Chronic myelogenous leukemia (CML) is characterized by a t(9;22) chromosome translocation
(Philadelphia chromosome-positive [Ph+]) that creates the BCR/ABL oncogene. This fusion
protein displays constitutive tyrosine kinase activity, leading to the induction of
aberrant proliferation and neoplastic transformation [1]. The Ph+ chromosome is found
in more than 95% of CML and in Ph+ acute lymphoblastic leukemia.
ABL tyrosine kinase inhibitors (AKIs) are utilized for the treatment of Ph+ leukemia;
the initial response is beneficial [2–4] but unfortunately, the clinical efficacy
of this treatment decreases steadily as the disease progresses [5]. CML blast crisis
or Ph+ acute lymphoblastic leukemia patients only benefit from TKI treatment temporarily,
if at all [6, 7].
Resistance to drug therapy in Ph+ leukemia is mediated by mutations within BCR/ABL
or by BCR/ABL-independent mechanisms, such as tumor microenvironment-mediated drug
resistance [8]. The bone marrow (BM) plays a vital role in hematopoiesis, as well
as in different aspects of disease progression in hematological malignancies [8, 9].
The BM microenvironment is also critical for long-term hematopoiesis, and for the
maintenance and regulation of stem cells and their progeny [9]. It is a rich source
of paracrine- and autocrine-derived factors which have also been implicated in drug
resistance for both hematologic malignancies and solid tumors that metastasize to
the BM [10–12]. Conditions within the BM niche, including soluble factors (SFs), interleukins
(ILs), stromal cells, and extracellular components, contribute to reduced drug sensitivity
of cancer cells [10, 13, 14], including drug resistance to multiple TKIs, such as
imatinib, nilotinib, and dasatinib [15–19].
To explore the BCR/ABL-independent mechanisms underlying Ph+ leukemia drug resistance,
we investigated the ability of SFs collected from mesenchymal stem cells (MSCs) to
confer drug resistance in CML cell lines in vitro. Exposure of CML cells to mesenchymal
SFs conferred resistance to imatinib, but not crizotinib, which was mediated in part
by the Janus kinase/signal transducers and activators of transcription (JAK/STAT)
pathway. Inhibition of JAK2 by ruxolitinib restored sensitivity to imatinib in CML
cells exposed to mesenchymal SFs. Moreover, the multi-TKI crizotinib, which has been
reported to also inhibit ABL tyrosine kinase activity, was also able to abrogate JAK2
activity, thereby overcoming SF-mediated drug resistance in Ph+ leukemia.
Blast crisis human chronic myelogenous leukemia cell line K562 and megakaryocytic
leukemia cell line MEG-01 (American Type Culture Collection (ATCC), VA, USA), and
Ba/F3 JAK2 V617F cells expressing activated JAK2 and harboring the V617F mutation,
have been described previously [20]. Cells were cultured in RPMI 1640 complete medium
supplemented with 10% (w/v) fetal bovine serum, 1% (w/v) L-glutamine, and penicillin/streptomycin.
The human and murine stromal cells HS-5 and MS-5, respectively (ATCC), were maintained
under the same conditions. All cells were grown at 37°C in a humidified atmosphere
with 5% CO2.
HS-5 or MS-5 cells were grown in complete RPMI medium and CM were collected after
72 h. Contaminating cells were cleared by centrifugation at 1,000 rpm for 1 min; the
collected supernatant was used fresh in each experiment.
Cells (2 × 105/well) were plated in six-well plates. After 24 h, cells were treated
with the specified agents. Solvent-treated samples were incubated with 1% (w/v) dimethyl
sulfoxide. Cells were collected 72 h later, stained with 0.4% (w/v) trypan blue solution
(1 : 1, v/v), and counted using a hemacytometer [20].
Cytokine levels were measured in the CM of MS-5 cells using mouse cytokine array panel
A (Proteome Profiler Array, R&D Systems, MN, USA). Briefly, CM were mixed with a cocktail
of biotinylated detection antibodies. The CM–antibody mixture was then incubated with
the mouse cytokine array membrane. Following a wash to remove unbound material, streptavidin
conjugated to horseradish peroxidase and chemiluminescent detection reagents were
added sequentially. Data were visualized using X-ray film. Optical densities were
determined using ImageJ software.
