted November 7, 2001; accepted January 11, 2002.
Supported in part by Public Health Services grant CA74455 from the National Cancer Institute, American Cancer Society grant RPG-99-202-01-LBC, and The Leukemia and Lymphoma Society Translational Research Grant 6468 to R.B. and by Public Health Services grant CA33572 to M.L.S.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Ravi Bhatia, Division of Hematology and Bone Marrow Transplantation, City of Hope National Medical Center, Duarte, CA 91010; e-mail: rbhatia{at}coh.org.
1. Fialkow PJ, Jacobson RJ, Papayannopoulou T. Chronic myelocytic leukemia: clonal origin in a stem cell common to the granulocyte, erythrocyte, platelet and monocyte/macrophage. Am J Med. 1977;63:125-130[CrossRef][Medline] [Order article via Infotrieve]. 2. Rowley JD. A new consistent chromosomal abnormality in chronic myelogenous leukaemia identified by quinacrine fluorescence and Giemsa staining [letter]. Nature. 1973;243:290-293[CrossRef][Medline] [Order article via Infotrieve].
3.
Kelliher MA, McLaughlin J, Witte ON, Rosenberg N.
Induction of a chronic myelogenous leukemia-like syndrome in mice with v-abl and BCR/ABL.
Proc Natl Acad Sci U S A.
1990;87:6649-6653
4.
Daley GQ, Van Etten RA, Baltimore D.
Induction of chronic myelogenous leukemia in mice by the P210bcr/abl gene of the Philadelphia chromosome.
Science.
1990;247:824-830 5. Van Etten RA. Disease progression in a murine model of bcr/abl leukemogenesis. Leuk Lymphoma. 1993;11:239-242.
6.
Pear WS, Miller JP, Xu L, et al.
Efficient and rapid induction of a chronic myelogenous leukemia-like myeloproliferative disease in mice receiving P210 bcr/abl-transduced bone marrow.
Blood.
1998;92:3780-3792
7.
Zhang X, Ren R.
Bcr-Abl efficiently induces a myeloproliferative disease and production of excess interleukin-3 and granulocyte-macrophage colony-stimulating factor in mice: a novel model for chronic myelogenous leukemia.
Blood.
1998;92:3829-3840
8.
Lugo TG, Pendergast AM, Muller AJ, Witte ON.
Tyrosine kinase activity and transformation potency of bcr-abl oncogene products.
Science.
1990;247:1079-1082
9.
Buchdunger E, Zimmermann J, Mett H, et al.
Inhibition of the Abl protein tyrosine kinase in vitro and in vivo by a 2-phenylaminopyrimidine derivative.
Cancer Res.
1996;56:100-104 10. Druker BJ, Tamura S, Buchdunger E, et al. Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nat Med. 1996;2:561-566[CrossRef][Medline] [Order article via Infotrieve].
11.
Heinrich MC, Griffith DJ, Druker BJ, Wait CL, Ott KA, Zigler AJ.
Inhibition of c-kit receptor tyrosine kinase activity by STI 571, a selective tyrosine kinase inhibitor.
Blood.
2000;96:925-932
12.
Okuda K, Weisberg E, Gilliland DG, Griffin JD.
ARG tyrosine kinase activity is inhibited by STI571.
Blood.
2001;97:2440-2448
13.
Carroll M, Ohno-Jones S, Tamura S, et al.
CGP 57148, a tyrosine kinase inhibitor, inhibits the growth of cells expressing BCR-ABL, TEL-ABL, and TEL-PDGFR fusion proteins.
Blood.
1997;90:4947-4952
14.
Druker BJ, Talpaz M, Resta DJ, et al.
Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia.
N Engl J Med.
2001;344:1031-1037
15.
Druker BJ, Sawyers CL, Kantarjian H, et al.
Activity of a specific inhibitor of the BCR-ABL tyrosine kinase in the blast crisis of chronic myeloid leukemia and acute lymphoblastic leukemia with the Philadelphia chromosome.
N Engl J Med.
2001;344:1038-1042
16.
Le Coutre P, Mologni L, Cleris L, et al.
In vivo eradication of human BCR/ABL-positive leukemia cells with an ABL kinase inhibitor.
