Identification of mTOR as a novel bifunctional target in chronic myeloid leukemia: dissection of growth-inhibitory and VEGF-suppressive effects of rapamycin in leukemic cells

©2005 FASEB

The FASEB Journal express article 10.1096/fj.04-1973fje. Published online March 22, 2005.

Identification of mTOR as a novel bifunctional target in chronic myeloid leukemia: dissection of growth-inhibitory and VEGF-suppressive effects of rapamycin in leukemic cells Matthias Mayerhofer,* Karl J. Aichberger,* Stefan Florian,* Maria-Theresa Krauth,* Alexander W. Hauswirth,* Sophia Derdak,� Wolfgang R. Sperr,* Harald Esterbauer,� Oswald Wagner,� Christine Marosi,§ Winfried F. Pickl,� Michael Deininger,║

Ellen Weisberg,¶ Brian J. Druker,# James D. Griffin,¶ Christian Sillaber,* and Peter Valent* *Department of Internal Medicine I, Division of Hematology & Hemostaseology; �Institute of Immunology, �Clinical Institute of Medical and Chemical Laboratory Diagnostics, and §Department of Internal Medicine I, Division of Oncology, Medical University of Vienna, Austria; ║Oregon Health and Science University Cancer Institute, Center for Hematological Malignancies, Portland, Oregon; ¶Department of Adult Oncology, Dana Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts; and #Howard Hughes Medical Institute, Portland, Oregon

Corresponding author: M. Mayerhofer, Department of Internal Medicine I, Division of Hematology & Hemostaseology, Medical University of Vienna, Waehringer Guertel 18-20, A-1097 Vienna, Austria. E-mail: [email protected]

ABSTRACT

The mammalian target of rapamycin (mTOR) has recently been described to be constitutively activated in Bcr-Abl-transformed cells and to mediate rapamycin-induced inhibition of growth in respective cell lines. We have recently shown that rapamycin down-regulates expression of vascular endothelial growth factor (VEGF), a mediator of leukemia-associated angiogenesis, in primary CML cells. In the present study, we analyzed growth-inhibitory in vitro and in vivo effects of rapamycin on primary CML cells and asked whether rapamycin-induced suppression of VEGF in leukemic cells is related to growth inhibition. Rapamycin dose dependently inhibited growth of primary CML cells obtained from patients with imatinib-responsive or imatinib-resistant disease as well as growth of Bcr-Abl-transformed imatinib-resistant cell lines. Moreover, we observed potent cytoreductive effects of rapamycin in a patient with imatinib-resistant Bcr-Abl+ leukemia. The growth-inhibitory effects of rapamycin on CML cells were found to be associated with G1 cell cycle arrest and with induction of apoptosis. In all cell types tested, rapamycin was found to down-regulate expression of VEGF. However, exogenously added VEGF did not counteract the rapamycin-induced decrease in proliferation. In conclusion, rapamycin inhibits growth of CML cells in vitro and in vivo and, in addition, down-regulates expression of VEGF. Both effects may contribute to the antileukemic activity of the drug in CML.

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Key words: CML � Bcr-Abl � imatinib � vascular endothelial growth factor

hronic myeloid leukemia (CML) is a myeloproliferative disease characterized by excessive accumulation of neoplastic cells exhibiting the disease-related chromosomal translocation t(9;22) (1�3). The respective oncogene, bcr-abl, encodes a 210 kDa

oncoprotein (Bcr-Abl) that displays constitutive tyrosine kinase activity and activates a number of signaling molecules including Ras, phosphoinositide 3-kinase (PI3-kinase), and STAT5 (4�7). A particularly important role in Bcr-Abl-dependent growth and survival of leukemic cells has been proposed for PI3-kinase and downstream effector molecules (8). Thus, several signaling molecules acting downstream of PI3-kinase have been suggested to contribute essentially to Bcr-Abl-dependent growth of leukemic cells (9, 10). Among them, the serine/threonine kinase mTOR (mammalian target of rapamycin) may be of considerable interest (11). In particular, this molecule was found to be activated in a Bcr-Abl-dependent manner in CML cells (12, 13).

During the past few years, the Bcr-Abl tyrosine kinase inhibitor imatinib has successfully been introduced in the treatment of CML. Currently available data show that imatinib is a superior compound compared with other drugs in producing complete cytogenetic and molecular responses in patients with newly diagnosed CML (14, 15). In addition, imatinib exhibits antileukemic activity in advanced disease, i.e., patients with accelerated or blast phase of CML (16�19). However, despite encouraging initial data and high expectations for an effect on survival, long-term results are not available, and more recent data suggest that resistance against imatinib can occur during therapy and can represent a serious clinical problem (20�22).

A number of different mechanisms and gene defects can underlie resistance against imatinib in patients with CML (23�27). In several of these patients, mutations in the catalytic domain of the Bcr-Abl oncogene lead to drug resistance (23�27). In other patients, Bcr-Abl gene amplifications with consecutive overexpression of Bcr-Abl are found. Moreover, in many patients with accelerated or blast phase, CML cells often acquire additional pro-oncogenic hits apart from Bcr-Abl, and thus become imatinib resistant (23�27).