Clonogenicity assay was performed as previously described [21]. The plates were stained
for 4 h with 5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide,
and the dye was extracted with solubilization buffer (20% w/v sodium dodecyl sulfate
(SDS), 50% w/v N,N-dimethylformamide, 25 mM HCl) for 24 h. Optical density was measured
at 570 nm with a reference wavelength of 630 nm.
Immunoblotting was performed as described previously [22]. Anti-pSTATs (phosphor-Stat
antibody sampler kit cat #9914), anti-JAK2 (cat #3230), anti-pJAK2 (Tyr 1007/1008)(cat
#3776), anti-phosphor Abl (Tyr 245) (cat #2861) and anti-cleaved PARP (cat #5625)
antibodies were from Cell Signaling Technology (MA, USA). Anti-PARP (cat #SC-8007)
antibodies were from Santa Cruz Biotechnology (CA, USA).
Student’s t-test was used for the statistical analyses, with the significance level
set at p < 0.01 or p < 0.001.
To address drug sensitivity of CML cell lines to AKIs in the presence of CM collected
from MSC cells, we utilized human K562 [23] and MEG-01 cells [24] as an in-vitro model
of CML. We followed the effect of imatinib on proliferation and triggering of PARP
cleavage as a marker of apoptosis induction [25] in MEG-01 CML cells in the presence
and absence of CM collected from MS-5 cells. Exposure of MEG-01 cells to various concentrations
of MS-5 CM (6.25–25%) resulted in increased cell survival in the presence of imatinib
(Figure 1 A). In fact, as little as 3% MS-5 CM was sufficient to confer partial drug
resistance in MEG-01 cells treated with imatinib (data not shown). Next, we measured
the effect of MS-5 CM on imatinib-induced growth inhibition using K562 and MEG-01
CML cells. MS-5 CM protected cells of both CML lines from imatinib-induced growth
inhibition, albeit with varying potency. Overall, the magnitude of the drug resistance
observed in MEG-01 cells was more than 2-fold greater than that in K562 cells (Figure
1 B). This suggested that murine SFs are active against human CML cells, although
the basis for the different responses of K562 and MEG-01 cells to MS-5 CM is unclear.
Nevertheless, we chose to further focus on drug sensitivity of MEG-01 cells in response
to exposure to MS-5 CM.
Figure 1
Mesenchymal stem cell (MSC) conditioned medium (CM) confers drug resistance to imatinib
in CML cells. A – MEG-01 and K562 cells were exposed to increasing concentrations
of CM (0, 6.25, 12.5, 25%) collected from MS-5 or HS-5, and viability of remaining
cells after treatment with 1 μM imatinib for 72 h was determined. CM collected from
MS-5 or HS-5 supplied to MEG-01 and K562, respectively. B – MEG-01 and K562 cells
were exposed to 12.5% CM collected from MSC cells in the presence or absence of 1
μM imatinib for 72 h. Negative control – 12.5% RPMI medium +1% FCS. *,**Significantly
different from untreated cells at p < 0.01 and 0.001, respectively. The experiment
was repeated twice, with a comparable outcome
We asked whether CM collected from other cells might promote resistance to imatinib
in MEG-01 cells. CM collected from A2780 and B16-F10 cell lines were not efficient
at conferring drug resistance to imatinib (Supplementary Figure S1 A). Additional
data (Supplementary Figure S1 B) supported involvement of SFs, but not microvessels,
as mediators of drug resistance in CML exposed to MSC CM [26].
We recently demonstrated that crizotinib, an anaplastic lymphoma kinase (ALK)/ROS1
inhibitor [27, 28], efficiently and selectively suppresses growth of Ph+ cells, and
inhibits activity of native and T315I-mutated BCR/ABL [29, 30]. Here, we also explored
the ability of CM collected from MSCs to induce drug resistance in MEG-01 cells exposed
to crizotinib. Figure 2 shows that MS-5 CM conferred significant drug resistance to
imatinib in MEG-01 cells, but had a minimal protective effect in cells exposed to
either ponatinib [31] or crizotinib (Figure 2 A). Next, we monitored the ability of
MSC CM to interfere with imatinib- and crizotinib-induced apoptosis of MEG-01 cells.