J Natl Cancer Inst.
1999;91:163-168
17.
Deininger MW, Goldman JM, Lydon N, Melo JV.
The tyrosine kinase inhibitor CGP57148B selectively inhibits the growth of BCR-ABL-positive cells.
Blood.
1997;90:3691-3698
18.
Marley SB, Deininger MW, Davidson RJ, Goldman JM, Gordon MY.
The tyrosine kinase inhibitor STI571, like interferon- 19. Kasper B, Fruehauf S, Schiedlmeier B, Buchdunger E, Ho AD, Zeller WJ. Favorable therapeutic index of a p210(BCR-ABL)-specific tyrosine kinase inhibitor; activity on lineage-committed and primitive chronic myelogenous leukemia progenitors. Cancer Chemother Pharmacol. 1999;44:433-438[CrossRef][Medline] [Order article via Infotrieve]. 20. Traycoff CM, Halstead B, Rice S, McMahel J, Srour EF, Cornetta K. Chronic myelogenous leukaemia CD34+ cells exit G0/G1 phases of cell cycle more rapidly than normal marrow CD34+ cells. Br J Haematol. 1998;102:759-767[CrossRef][Medline] [Order article via Infotrieve].
21.
Eaves AC, Cashman JD, Gaboury LA, Kalousek DK, Eaves CJ.
Unregulated proliferation of primitive chronic myeloid leukemia progenitors in the presence of normal marrow adherent cells.
Proc Natl Acad Sci U S A.
1986;83:5306-5310
22.
Bedi A, Zehnbauer BA, Barber JP, Sharkis SJ, Jones RJ.
Inhibition of apoptosis by BCR-ABL in chronic myeloid leukemia.
Blood.
1994;83:2038-2044
23.
McGahon A, Bissonnette R, Schmitt M, Cotter KM, Green DR, Cotter TG.
BCR-ABL maintains resistance of chronic myelogenous leukemia cells to apoptotic cell death.
Blood.
1994;83:1179-1187 24. Amos TA, Lewis JL, Grand FH, Gooding RP, Goldman JM, Gordon MY. Apoptosis in chronic myeloid leukaemia: normal responses by progenitor cells to growth factor deprivation, X-irradiation and glucocorticoids. Br J Haematol. 1995;91:387-393[Medline] [Order article via Infotrieve]. 25. Clarkson BD, Strife A, Wisniewski D, Lambek C, Carpino N. New understanding of the pathogenesis of CML: a prototype of early neoplasia. Leukemia. 1997;11:1404-1428[CrossRef][Medline] [Order article via Infotrieve].
26.
Horita M, Andreu EJ, Benito A, et al.
Blockade of the Bcr-Abl kinase activity induces apoptosis of chronic myelogenous leukemia cells by suppressing signal transducer and activator of transcription 5-dependent expression of Bcl-xL.
J Exp Med.
2000;191:977-984
27.
Fang G, Kim CN, Perkins CL, et al.
CGP57148B (STI-571) induces differentiation and apoptosis and sensitizes Bcr-Abl-positive human leukemia cells to apoptosis due to antileukemic drugs.
Blood.
2000;96:2246-2253
28.
Bhatia R, McGlave PB, Dewald GW, Blazar BR, Verfaillie CM.
Abnormal function of the bone marrow microenvironment in chronic myelogenous leukemia: role of malignant stromal macrophages.
Blood.
1995;85:3636-3645
29.
Gupta P, McCarthy JB, Verfaillie CM.
Stromal fibroblast heparan sulfate is required for cytokine-mediated ex vivo maintenance of human long-term culture-initiating cells.
Blood.
1996;87:3229-3236
30.
Jiang Y, Zhao RC, Verfaillie CM.
Abnormal integrin-mediated regulation of chronic myelogenous leukemia CD34+ cell proliferation: BCR/ABL up-regulates the cyclin-dependent kinase inhibitor, p27Kip, which is relocated to the cell cytoplasm and incapable of regulating cdk2 activity.
Proc Natl Acad Sci U S A.
2000;97:10538-10543
31.
Bhatia R, Wayner EA, McGlave PB, Verfaillie CM.