During the past few years, a number of efforts have been made to identify novel drug targets in CML cells and to develop strategies preventing or counteracting imatinib-resistance by administration of respective (targeted) drugs (28�33). As mentioned above, mTOR has recently been proposed as a novel potential drug target in CML (12, 13). Thus, exposure of CML cells to rapamycin is associated with a significant decrease in production of VEGF (34). Moreover, rapamycin reportedly inhibits growth of Bcr-Abl-positive cell lines including Ba/F3 cells exhibiting the imatinib-resistant T315I mutant of Bcr-Abl (12, 13). However, so far, little is known about the effects of rapamycin on growth of primary CML cells in vitro and in vivo. In addition, little is known about the mechanisms of action of rapamycin on CML cells.

In this study, we analyzed growth-inhibitory effects of rapamycin on primary CML cells and asked whether rapamycin-induced suppression of VEGF in leukemic cells is related to growth inhibition.

C


MATERIALS AND METHODS

Reagents

RPMI 1640 medium was purchased from Mediatech Cellgrow (Herndon, VA), fetal calf serum (FCS) and Trizol® from Gibco (Carlsbad, CA), and rapamycin from Calbiochem (San Diego, CA). Rapid-hyb® buffer, [3H]thymidine, and MegaprimeTM kit were from Amersham (Aylesbury, UK). Polyclonal antibodies against human VEGF, VEGF receptor type 1 (VEGF-R1, flt-1), and VEGF receptor type 2 (VEGF-R2, flk-1) as well as recombinant human VEGF165 were from R&D Systems (Abingdon, UK). All other reagents were purchased from Sigma (St. Louis, MO). Imatinib was kindly provided by Novartis Pharma AG (Basel, Switzerland).

Primary cells and cell lines

Primary leukemic cells were obtained from five patients with untreated chronic phase CML and one with accelerated phase CML in whom imatinib resistance had occurred (and the Bcr-Abl mutation G250E was found). Informed consent was obtained before blood donation. Peripheral blood (pb) mononuclear cells (MNC) were isolated using Ficoll. The CML-derived (Bcr-Abl+) cell lines K562 and KU812 were maintained in RPMI 1640 medium with 10% FCS. Typical phenotypic and molecular features of the cell lines are depicted in Table 1. Imatinib-resistant K562 cells (20) were maintained in 1 µM of imatinib. In select experiments, these cells were split and grown in the presence or absence of imatinib for 24 h before being analyzed. Ba/F3 cells expressing wild-type Bcr-Abl (Ba/F3p210WT) or imatinib-resistant mutants of Bcr-Abl (35) (Ba/F3p210T315I, Ba/F3p210E255K, Ba/F3p210M351T, Ba/F3p210Y253F, Ba/F3p210Q252H, Ba/F3p210H396P) were cultured in RPMI 1640 medium and 10% FCS.

Detection of point mutations in Bcr-Abl

RNA from peripheral blood leukocytes was isolated using the RNeasy Mini Kit (Qiagen, Hilden, Germany). After reverse transcription, the rearranged Bcr-Abl tyrosine kinase domain was amplified by hemi-nested PCR (36). Resulting PCR products harboring the Bcr-Abl ATP binding pocket and the activation loop were directly sequenced in both directions by dye terminator cycle sequencing. Obtained sequences were aligned to wild-type Bcr (GenBank NM_004327) and Abl (GenBank NM_005157) sequences and analyzed for the presence of mutations using Seqscape (Applied Biosystems, Foster City, CA).

[3H]thymidine incorporation assay

To examine antiproliferative effects of rapamycin, Bcr-Abl-positive cell lines (K562, imatinib-resistant K562, KU812, Ba/F3p210-clones) as well as primary CML cells were cultured in 96-well microtiter plates (5×104 cells per well) in the absence or presence of graded concentrations of rapamycin (10 µM-1 fM) for 48 h. In time-course experiments, K562 cells were cultured in rapamycin for 24-72 h at 37°C. Primary CML cells were either exposed to rapamycin (48 h) without cytokine-stimulation or were kept in GM-CSF (100 ng/ml) for 7 days before being exposed to rapamycin. To analyze potential synergistic effects between rapamycin and imatinib, imatinib-resistant K562 cells, and Ba/F3p210 cells were coincubated with increasing concentrations of imatinib and rapamycin.


In a separate set of experiments, KU812 and K562 cells were cultured in various concentrations of rapamycin in the presence or absence of recombinant VEGF (10-100 ng/ml) at 37°C for 24-48 h. In antibody-inhibition experiments, K562 cells were incubated with neutralizing anti-VEGF- (1 µg/ml), anti-VEGF-R1- (1 µg/ml), or anti-VEGF-R2 (1 µg/ml) antibodies for 5 h (37°C) and then exposed to control medium or VEGF (10 ng/ml) for another 5 h at 37°C. In another set of experiments, VEGF was preincubated with anti-VEGF antibody (1 µg/ml) before it was added to K562 cells.

After treatment of cells with cytokines, inhibitors, or antibodies, 1 µCi [3H]thymidine was added to each well. Twelve hours later, cells were harvested on filter membranes (Packard Bioscience, Meriden, CT) in a Filtermate 196 harvester (Packard Bioscience). Filters were air-dried, and the bound radioactivity was measured in a β-counter (Top-Count NXT, Packard Bioscience). All experiments were performed in triplicates.