Initially, MEG-01 cells were treated with crizotinib or imatinib for 24 h in the presence
or absence of MS-5 CM. Exposure to imatinib/crizotinib significantly increased the
levels of cleaved PARP enzyme (Figure 2 B) [25]. Interestingly, the presence of MS-5
CM prevented PARP cleavage, again suggesting that it confers drug resistance to imatinib
in MEG-01 cells (Figure 2 B). In contrast, exposure of MEG-01 cells to crizotinib
resulted in significant cleavage of PARP, indicating induction of apoptosis (Figure
2 B). Next, we examined the effect of MS-5 CM on the activity of BCR/ABL in MEG-01
cells exposed to imatinib or crizotinib. Data presented in Figures 2 C and D show
that imatinib was active in inhibiting BCR/ABL auto-phosphorylation in the presence
and absence of MS-5 CM. In addition, the high concentration of crizotinib was active
at inhibiting BCR/ABL auto-phosphorylation, but showed only marginal activity at the
low concentration, in the presence and absence of MS-5 CM. Imatinib was active at
inducing PARP cleavage in MEG-01 cells, and induction of PARP cleavage was largely
reduced (by about 50%) in the presence of MS-5 CM. In contrast, crizotinib induction
of PARP cleavage was not reduced, but rather enhanced in the presence of MS-5 CM.
Thus, the apoptosis-inducing activity of imatinib, but not of crizotinib, was reduced
in the presence of MS-5 CM with no significant change in imatinib’s ability to inhibit
BCR/ABL auto-phosphorylation.
Figure 2
Crizotinib overcomes mesenchymal stem cell (MSC) conditioned medium (CM)-mediated
drug resistance. A – MEG-01 cells were exposed to 1 µM imatinib, ponatinib or crizotinib
in the presence or absence of 12.5% HS-5 CM and the number of viable cells was counted
after 72 h exposure. B – MEG-01 cells were exposed to 1 µM imatinib, ponatinib or
crizotinib in the presence or absence of 12.5% MS-5 CM and the number of viable cells
was counted after 72 h exposure. C – Immunoblot of MEG-01 cells exposed to 1 µM imatinib
or crizotinib in the presence or absence of MS-5 CM. In addition, 1 mM imatinib or
crizotinib was added to MEG-01 cells co-cultured with MS-5 cells. Filters were probed
with anti-cleaved (c)-PARP antibody and a-tubulin was used as a loading control. D
– Immunoblot of K562 cells exposed to 0.5 µM and 2 µM imatinib or crizotinib in the
presence or absence of MS-5 CM. Filters were probed with anti-c-PARP antibody, anti-phospho
Abl (Tyr 245) and anti-GAPDH was used as a loading control. E – Quantitation of experiment
in D showing relative levels of cleaved PARP and phospho BCR/ABL (relative to GAPDH).
*Significantly different from untreated cells at p < 0.01. The experiment was repeated
twice, with a comparable outcome
To identify potential SFs in the MS-5 CM that are involved in mediating drug resistance
to imatinib, we monitored the levels of a variety of SFs using a cytokine array assay
kit. Figures 3 A and B shows a number of cytokines and ILs, in particular IL-3 and
IL-17 (Figures 3 A, B), that were present at high levels in the MS-5 CM, whereas others,
such as IL-1Rα and IL-12 p70, were present at low levels (Figures 3 A, B). We hypothesized
that one or a few cytokines are involved in mediating imatinib drug resistance in
CML cells, and we selected IL-3 to further investigate this. Increasing concentrations
of murine IL-3 conferred resistance to imatinib in MEG-01 cells in a dose-dependent
manner (Figure 3 C).
Figure 3
Identification of soluble factors (SFs) in MS-5 conditioned medium (CM) as potential
mediators of imatinib resistance in MEG-01 cells. CM was collected from MS-5 cells
after 72 h of culture. A – Immunoblot test for relative expression levels of 40 factors
in MS-5 CM was carried out according to the manufacturer’s instructions. B – Optic
absorption was measured by TINA software and levels of soluble factors in MS-5 CM
relative to RPMI are presented. C – Cell viability (number of remaining cells) of
K562 cells treated with 1 µM imatinib and increasing concentrations of murine (m)
IL-3 for 72 h was determined. *Significantly different from untreated cells at p <
0.01. The experiment was repeated twice, with a comparable outcome
JAK/STAT pathways are downstream regulators of a number of ILs, such as IL-6 and IL-3,
which have also been implicated in drug resistance in CML [15, 32, 33]. Moreover,
emerging data also implicate the JAK/STAT pathway in BM-mediated drug resistance [15,
16, 19]. Thus, we investigated the involvement of the JAK/STAT pathway in the observed
imatinib resistance in CML cells exposed to MS-5 CM. MEG-01 cells were exposed to
MSC CM and levels of various phosphorylated STATs were monitored.