Interferon- 32. Bhatia R, Munthe HA, Williams AD, Zhang F, Forman SJ, Slovak ML. Chronic myelogenous leukemia primitive hematopoietic progenitors demonstrate increased sensitivity to growth factor-induced proliferation and maturation. Exp Hematol. 2000;28:1401-1412[CrossRef][Medline] [Order article via Infotrieve].
33.
Bedi A, Griffin CA, Barber JP, et al.
Growth factor-mediated terminal differentiation of chronic myeloid leukemia.
Cancer Res.
1994;54:5535-5538
34.
Petzer AL, Eaves CJ, Barnett MJ, Eaves AC.
Selective expansion of primitive normal hematopoietic cells in cytokine-supplemented cultures of purified cells from patients with chronic myeloid leukemia.
Blood.
1997;90:64-69 35. Verfaillie CM, McCarthy JB, McGlave PB. Mechanisms underlying abnormal trafficking of malignant progenitors in chronic myelogenous leukemia: decreased adhesion to stroma and fibronectin but increased adhesion to the basement membrane components laminin and collagen type IV. J Clin Invest. 1992;90:1232-1239.
36.
Bhatia R, McCarthy JB, Verfaillie CM.
Interferon-
37.
Bhatia R, Munthe HA, Verfaillie CM.
Role of abnormal integrin-cytoskeletal interactions in impaired
38.
Cashman JD, Eaves CJ, Sarris AH, Eaves AC.
MCP-1, not MIP-1
39.
Eaves CJ, Cashman JD, Wolpe SD, Eaves AC.
Unresponsiveness of primitive chronic myeloid leukemia cells to macrophage inflammatory protein 1 40. Veena P, Cornetta K, Davidson A, et al. Preferential sequestration in vitro of BCR/ABL negative hematopoietic progenitor cells among cytokine nonresponsive CML marrow CD34+ cells. Bone Marrow Transplant. 1997;19:1213-1221[CrossRef][Medline] [Order article via Infotrieve]. 41. Verfaillie CM. Biology of chronic myelogenous leukemia. Hematol Oncol Clin North Am. 1998;12:1-29[CrossRef][Medline] [Order article via Infotrieve].
42.
Afar DE, McLaughlin J, Sherr CJ, Witte ON, Roussel MF.
Signaling by ABL oncogenes through cyclin D1.
Proc Natl Acad Sci U S A.
1995;92:9540-9544
43.
Zhao RC, Jiang Y, Verfaillie CM.
A model of human p210(bcr/ABL)-mediated chronic myelogenous leukemia by transduction of primary normal human CD34+ cells with a BCR/ABL-containing retroviral vector.
Blood.
2001;97:2406-2412
44.
Parada Y, Banerji L, Glassford J, et al.
BCR-ABL and interleukin 3 promote haematopoietic cell proliferation and survival through modulation of cyclin D2 and p27Kip1 expression.
J Biol Chem.
2001;276:23572-23580
45.
Jonuleit T, van der Kuip H, Miething C, et al.
Bcr-Abl kinase down-regulates cyclin-dependent kinase inhibitor p27 in human and murine cell lines.
Blood.
2000;96:1933-1939
46.
Gesbert F, Sellers WR, Signoretti S, Loda M, Griffin JD.
BCR/ABL regulates expression of the cyclin-dependent kinase inhibitor p27Kip1 through the phosphatidylinositol 3-kinase/AKT pathway.
J Biol Chem.
2000;275:39223-39230
© 2002 by The American Society of Hematology.