Immunometric determination of VEGF

Ba/F3 cells carrying imatinib-resistant mutants of Bcr-Abl as well as Ba/F3p210WT cells were exposed to rapamycin (20 nM) at 37°C for 48 h. Then, cells were centrifuged, and the cell-free supernatants were examined for the presence of VEGF by a commercial ELISA (Quantikine M® mouse VEGF, R&D Systems, Minneapolis, MN). The detection limit of the ELISA amounted to 3 pg VEGF per ml.

Cell cycle analysis

K562 cells were synchronized in G1 phase by exposure to imatinib (1 µM) for 12 h. After synchronization, cells were washed twice and cultured in the presence or absence of rapamycin (20 nM) for another 12 h. Then, cells were washed once and fixed in 96% ethanol for 30 min at 4°C. Cells were then centrifuged and incubated in staining solution containing propidium iodide (50 µg/ml), RNase A (0.05 mg/ml), Triton-X100 (0.1%), and EDTA (0.1 mM) for 30 min. DNA content and hence cell cycle distribution was determined by flow cytometry on a FACScan (Becton-Dickinson, Heidelberg, Germany) using FlowJo software (Tree Star, Ashland, OR).

Northern blot experiments and RT-PCR

For Northern blot experiments, primary pb MNC and K562 cells were used. Total RNA was extracted from cells using Trizol® according to the instructions of the manufacturers. In select experiments, cells were preincubated with rapamycin (20 nM) for 16 h before RNA isolation. Northern blotting was performed essentially as described previously (34). In brief, 20 µg of total RNA were size-fractionated on 1.0% formaldehyde-agarose gels and transferred to nylon membranes (Hybond N, Amersham, Aylesbury, UK) as described by Chomczynski (37). Membranes were hybridized in rapid-hyb® buffer (Amersham). Hybridization was performed with 32P-labeled cDNAs specific for VEGF and β-actin. Primers for PCR amplification of probes were as follows: VEGF: 5′-ATGAACTTTCTGCTGTCTTGGG-3′ (forward) and 5′-CCGCCTCGGCTTGTCACATCTGC-3′ (reverse), and β-actin: 5′-ATGGATGATGATATCGCCGCG-3′ (forward) and 5′-CTAGAAGCATTTGCGGTGGACGATGGAGGGGCC-3′ (reverse). Labeling was performed using the MegaprimeTM kit. Blots were washed in 0.2× SSC (1× SSC = 150 mM NaCl and 15 mM sodium citrate, pH 7.0) with 0.1% sodium dodecyl sulfate (SDS) at 42°C for 1 h, and at 62°C for another 30 min.


Bound radioactivity was visualized by exposure to Biomax MS film® (Kodak, Rochester, NY) at -80°C using intensifying screens (Eastman Kodak, Rochester, NY).

One-step RT-PCR reactions (Titan; Roche, Mannheim, Germany) (30 cycles; annealing temperature: 55°C) were performed on Trizol-isolated total RNA (50 ng per reaction) of KU812 cells and K562 cells. Primer sequences were as follows: VEGF (see above); VEGF-R1 (flt-1): 5′-GCCCATAAATGGTCTTTGCCT-3′ (forward) and 5′-GTTTTATGCTCAGCAAGATTGTATAAT-3′ (reverse); VEGF-R2 (flk-1): 5′-GTGTAACCCGGAGTGACCAAGGAT-3′ (forward) and 5′-GATGTGATGCGGGGGAGGAA-3′ (reverse); mTOR: 5′-GCACATTGACTTTGGGGACT-3′ (forward) and 5′-CTGGTTTCACCAAACCGTCT-3′ (reverse).

Determination of cell viability and evaluation of apoptosis

Primary CML cells were incubated with rapamycin (20 nM) in the presence or absence of imatinib (1 µM) at 37°C and 5% CO2 for 3 wk. Rapamycin as well as imatinib was replaced every 48 h. Cell viability was determined by trypan blue exclusion. The percentage of apoptotic cells was quantified on Wright-Giemsa-stained cytospin preparations, according to conventional cytomorphological criteria.

Immunologic characterization of leukemic cells by flow cytometry

Phenotyping was performed on primary CML cells (patient with blast phase of CML) as well as K562 and KU812 cells using monoclonal antibodies (mAbs) against various leukocyte differentiation antigens and flow cytometry. The following mAbs were applied: H-43-5 (anti-myeloperoxidase) from An-der-Grub (Kaumberg, Austria); mAb SJ1D1 (CD13) from Immunotech (Marseille, France); WM15 (CD13), MΦP9 (CD14), C3D-1 (CD15), MMA (CD15), SJ25C1 (CD19), P67.6 (CD33), WM53 (CD33), 8G11 (CD34), 581 (CD34), GAR-2(HIR-2) (anti-glycophorin A), and L243 (anti-HLA-DR) from Becton Dickinson; anti-HTDT-Mix (TdT) from Supertechs (Bethesda, MD); ICRF44 (CD11b) from Serotec (Oxford, UK); and SS2/36 (CD10) from DAKO (Glostrup, Denmark). Expression of glycophorin A was determined after incubation of K562 cells in imatinib (0.2 µM), rapamycin (10 nM), or control medium for 96 h (37°C). Flow cytometry was performed on a FACScan or FACSCalibur (Becton-Dickinson).