The levels of both pSTAT1 and pSTAT2 were undetectable in the MEG-01 cells, with no
change after exposure to either MS-5 CM (Figure 4 A) or HS-5 CM (data not shown).
Moreover, STAT6 and STAT3 were not phosphorylated in MEG-01 cells, and exposure to
MS-5 or HS-5 CM significantly stimulated their phosphorylation levels. In contrast,
STAT5, which is a direct downstream target of BCR/ABL [34–36], was significantly phosphorylated
in MEG-01 cells and the phosphorylation levels were further increased upon exposure
to both HS-5 and MS-5 CM (Figure 4 A).
Figure 4
MS-5 conditioned medium (CM) stimulates JAK/STAT pathway in MEG-01 cells. A – MEG-01
cells exposed to 12.5% CM collected from MS-5 and HS-5 cultures were used to monitor
levels of activated STATs by immunoblotting. MEG-01 cells were seeded and exposed
to 12.5% CM from MS-5/HS-5 cells for 2 h. Cells were collected, and the phosphorylation
of STAT3/4/6 was monitored by immunoblotting. Tubulin was used as a loading control.
B – MEG-01 cell viability was monitored after exposure to 12.5% MS-5 CM in the presence
of 1 μM imatinib, imatinib + ruxolitinib or crizotinib alone. Cell viability was monitored
72 h after treatment. C – Ability of different kinase inhibitors to induce apoptosis
in MEG-01 cells in the presence of MS-5 CM. MEG-01 cells were treated with 1 µM of
the different Abl kinase inhibitors in the presence or absence of 12.5% CM for 24
h. The complete PARP protein (120 kDa) was used as a positive control. *,**Significantly
different from untreated cells at p < 0.01 and 0.001, respectively. The experiment
was repeated twice, with a comparable outcome
STATs are downstream transcription factors that are activated by JAKs in response
to cytokine activation. Thus, we investigated the influence of JAK inhibitors on imatinib
resistance mediated by MS-5 CM. Figure 4 B shows the viability of MEG-01 cells exposed
to MS-5 CM in the presence of imatinib and the clinically approved JAK1/2 inhibitor
ruxolitinib [37]. As expected, exposure of MEG-01 cells to MS-5 CM conferred drug
resistance to imatinib, but not crizotinib. When ruxolitinib was added along with
imatinib, MEG-01 sensitivity to the latter was partially restored (Figure 4 B), supporting
the involvement of JAK1/2 in mediating the MSC CM-induced drug resistance. Further
support for the ability of ruxolitinib to restore sensitivity to imatinib in the presence
of MS-5 CM was provided by following the levels of cleaved PARP in MEG-01 cells treated
with imatinib, crizotinib, ruxolitinib, or imatinib + ruxolitinib in the presence
and absence of MS-5 CM. Imatinib and crizotinib induced PARP cleavage in MEG-01 cells.
However, no PARP cleavage was observed in MEG-01 cells treated with either imatinib
or ruxolitinib alone in the presence of MS-5 CM (Figure 4 C). On the other hand, ruxolitinib
+ imatinib effectively caused significant PARP cleavage, indicating restoration of
partial sensitivity of MEG-01 cells to imatinib in the presence of MS-5 CM (Figure
4 C).
Data presented in Figures 4 B and C show that inhibition of JAK1/2 (by ruxolitinib)
can restore sensitivity to imatinib in CML cells exposed to MS-5 CM. We therefore
speculated that crizotinib’s ability to overcome mesenchymal SF-mediated drug resistance
in CML might be related to JAK1/2 activity. To address this point, we utilized a Ba/F3
model system in which cells become IL-3-independent upon introduction of activated
JAK2 (JAK2 V617F) [20]. Use of this system allowed us to monitor the direct effect
of crizotinib on JAK2 activity. Initially, we monitored the effects of crizotinib
and other relevant TKIs on the clonogenicity of Ba/F3 JAK2 V617F cells. Figures 5
A and B shows that the colony-forming ability of Ba/F3 JAK2 V617F cells was not affected
by imatinib or other ABL inhibitors such as GNF-2 or GNF-5 [38]. However, crizotinib
inhibited clonogenicity of Ba/F3 JAK2 V617F cells with a potency comparable to that
of ruxolitinib. Interestingly, ponatinib was also a potent inhibitor of Ba/F3 JAK2
V617F clonogenicity.