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  F. Pellicano, M. Copland, H. G. Jorgensen, J. Mountford, B. Leber, and T. L. Holyoake BMS-214662 induces mitochondrial apoptosis in chronic myeloid leukemia (CML) stem/progenitor cells, including CD34+38- cells, through activation of protein kinase C{beta} Blood, November 5, 2009; 114(19): 4186 - 4196. [Abstract] [Full Text] [PDF]
|
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 D. H. Mak, W. D. Schober, W. Chen, M. Konopleva, J. Cortes, H. M. Kantarjian, M. Andreeff, and B. Z. Carter Triptolide induces cell death independent of cellular responses to imatinib in blast crisis chronic myelogenous leukemia cells including quiescent CD34+ primitive progenitor cells Mol. Cancer Ther., September 1, 2009; 8(9): 2509 - 2516. [Abstract] [Full Text] [PDF]
|
|
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|
|
S. Grosso, A. Puissant, M. Dufies, P. Colosetti, A. Jacquel, K. Lebrigand, P. Barbry, M. Deckert, J. P. Cassuto, B. Mari, et al. Gene expression profiling of imatinib and PD166326-resistant CML cell lines identifies Fyn as a gene associated with resistance to BCR-ABL inhibitors Mol. Cancer Ther., July 1, 2009; 8(7): 1924 - 1933. [Abstract] [Full Text] [PDF]
|
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|
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|
 J. Mukherjee, D. Kamnasaran, A. Balasubramaniam, I. Radovanovic, G. Zadeh, T.-R. Kiehl, and A. Guha Human Schwannomas Express Activated Platelet-Derived Growth Factor Receptors and c-kit and Are Growth Inhibited by Gleevec (Imatinib Mesylate) Cancer Res., June 15, 2009; 69(12): 5099 - 5107. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Quintas-Cardama and J. Cortes Molecular biology of bcr-abl1-positive chronic myeloid leukemia Blood, February 19, 2009; 113(8): 1619 - 1630. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Konig, M. Copland, S. Chu, R. Jove, T. L. Holyoake, and R. Bhatia Effects of Dasatinib on Src Kinase Activity and Downstream Intracellular Signaling in Primitive Chronic Myelogenous Leukemia Hematopoietic Cells Cancer Res., December 1, 2008; 68(23): 9624 - 9633. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T.-S. Lee, W. Ma, X. Zhang, F. Giles, J. Cortes, H. Kantarjian, and M. Albitar BCR-ABL alternative splicing as a common mechanism for imatinib resistance: evidence from molecular dynamics simulations Mol. Cancer Ther., December 1, 2008; 7(12): 3834 - 3841. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Copland, F. Pellicano, L. Richmond, E. K. Allan, A. Hamilton, F. Y. Lee, R. Weinmann, and T. L. Holyoake BMS-214662 potently induces apoptosis of chronic myeloid leukemia stem and progenitor cells and synergizes with tyrosine kinase inhibitors Blood, March 1, 2008; 111(5): 2843 - 2853. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Konig, T. L. Holyoake, and R. Bhatia Effective and selective inhibition of chronic myeloid leukemia primitive hematopoietic progenitors by the dual Src/Abl kinase inhibitor SKI-606 Blood, February 15, 2008; 111(4): 2329 - 2338. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Jin, Y. Tabe, S. Konoplev, Y. Xu, C. E. Leysath, H. Lu, S. Kimura, A. Ohsaka, M.-B. Rios, L. Calvert, et al. CXCR4 up-regulation by imatinib induces chronic myelogenous leukemia (CML) cell migration to bone marrow stroma and promotes survival of quiescent CML cells Mol. Cancer Ther., January 1, 2008; 7(1): 48 - 58. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. D. Klion, J. Robyn, I. Maric, W. Fu, L. Schmid, S. Lemery, P. Noel, M. A. Law, M. Hartsell, C. Talar-Williams, et al. Relapse following discontinuation of imatinib mesylate therapy for FIP1L1/PDGFRA-positive chronic eosinophilic leukemia: implications for optimal dosing Blood, November 15, 2007; 110(10): 3552 - 3556. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. N. Anderson, D. L. Towne, D. J. Burns, and U. Warrior A High-Throughput Soft Agar Assay for Identification of Anticancer Compound J Biomol Screen, October 1, 2007; 12(7): 938 - 945. [Abstract] [PDF]