In vivo administration of rapamycin in a patient with imatinib-resistant CML

Rapamycin was applied to a patient with imatinib-resistant Bcr-Abl+ leukemia following the guidelines of the local institutional review board and in accordance with the Declaration of Helsinki after written informed consent was given by the patient. The patient, a 72-year-old male, was first diagnosed to have CML blast phase (blasts expressed MPO, CD13, CD33, CD34, TdT, HLA-DR, CD10, and CD19) in October 2001. In response to induction polychemotherapy (daunorubicin iv, 45 mg/m2/day, days 1-3; etoposide iv, 100 mg/m2/day, days 1-5, and cytarabine iv, 100 mg/m2/days, days 1-7 = 3+5+7), he entered complete remission. He then received one cycle of consolidation chemotherapy (daunorubicin i., 45 mg/m2/day, days 1-2; etoposide iv, 100 mg/m2/day, days 1-5, and cytarabine iv, 100 mg/m2/d, days 1-5) followed by �maintenance therapy� with imatinib (600 mg per os daily). In response to this therapy, a complete cytogenetic response as well as a decrease in Bcr-Abl (expressed as % of Abl) to <0.1% determined by real


time PCR (38, 39) were recorded. In October 2002, a relapse occurred during imatinib therapy. He then received reinduction chemotherapy using high-dose ARA-C and mitoxantrone, followed by imatinib. In response to this therapy, he again entered remission with a decrease of Bcr-Abl to 0.4%. However, in March 2003, a second relapse was diagnosed. At that time, chromosome analysis revealed a typical t(9;22)(q34;q11) and monosomy 7 (-7). Moreover, a Bcr-Abl mutation (F359I) was detected, and Bcr-Abl transcripts increased to 60%. Based on these results, the disease was judged as being �imatinib-resistant.� Imatinib therapy was stopped, and the patient was treated with hydroxyurea to control leukocytosis. In the absence of any curative therapeutic option and because no further chemotherapy could be administered (fungal pneumonia after chemotherapy, performance status), we decided to start experimental therapy with rapamycin (2 mg per os daily). At that time, leukocyte counts ranged between 15,000-20,000/µl.

Statistical analysis

To determine the significance in differences between proliferation rates in CML cells after exposure to inhibitors, the student�s t test for dependent samples was applied. Results were considered statistically significant when P was <0.05. To determine synergistic effects of rapamycin and imatinib on growth of Bcr-Abl-positive cell lines, combination index values were calculated using a commercially available software program (Calcusyn; Biosoft, Ferguson, MO) (40).

RESULTS

Rapamycin inhibits growth of imatinib-sensitive CML cells in vitro

As assessed by [3H]thymidine incorporation, rapamycin inhibited the spontaneous as well as the GM-CSF-induced proliferation of primary CML cells in a dose-dependent manner. The IC50 values for untreated cells ranged between 1 and 10 pM. Figure 1A shows the dose-dependent effect of rapamycin in one representative donor (chronic phase CML). The inhibitory effect of rapamycin on proliferation was also seen in GM-CSF-treated CML cells, although the IC50 values were higher compared with untreated cells (>10 pM) (Fig. 1B). Nevertheless, even when grown in the presence of GM-CSF, the proliferation of primary CML cells was significantly inhibited by rapamycin at pharmacologic concentrations. Figure 1C shows the rapamycin-induced decrease in [3H]thymidine uptake by CML cells in four donors (control: 100% vs. rapamycin, 10 nM: 50±17%; P<0.05). A similar inhibition of growth was seen when the CML-derived cell lines K562 and KU812 were incubated with rapamycin. In fact, inhibition was seen in both cell lines at a rapamycin concentration of 10 nM (Fig. 1D). Interestingly, rapamycin appeared to counteract proliferation of KU812 cells more effectively (IC50: 1-10 pM) compared with K562 cells (IC50: 1 µM). Figure 1E shows the dose- and time-dependent effects of rapamycin on proliferation of K562 cells, and Fig. 1F shows the dose-dependent effect of rapamycin on KU812 cells.

Rapamycin inhibits growth of imatinib-resistant CML cells

We next asked whether rapamycin would inhibit growth of imatinib-resistant CML cells. As assessed by [3H]thymidine incorporation experiments, rapamycin inhibited the proliferation of


primary CML cells obtained from patients with imatinib-resistant disease (Fig. 2A). Rapamycin also produced dose-dependent inhibition of proliferation in imatinib-resistant K562 cells (Fig. 2, B and C). At a concentration of 10 nM, rapamycin decreased the growth of these cells to 64 ± 6% in the absence of imatinib (1 µM) (Fig. 2B) and to 46 ± 7% in the presence of imatinib (Fig. 2C). In a next step, Ba/F3 cells containing imatinib-resistant mutants of Bcr-Abl were analyzed. As visible in Fig. 2D, rapamycin inhibited [3H]thymidine incorporation in Ba/F3 cells containing various imatinib-resistant mutants of Bcr-Abl.