Figure 5
Crizotinib inhibits JAK2 activity. A – Ba/F3 JAK2 V617F cells were used to monitor
different kinase inhibitors’ effects on clonogenicity as described in Material and
methods. B – Areas of each sample were determined by monitoring absorbance using Image
J and values obtained in samples treated with 0.1 µM and 1 µM kinase inhibitor were
plotted. C – Ba/F3 JAK2 V617F cells were exposed for 1 h to different concentrations
of imatinib, crizotinib (1, 3 and 10 µM) or GNF-5 (3 and 10 µM). Ruxolitinib at 1
µM was used as a positive control. Cells were collected and lysed and their proteins
were separated by SDS-PAGE. Levels of pJAK2 were monitored relative to JAK2 and a-tubulin
was used as a loading control. D – Levels of pJAK2 relative to a-tubulin. The experiment
was repeated twice, with a comparable outcome
Next, we utilized the same cells to monitor direct inhibition of JAK2 auto-phosphorylation
by crizotinib. Ruxolitinib actively inhibited auto-phosphorylation of JAK2 V617F,
and crizotinib also exhibited strong inhibition of JAK2 auto-phosphorylation. In contrast,
neither imatinib nor GNF-5 inhibited JAK2 auto-phosphorylation (Figures 5 C, D).
CML-targeted therapy has proven to be very effective in managing the disease and has
led to extended lifespans for many CML patients. However, about 30% of those patients
fail to respond, respond suboptimally, or experience disease relapse after treatment
with CML-targeted therapy due, in part, to drug resistance [5, 39]. In general, Ph+
leukemia resistance to drug therapy might be due to mutations in the cancer cells,
including alterations in the BCR/ABL fusion gene, or associated with interactions
between Ph+ leukemia cancer cells and the BM microenvironment, leading to tumor microenvironment-mediated
drug resistance and consequently a low level of residual disease and disease progression
[8].
Intensive effort has been invested in developing strategies to address drug resistance
in Ph+ leukemia by introducing new TKIs that are active against ABL1 mutations, including
the gatekeeper T315I mutation [40]. Moreover, effort is still being made to target
additional regulatory elements within the ABL1 kinase, such as the myristoyl-binding
pocket in the BCR/ABL protein [20, 41], or to use a drug combination [21, 42].
The BM microenvironment contributes significantly to drug resistance in both hematologic
malignancies and solid tumors that metastasize to the BM [10–12]. Conditions in the
BM niche that contribute to reduced drug sensitivity might include SFs, stromal cells,
and extracellular components [10, 13, 14, 43]. Thus, modulation of signaling pathways
involved in mediating the interaction, adhesion, or homing of hematopoietic cancer
cells to BM stromal cells is expected to influence drug sensitivity in hematopoietic
malignancies [13–16].
The BM microenvironment-mediated drug resistance might be due to SFs or to cell adhesion
to the microenvironment compartment. To address BM microenvironment-mediated drug
resistance, we monitored the ability of CM collected from MSCs to affect the sensitivity
of CML cells to imatinib and crizotinib. We monitored the viability of CML cells as
well as induction of PARP cleavage (Figures 1, 2), a marker of apoptosis induction
[25].