|
|
|
|
|
|
S. Chu, L. Li, H. Singh, and R. Bhatia BCR-Tyrosine 177 Plays an Essential Role in Ras and Akt Activation and in Human Hematopoietic Progenitor Transformation in Chronic Myelogenous Leukemia Cancer Res., July 15, 2007; 67(14): 7045 - 7053. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Rink, A. Slupianek, T. Stoklosa, M. Nieborowska-Skorska, K. Urbanska, I. Seferynska, K. Reiss, and T. Skorski Enhanced phosphorylation of Nbs1, a member of DNA repair/checkpoint complex Mre11-RAD50-Nbs1, can be targeted to increase the efficacy of imatinib mesylate against BCR/ABL-positive leukemia cells Blood, July 15, 2007; 110(2): 651 - 660. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Modi, T. McDonald, S. Chu, J.-K. Yee, S. J. Forman, and R. Bhatia Role of BCR/ABL gene-expression levels in determining the phenotype and imatinib sensitivity of transformed human hematopoietic cells Blood, June 15, 2007; 109(12): 5411 - 5421. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Jiang, K. M. Saw, A. Eaves, and C. Eaves Instability of BCR-ABL Gene in Primary and Cultured Chronic Myeloid Leukemia Stem Cells J Natl Cancer Inst, May 2, 2007; 99(9): 680 - 693. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Miething, R. Grundler, C. Mugler, S. Brero, J. Hoepfl, J. Geigl, M. R. Speicher, O. Ottmann, C. Peschel, and J. Duyster Retroviral insertional mutagenesis identifies RUNX genes involved in chronic myeloid leukemia disease persistence under imatinib treatment PNAS, March 13, 2007; 104(11): 4594 - 4599. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Wang, D. Cai, C. Brendel, C. Barett, P. Erben, P. W. Manley, A. Hochhaus, A. Neubauer, and A. Burchert Adaptive secretion of granulocyte-macrophage colony-stimulating factor (GM-CSF) mediates imatinib and nilotinib resistance in BCR/ABL+ progenitors via JAK-2/STAT-5 pathway activation Blood, March 1, 2007; 109(5): 2147 - 2155. [Abstract] [Full Text] [PDF]
|
|
|
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|
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M. Holtz, S. J. Forman, and R. Bhatia Growth Factor Stimulation Reduces Residual Quiescent Chronic Myelogenous Leukemia Progenitors Remaining after Imatinib Treatment Cancer Res., February 1, 2007; 67(3): 1113 - 1120. [Abstract] [Full Text] [PDF]
|
|
|
|
|
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Y. Hu, S. Swerdlow, T. M. Duffy, R. Weinmann, F. Y. Lee, and S. Li Targeting multiple kinase pathways in leukemic progenitors and stem cells is essential for improved treatment of Ph+ leukemia in mice PNAS, November 7, 2006; 103(45): 16870 - 16875. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Copland, A. Hamilton, L. J. Elrick, J. W. Baird, E. K. Allan, N. Jordanides, M. Barow, J. C. Mountford, and T. L. Holyoake Dasatinib (BMS-354825) targets an earlier progenitor population than imatinib in primary CML but does not eliminate the quiescent fraction Blood, June 1, 2006; 107(11): 4532 - 4539. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. G. Jorgensen, M. Copland, E. K. Allan, X. Jiang, A. Eaves, C. Eaves, and T. L. Holyoake Intermittent Exposure of Primitive Quiescent Chronic Myeloid Leukemia Cells to Granulocyte-Colony Stimulating Factor In vitro Promotes their Elimination by Imatinib Mesylate Clin. Cancer Res., January 15, 2006; 12(2): 626 - 633. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. Huff, W. Matsui, B. D. Smith, and R. J. Jones The paradox of response and survival in cancer therapeutics Blood, January 15, 2006; 107(2): 431 - 434. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. S. Ginsburg, R. P. Konstance, J. S. Allsbrook, and K. A. Schulman Implications of Pharmacogenomics for Drug Development and Clinical Practice Arch Intern Med, November 14, 2005; 165(20): 2331 - 2336. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. C. Wolff, D. R. Veach, W. P. Tong, W. G. Bornmann, B. Clarkson, and R. L. Ilaria Jr PD166326, a novel tyrosine kinase inhibitor, has greater antileukemic activity than imatinib mesylate in a murine model of chronic myeloid leukemia Blood, May 15, 2005; 105(10): 3995 - 4003. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Deininger, E. Buchdunger, and B. J. Druker The development of imatinib as a therapeutic agent for chronic myeloid leukemia Blood, April 1, 2005; 105(7): 2640 - 2653. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Chu, H. Xu, N. P. Shah, D. S. Snyder, S. J. Forman, C. L. Sawyers, and R. Bhatia Detection of BCR-ABL kinase mutations in CD34+ cells from chronic myelogenous leukemia patients in complete cytogenetic remission on imatinib mesylate treatment Blood, March 1, 2005; 105(5): 2093 - 2098. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. L. Ilaria Jr. Pathobiology of Lymphoid and Myeloid Blast Crisis and Management Issues Hematology, January 1, 2005; 2005(1): 188 - 194. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Yin, Y.-L. Wu, H.-P. Sun, G.-L. Sun, Y.-Z. Du, K.-K. Wang, J. Zhang, G.-Q. Chen, S.-J. Chen, and Z. Chen Combined effects of As4S4 and imatinib on chronic myeloid leukemia cells and BCR-ABL oncoprotein Blood, December 15, 2004; 104(13): 4219 - 4225. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Parmar, E. Katsoulidis, A. Verma, Y. Li, A. Sassano, L. Lal, B. Majchrzak, F. Ravandi, M. S. Tallman, E. N. Fish, et al. Role of the p38 Mitogen-activated Protein Kinase Pathway in the Generation of the Effects of Imatinib Mesylate (STI571) in BCR-ABL-expressing Cells J. Biol. Chem., June 11, 2004; 279(24): 25345 - 25352. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. M. Kvasnicka, J. Thiele, P. Staib, A. Schmitt-Graeff, M. Griesshammer, J. Klose, K. Engels, and S. Kriener Reversal of bone marrow angiogenesis in chronic myeloid leukemia following imatinib mesylate (STI571) therapy Blood, May 1, 2004; 103(9): 3549 - 3551. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. J. Jones, W. H. Matsui, and B. D. Smith Cancer Stem Cells: Are We Missing the Target? J Natl Cancer Inst, April 21, 2004; 96(8): 583 - 585. [Full Text] [PDF]
|
|
|
|
|
|
S. Chu, M. Holtz, M. Gupta, and R. Bhatia BCR/ABL kinase inhibition by imatinib mesylate enhances MAP kinase activity in chronic myelogenous leukemia CD34+ cells Blood, April 15, 2004; 103(8): 3167 - 3174. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Rosti, G. Martinelli, S. Bassi, M. Amabile, E. Trabacchi, B. Giannini, D. Cilloni, B. Izzo, A. De Vivo, N. Testoni, et al. Molecular response to imatinib in late chronic-phase chronic myeloid leukemia Blood, March 15, 2004; 103(6): 2284 - 2290. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Matei, D. D. Chang, and M.-H. Jeng Imatinib Mesylate (Gleevec) Inhibits Ovarian Cancer Cell Growth through a Mechanism Dependent on Platelet-Derived Growth Factor Receptor {alpha} and Akt Inactivation Clin. Cancer Res., January 15, 2004; 10(2): 681 - 690. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Bartolovic, S. Balabanov, U. Hartmann, M. Komor, A. M. Boehmler, H.-J. Buhring, R. Mohle, D. Hoelzer, L. Kanz, W.-K. Hofmann, et al. Inhibitory effect of imatinib on normal progenitor cells in vitro Blood, January 15, 2004; 103(2): 523 - 529. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Appel, A. M. Boehmler, F. Grunebach, M. R. Muller, A. Rupf, M. M. Weck, U. Hartmann, V. L. Reichardt, L. Kanz, T. H. Brummendorf, et al. Imatinib mesylate affects the development and function of dendritic cells generated from CD34+ peripheral blood progenitor cells Blood, January 15, 2004; 103(2): 538 - 544. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. La Rosee, K. Johnson, A. S. Corbin, E. P. Stoffregen, E. M. Moseson, S. Willis, M. M. Mauro, J. V. Melo, M. W. Deininger, and B. J. Druker In vitro efficacy of combined treatment depends on the underlying mechanism of resistance in imatinib-resistant Bcr-Abl-positive cell lines Blood, January 1, 2004; 103(1): 208 - 215. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Oehler, E. Jaeger, A. Eser, C. Sillaber, H. Gisslinger, and K. Geissler Imatinib mesylate inhibits autonomous erythropoiesis in patients with polycythemia vera in vitro Blood, September 15, 2003; 102(6): 2240 - 2242. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. W. N. Deininger and B. J. Druker Specific Targeted Therapy of Chronic Myelogenous Leukemia with Imatinib Pharmacol. Rev., September 1, 2003; 55(3): 401 - 423. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Bhatia, M. Holtz, N. Niu, R. Gray, D. S. Snyder, C. L. Sawyers, D. A. Arber, M. L. Slovak, and S. J. Forman Persistence of malignant hematopoietic progenitors in chronic myelogenous leukemia patients in complete cytogenetic remission following imatinib mesylate treatment Blood, June 15, 2003; 101(12): 4701 - 4707. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Decker, S. Hipp, I. Ringshausen, C. Bogner, M. Oelsner, F. Schneller, and C. Peschel Rapamycin-induced G1 arrest in cycling B-CLL cells is associated with reduced expression of cyclin D3, cyclin E, cyclin A, and survivin Blood, January 1, 2003; 101(1): 278 - 285. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. J. Druker, S. G. O'Brien, J. Cortes, and J. Radich Chronic Myelogenous Leukemia Hematology, January 1, 2002; 2002(1): 111 - 135. [Abstract] [Full Text]
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Top
Abstract
Introduction
) and committed
(CD34+CD38+) progenitors to a much greater
extent than normal cells. Conversely, treatment with 1 to 2 µM
imatinib mesylate did not significantly increase the percentage of
cells undergoing apoptosis. Although a higher concentration of imatinib
mesylate (5 µM) led to an increase in apoptosis of CML cells,
apoptosis also increased in normal samples. In summary, at clinically
relevant concentrations, imatinib mesylate selectively suppresses CML
primitive progenitors by reversing abnormally increased proliferation
but does not significantly increase apoptosis. These results suggest
that inhibition of Bcr-Abl tyrosine kinase by imatinib mesylate
restores normal hematopoiesis by removing the proliferative advantage
of CML progenitors but that elimination of all CML progenitors may not occur.
(Blood. 2002;99:3792-3800)
- -) samples, is based on replicate experiments (CML,
n = 5; normal, n = 4). Concentrations of imatinib mesylate at which
CML progenitors were significantly suppressed compared with no exposure
to imatinib mesylate are indicated by asterisks below the curve (3 asterisks, P < .001; 2 asterisks, P < .01;
and 1 asterisk, P < .05). Concentrations of imatinib
mesylate at which CML progenitor frequency was significantly different
from normal progenitor frequency are indicated by daggers above the
curve (3 daggers, P < .001; 2 daggers,
P < .01; and 1 dagger, P < .05). (A) LTCIC
frequency was calculated in limiting-dilution assays. The progenitor
frequency normalized to the frequency of progenitors in the absence of
imatinib mesylate is shown for each concentration of the drug. LTCIC
frequency (mean ± SEM) in the absence of imatinib mesylate was
15 ± 10/1000 input CD34+ cells for CML samples and
7 ± 3/1000 cells for normal samples. (B) CFC frequency is plotted
for each concentration of imatinib mesylate, normalized to the colony
number obtained in the absence of imatinib mesylate. CFC frequency
(mean ± SEM) in the absence of imatinib mesylate was
366 ± 87/1000 input CD34+ cells for CML samples and
101 ± 29/1000 cells for normal samples.
- -), and 5 µM (
) and
BCR/ABL-positive (
) progenitor frequencies (LTCICs and
CFCs) for CML CD34+ cells exposed to imatinib mesylate for
96 hours are shown. The frequency of BCR/ABL-positive
progenitors equals the total number of progenitors times the percentage
of cells that were BCR/ABL positive on FISH analysis of
pooled colonies. The data plotted are normalized to the number of total
progenitors observed in the absence of imatinib mesylate and are shown
as the mean ± SEM.
). Significant changes in the percentage of cells in any
generation in response to imatinib mesylate exposure are indicated (2 asterisks, P < .01; and 1 asterisk,
P < 0.05). (D) The PI for CML (
1 integrin receptor function.
J Clin Invest.
1994;94:384-391.