In vivo effects of rapamycin in a patient with imatinib-resistant leukemia

Based on our in vitro data, we decided to treat a patient with imatinib-resistant blast phase of CML with rapamycin. Rapamycin was administered at 2 mg po daily for 17 consecutive days. To determine the in vivo inhibitory effects of the drug, the numbers of pb leukocytes and absolute numbers of blasts were measured. During treatment with rapamycin, a decrease in leukocytes and blast cells as well as a decrease in the LDH level were recorded (Fig. 3A). In addition, a decrease in Bcr-Abl transcript levels in the patient�s pb leukocytes was observed during treatment with imatinib (Fig. 3B). After discontinuation of rapamycin (day 17), no increase in blast cells or LDH was seen during the following 4 wk. Thereafter, blast cells again increased. The patient then received again imatinib and experienced a short-lived response. Plasma levels of rapamycin were undetectable before treatment and increased to a maximum level of 8.2 ng/ml on day 8 after the start of therapy. Together, our data suggest that rapamycin can reduce growth of CML cells in vivo.

Rapamycin induces G1 cell cycle arrest and apoptosis in CML cells

A number of previous and more recent data suggest that depending on the cell type analyzed, rapamycin inhibits cell growth through induction of cell cycle arrest and/or induction of apoptosis (41). To explore the mechanism(s) through which rapamycin inhibits growth of Bcr-Abl-transformed cells, K562 cells and primary CML cells were examined. In a first step, we investigated the effects of rapamycin on cell cycle distribution in K562 cells. As expected, rapamycin induced a G1 cell cycle arrest in these cells (Fig. 4). In a next step, the effects of rapamycin on viability of primary CML cells were examined. As shown in Fig. 5A, rapamycin (20 nM) decreased the viability in these cells in a time-dependent manner. This inhibitory effect of rapamycin was found to be due to induction of apoptosis. In fact, the number of apoptotic cells was substantially higher in rapamycin-treated cultures compared with cells kept in control medium (Fig. 5B).

Since growth inhibition may be associated with induction of differentiation, we also asked whether rapamycin induces differentiation in K562 cells as has been reported for imatinib (42). For this purpose, K562 cells were exposed to imatinib (0.5 µM) or rapamycin (20 nM) and analyzed for expression of glycophorin A by flow cytometry. However, whereas imatinib was found to promote expression of glycophorin-A, no effect of rapamycin was seen (Fig. 6). All in all, these data suggest that rapamycin suppresses the growth of CML cells through induction of cell cycle arrest and by increasing the rate of apoptosis without inducing differentation, whereas imatinib also induces differentiation in K562 cells.


Rapamycin does not inhibit growth of CML cells through down-regulation of expression of VEGF

We and others have previously shown that VEGF is expressed in primary CML cells (34, 43�45) and that rapamycin counteracts Bcr-Abl-dependent production of VEGF in these cells (34). In the current study, we confirmed our previous data on primary CML cells and found that rapamycin down-regulates expression of VEGF mRNA in imatinib-sensitive (not shown) as well as imatinib-resistant K562 cells (Fig. 7A). Moreover, rapamycin decreased the levels of VEGF in the supernatants of Ba/F3p210WT cells as well as in Ba/F3p210 subclones carrying various imatinib-resistant mutants of Bcr-Abl (Fig. 7B). Finally, we were able to show that rapamycin down-regulates expression of VEGF mRNA in CML cells in vivo. In fact, as shown in Fig. 7C, a substantial decrease in expression of VEGF mRNA was recorded in ex vivo-analyzed pb MNC in our CML patient during treatment with rapamycin. We next asked whether VEGF can act as an (rapamycin-sensitive) autocrine growth regulator in CML cells. As assessed by RT-PCR, K562 cells were found to express VEGF-R1 (flt-1) mRNA but not VEGF-R2 (flk-1) mRNA, thus confirming recent data obtained with CML-derived cell lines and primary CML cells (Table 2) (46, 47). We also found that VEGF slightly up-regulates [3H]thymidine incorporation in K562 cells (Fig. 7D). However, exogenously added VEGF did not counteract rapamycin-induced inhibition of proliferation of K562 cells (Fig. 7E) or KU812 cells (not shown) in our experiments. Moreover, autonomous growth of K562 cells was neither inhibited by a neutralizing anti-VEGF antibody nor by antibodies against VEGF-R1 or VEGF-R2 (not shown). All in all, these data suggest that the rapamycin-induced inhibition of growth of CML cells was not caused by suppression of an autocrine loop involving production and secretion of VEGF in CML cells.

Effects of combinations of rapamycin and imatinib on growth of CML cells

To investigate potential additive or synergistic effects of rapamycin and imatinib on leukemic cell growth, Ba/F3p210 cells were exposed to various combinations of these two drugs. In these experiments, rapamycin and imatinib were found to exert cooperative inhibitory effects on growth of Ba/F3p210WT cells as well as on Ba/F3p210 subclones containing imatinib-resistant mutants of Bcr-Abl (Fig. 8A�C). To determine whether drug interactions are synergistic, combination index (CI) values were determined for each fraction affected. A CI value of 1.0 indicates an additive effect, a CI >1.0 indicates antagonism, whereas a CI <1.0 indicates synergism. In all Ba/F3 subclones examined, CI values of less than 1 were obtained (Table 3). Corresponding results were obtained for the imatinib-resistant subclone of K562 (Fig. 8D, Table 3). All in all, these data suggest that rapamycin synergistically augments the effects of imatinib on growth of imatinib-resistant leukemic cells.