MEG-01 cells were sensitive to imatinib and crizotinib as evidenced by inhibition
of CML cell viability (Figures 1 A, B) and induction of PARP cleavage (Figures 2 B,
C). However, CM collected from MSCs conferred resistance to imatinib, consistent with
previous observations [16]. Interestingly, MEG-01 cells were more sensitive to MS-5
CM than K562 cells. The presence of CM from MSCs reduced the sensitivity of the CML
cells to imatinib-induced apoptosis, but not to crizotinib, suggesting that mesenchymal
SFs can promote imatinib resistance with continued sensitivity to the activity of
crizotinib (Figure 2). We speculated that drug resistance stimulated by the CM is
mediated by SFs secreted by the MSCs. We identified a number of SFs that were present
in significant amounts in the CM, such as IL-3, IL-1Rα, IL-17 and IL-12 p70, and speculated
that one or a combination of these is responsible for mediating imatinib resistance
in CML cells. Our speculation was supported by previous reports showing that IL-7
secreted by MSCs in the BM might protect leukemic cells from apoptosis induced by
imatinib through the JAK/STAT-signaling pathway [44]. In addition, Jiang et al. [45]
demonstrated autocrine production and activity of IL-3 and granulocyte colony-stimulating
factor (G-CSF) in CML [45]. Moreover, culturing K562 cells with HS-5-derived CM significantly
inhibited apoptosis induced by imatinib via a STAT3-dependent mechanism [18], and
imatinib sensitivity was restored by exposure to STAT3 inhibitor [46]. Our results
are also corroborated by Zhang et al. [43], who found that CML stem cells demonstrate
increased IL-1 receptor expression and an enhanced signaling response; however, inhibition
of IL-1 signaling when combined with TKIs enhanced anti-CML efficacy. In our study,
we also monitored levels of activated STAT proteins and found that the presence of
MSC CM results in activation of STAT3 and STAT6 (Figure 4). Increased activation of
STAT3 has been associated with malignant cell transformation and drug-resistant tumors
[44, 46, 47]. Moreover, exposure of CML cells to MSC CM caused an increase in pSTAT3
and consequently increased the expression of STAT3-regulated genes such as Bcl-xl,
Mcl-1, and survivin [46]. Thus, we argue that exposure to SFs that stimulate activation
of STAT3 or STAT6 promotes the expression of a variety of antiapoptotic genes [48,
49], and hence promotes drug resistance.
JAK family kinases are the upstream activators of STAT proteins and also participate
in mediating the function of a variety of cytokines [50]. Thus, we monitored the ability
of the JAK1/2 inhibitor ruxolitinib [37] to abolish the drug resistance mediated by
MSC CM. The presence of ruxolitinib restored partial imatinib sensitivity in CML cells
exposed to MS-5 CM. Our results are consistent with data reported by Mencalha et al.
[51] showing that STAT3 inhibitor has an additive effect with imatinib in inducing
apoptosis in CML cells, suggesting a potential therapeutic value to combining these
two drug regimens for the treatment of CML patients. Moreover, evidence for the potential
of drug combinations in CML therapy was also provided by Ma et al. [52], who showed
inhibition of growth and proliferation, cell-cycle blockade, and induction of apoptosis
in K562 cells transduced with STAT3 siRNA. Thus, the use of two components was recommended
to overcome SF-mediated drug resistance: one to inhibit BCR/ABL and the other to inhibit
JAK1/2 activity. However, with the approval of multitarget kinase inhibitors, one
might select those that are capable of inhibiting both relevant targets. Previously,
we demonstrated that crizotinib efficiently inhibits kinase activity of native and
T315I-mutated BCR/ABL [29]. In this study, we also found that crizotinib inhibits
JAK2 activity. However, crizotinib activity against JAK1 remains to be determined.
Crizotinib’s ability to inhibit JAK2 enables it to overcome JAK2-dependent SF-mediated
drug resistance. Similarly, ponatinib as a multi-kinase inhibitor was active in overcoming
SF-mediated drug resistance in MEG-01, in part by targeting JAK2 activity, probably
indirectly (Figure 5). The exact mechanism by which ponatinib inhibited clonogenicity
of Ba/F3 JAK2 V617F remains to be elucidated.
In conclusion, the study showed that SFs secreted from MSC, such IL-3 and IL-7, among
others, are capable of activating the JAK/STAT pathway and consequently compromise
the apoptosis-inducing activity of imatinib targeting CML cells. On the other hand,
the multi-kinase inhibitor crizotinib, an FDA-approved drug for ALK-positive non-small
cell lung carcinoma targeting ALK kinase, was found to inhibit BCR/ABL kinase activity
and is also active in inhibiting JAK2. Exposure to crizotinib actively overcame drug
resistance in CML mediated by SFs secreted from MSC. Our results raise the possibility
of therapeutic use of crizotinib for CML patients who also require a JAK2 inhibitor
to overcome BM microenvironment-mediated drug resistance.
Supplementary Material
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