DISCUSSION

A number of recent data suggest that rapamycin, apart from its immunosuppressive activities, also exhibits potent anti-neoplastic effects in various tumors (41, 48). We have recently shown that rapamycin suppresses expression of VEGF in neoplastic cells in patients with CML (34). In the current study, we report that rapamycin inhibits the growth and survival of primary CML cells in vitro and in vivo. Moreover, our data show that the growth-inhibitory and VEGF-down-regulating effects of rapamycin are separable. Thus, rapamycin may counteract growth of CML


cells in vivo by at least two independent mechanisms and may therefore represent a promising targeted drug. This notion was supported by the observation that rapamycin also counteracts growth of imatinib-resistant CML cells and that the drug synergized with imatinib in producing growth inhibition in leukemic cells.

Recent data suggest that rapamycin is a highly active and potent substance concerning inhibition of tumor cell growth (41, 48). Notably, in studies analyzing the actual potency of rapamycin in human tumor cell lines, sufficient inhibition occurred at very low concentrations with IC50 values of <10−8 M (49�51). In line with these data, rapamycin produced significant inhibitory effects on primary CML cells at low concentrations in the present study. In fact, the IC50 of rapamycin obtained with primary CML cells was found to average ~1-10 pM. A remarkable observation was that such low IC50 values were obtained with both imatinib-sensitive as well as imatinib-resistant CML cells. Another interesting observation was that although GM-CSF was found to counteract the effect of rapamycin on CML cells, the drug still produced potent anti-leukemic effects in GM-CSF-treated cells at pharmacological concentrations. All in all, our in vitro data show that anti-leukemic effects of rapamycin on primary CML cells can be obtained at doses that can be measured in transplant patients receiving rapamycin.

To demonstrate in vivo effects of rapamycin, we measured leukocyte counts and peripheral blast cells in a patient with chemotherapy-refractory and imatinib-resistant blast phase of CML. The patient received rapamycin at 2 mg daily per os for 17 consecutive days. In this particular patient, rapamycin was indeed found to decrease the leukocyte count and the numbers of myeloblasts. Interestingly, the effect of rapamycin was sustained and lasted for more than 4 weeks after cessation of rapamycin (without further cytoreductive therapy). This durable effect of rapamycin was unexpected and may be explained by inhibition of very immature clonal (Bcr-Abl-positive) progenitors. Thus, recent data suggest that rapamycin decreases the number of colony-forming cells (CFU-level) in patients with CML (52).

A number of studies have addressed the mechanisms underlying the inhibitory effects of rapamycin on growth of tumor cells. Based on these data, rapamycin primarily binds to FKBP12, an intracellular acceptor protein (53, 54). In a second step, the rapamycin-FKBP12 complex appears to inhibit the serine/threonine kinase mTOR, a major regulator of cell cycle progression (55�57). Thus, most studies have shown that the rapamycin-induced inhibition of cell growth is associated with cell cycle arrest in the G1 phase (51, 58, 59). Moreover, rapamycin leads to induction of apoptosis in certain cell types (50, 60, 61). In line with this notion, our data show that the rapamycin-dependent inhibition of growth of CML cells is associated with cell cycle arrest and induction of apoptosis.

Apart from direct inhibition of tumor cell growth, rapamycin may also inhibit tumor formation by suppressing the production (or release) of autocrine or paracrine growth regulators. Likewise, recent data have shown that rapamycin exerts antineoplastic effects in vivo in xenotransplanted mice by inhibiting the production of VEGF in tumor cells and hence tumor-associated angiogenesis (62). Since we have recently shown that rapamycin also inhibits VEGF production in CML cells (34), we were interested to learn whether this effect of rapamycin was directly associated with rapamycin-induced inhibition of cell growth. Specifically, we asked whether VEGF acts as a rapamycin-sensitive autocrine growth regulator. However, although the CML-derived cell lines analyzed (K562, KU812) were found to express VEGF-R1 mRNA at the PCR-


level and were found to grow slightly better in recombinant VEGF compared with control medium, the rapamycin-induced inhibition of growth could not be reverted by addition of exogenous VEGF in these cells. This observation argues against a rapamycin-sensitive autocrine loop in CML cells. On the other hand, it cannot be excluded with certainty that such a loop is restricted to intracellular compartments of CML (stem) cells. Thus, recent data obtained with murine hematopoietic stem cells have proposed an intracellular autocrine loop involving intracellular VEGF and intracellular VEGF receptors (63). A third possibility to be mentioned is that rapamycin might be able to block both VEGF production and signaling through VEGF receptors in CML cells.

Resistance to imatinib has become a major clinical problem in patients with CML. In fact, many patients appear to develop imatinib-resistant subclones during therapy (21, 22) with a number of different mechanisms and gene defects underlying such resistance (23, 26).

A number of different strategies have been proposed to prevent the development of imatinib resistance in these patients or to counteract growth of imatinib-resistant CML clones once they have developed. One strategy is to switch to an alternative drug for a certain time period and later to try imatinib again. Another strategy in patients with imatinib-resistant CML may be to combine imatinib with other (conventional or targeted) drugs. We were therefore also interested to find out whether imatinib and rapamycin would show additive or even synergistic effects on growth of CML cells. Interestingly, our data show that rapamycin and imatinib synergize in inhibiting growth of imatinib-resistant K562 cells as well as Ba/F3 cells expressing various imatinib-resistant mutants of Bcr-Abl.

In summary, our data show that rapamycin is a potent inhibitor of growth of imatinib-sensitive and imatinib-resistant CML cells in vitro. Moreover, we show that rapamycin has antileukemic activity in vivo in imatinib-resistant CML. Whether rapamycin shows beneficial in vivo effects in other patients with CML remains to be determined. Finally, it remains to be defined whether, how, and in which patients rapaymcin can be combined with imatinib to produce cooperative antileukemic effects on CML cells in clinical trials.

ACKNOWLEDGMENTS

We would like to thank Thomas Szekeres and Robert Mader for helpful discussions. This study was supported by the Hans and Blanca Moser�Stiftung and the Fonds zur Förderung der Wissenschaftlichen Forschung in Österreich (FWF) Grants # P-15487 and # P-16412. S. Derdak and W. F. Pickl were supported by Grant #20030 from CeMM Center of Molecular Medicine, Austrian Academy of Sciences.

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Received May 11, 2004; accepted January 28, 2005.


Table 1 Karyotypic and phenotypic properties of CML-derived cell lines*

Markers expressed in Marker K562 K562-R KU812 Histamine – – + Glycophorin-A + + – CD11b – – + CD13 + + + CD14 – – – CD15 + + – CD33 + + + CD34 – – – K562-R: Imatinib-resistant subclone of K562. *All markers were determined on cell lines used in this study. Expression of histamine was analyzed by radioimmunoassay (Immunotech) and expression of CD antigens by monoclonal antibodies and flow cytometry (see Materials and Methods).


Table 2 Target gene expression in CML cells and CML-derived cell lines

Expression of mRNA in Target CML-CP K562 K562-R KU812 VEGF + + + + VEGF-R1 (flt-1) + + + + VEGF-R2 (flk-1) – – – – mTOR + + + + Expression of mRNA specific for VEGF, VEGF receptors, and mTOR in CML-derived cells were examined by RT-PCR. CP, chronic phase; K562-R, Imatinib-resistant K562; VEGF, vascular endothelial growth factor; mTOR, mammalian target of rapamycin.


Table 3 Evaluation of synergistic drug effects in Bcr-Abl-transformed cells

Rapamycin (nM) Imatinib (µM) Fa CI Ba/F3p210wt 0.1 0.1 0.54 0.201 0.25 0.25 0.54 0.503 0.5 0.5 0.79 0.828 0.75 0.75 0.89 1.092 1 1 0.94 1.303 Ba/F3p210Y253F 0.5 0.5 0.39 0.223 1 1 0.48 0.405 1.5 1.5 0.48 0.608 2.5 2.5 0.6 0.899 5 5 0.82 1.398 Ba/F3p210E255K 1 1 0.61 0.264 2.5 2.5 0.78 0.202 5 5 0.78 0.415 7.5 7.5 0.81 0.503 10 10 0.94 0.178 Imatinib-resistant K562 10 0.1 0.6 0.169 10 0.3 0.73 0.206 10 0.6 0.76 0.366 10 0.8 0.8 0.409 10 1 0.89 0.301

Cells were incubated in the presence of drug combinations as indicated. Combination index values were calculated using a commercially available software program (Calcusyn Biosoft). Fa, fraction affected; CI, combination index; CI values <1 indicate synergism.


Fig. 1

Figure 1. Effects of rapamycin on growth of CML cells. A) Peripheral blood (pb) MNC from a patient with chronic phase CML were cultured in the presence of rapamycin (10−4-103 nM) for 48 h. Thereafter, proliferation was assessed by [3H]thymidine incorporation assay. Results are means ± SD of triplicates. B) Peripheral blood MNC from a patient with chronic phase CML were stimulated with GM-CSF (100 U/ml) for 7 days and then incubated with rapamycin (10−6-104 nM) for 48 h. Thereafter, [3H]thymidine incorporation was measured. Results are means ± SD of triplicates from 1 representative donor. C) Peripheral blood MNC from 4 CML patients were grown in GM-CSF (100 U/ml) for 7 days and then cultured in the absence (Control) or presence (Rapamycin) of rapamycin (10 nM) for 48 h before measuring [3H]thymidine uptake. Results are means ± SD of 4 donors (4 independent experiments). *P < 0.05. D) K562 cells and KU812 cells were cultured in the absence or presence of rapamycin (10 nM) for 48 h. Thereafter, [3H]thymidine uptake was measured. Results are means ± SD of 10 independent experiments for K562 cells and of 6 independent experiments for KU812 cells. *P < 0.05. E) K562 cells were incubated with various concentrations of rapamycin for 24, 48, and 72 h before measuring [3H]thymidine incorporation. Results are means ± SD of triplicates from 1 typical experiment. F) KU812 cells were incubated with rapamycin for 48 h and then subjected to [3H]thymidine incorporation. Results are means ± SD of triplicates.


Fig. 2

Figure 2. Effects of rapamycin on growth of imatinib-resistant CML cells. A) Peripheral blood MNC from a patient with imatinib-resistant CML were cultured in GM-CSF (100 U/ml) for 7 days and then incubated with rapamycin for 48 h. Thereafter, [3H]thymidine incorporation was measured. B) Imatinib-resistant K562 cells were grown in the absence of imatinib for 24 h and then incubated with various wild type Bcr-Abl (wt) or concentrations of rapamycin for 48 h. Thereafter, cells were subjected to [3H]thymidine incorporation assay. Results are means ± SD of triplicates obtained in 1 typical experiment. C) Imatinib-resistant K562 cells grown in the presence of imatinib (1 µM) were incubated with various concentrations of rapamycin for 48 h before subjected to [3H]thymidine incorporation assay. Results represent means ± SD of triplicates obtained in 1 typical experiment. D) Ba/F2p210 cells expressing imatinib-resistant various Bcr-Abl mutants were incubated with rapamycin (10 nM) or control medium for 48 h. Thereafter, cell growth was determined by [3H]thymidine incorporation assay. Results are relative inhibition of growth in percent (%) of control (= cells kept without rapamycin) and are means ± SD of 3 independent experiments.


Fig. 3

Figure 3. Antileukemic effects of rapamycin in a patient with imatinib-resistant CML. A) A patient with chemotherapy-refractory and imatinib-resistant CML in blast phase was treated with rapamycin (2 mg po per day) for 17 consecutive days. To assess the antileukemic effect of rapamycin, number of leukocytes in the peripheral blood (WBC, cells/µl), number of blast cells in peripheral blood (blasts, cells/µl), and serum levels of LDH (LDH, U/l) were recorded. Before rapamycin therapy, leukocytes were controlled using hydroxyurea (HU) at 1000 mg po daily. HU therapy was ended on day 5 of rapamycin. B) Time course of Bcr-Abl transcript levels (ratio: Bcr-Abl to Abl) in patient’s peripheral blood leukocytes. Chemotherapy (CT), treatment with imatinib, and treatment with rapamycin (Rapa) are indicated.


Fig. 4

Figure 4. Effects of rapamycin on cell cycle distribution of K562 cells. K562 cells were grown in the presence (Rapamycin) or absence (Control) of rapamycin (20 nM) for 12 h. Thereafter, cell cycle distribution was assessed by propidium iodide (PI) staining.


Fig. 5

Figure 5. Effects of rapamycin on survival of primary CML cells. A) Leukemic cells obtained from patients with untreated chronic phase CML (n=3) were cultured in the presence (rapamycin) or absence (control) of rapamycin (20 nM) for 20 days. Cell death was assessed by trypan blue exclusion test. B) In addition, percentage of apoptotic cells was quantified on Wright-Giemsa-stained cytospin preparations. Results are means ± SD of 3 independent experiments.


Fig. 6

Figure 6. Effects of rapamycin on differentiation in K562 cells. K562 cells were incubated with imatinib (0.2 µM) (bold line), rapamycin (20 nM) (dotted line), or control medium (shaded) for 96 h (37°C). Thereafter, expression of glycophorin A was determined by flow cytometry.


Fig. 7

Figure 7. Role of VEGF in rapamycin-dependent growth inhibition of CML cells. A) Imatinib-resistant K562 cells were cultured in the presence (Rapamycin) or absence (Control) of rapamycin for 16 h. Thereafter, RNA was isolated and subjected to Northern blotting. Expression of VEGF was analyzed with a cDNA probe pecific for human VEGF. Expression of β-actin (loading control) is also shown. B) Ba/F3p210wt cells as well as Ba/F3p210 cells carrying imatinib-resistant mutants of Bcr-Abl were cultured in the presence or absence of rapamycin (20 nM) for 48 h. Thereafter, immunogenic VEGF was determined in cell culture supernatants by ELISA. Results are means ± SD of triplicates. C) Peripheral blood MNC were obtained from a CML patient treated with rapamycin (see Fig. 3) prior (pre), during (days 1-16) and after (days 20 and 24) rapamycin therapy. After isolation of RNA and Northern blotting, expression of VEGF and β-actin was analyzed. D) K562 cells were cultured in control medium, VEGF (10 ng/ml), or VEGF (10 ng/ml) preincubated with a neutralizing anti-VEGF antibody (1 µg/ml) for 48 h. Thereafter, proliferation was assessed by [3H]thymidine incorporation assay. Results are means ± SD of 3 independent experiments. E) K562 cells were grown in the presence or absence of rapamycin (100 nM) or in the presence of rapamycin (100 nM) and VEGF (100 ng/ml) (Rapamycin + VEGF) for 48 h. Thereafter, proliferation was assessed by [3H]thymidine incorporation assay. Results are means ± SD of 3 independent experiments.


Fig. 8

Figure 8. Effects of rapamycin and imatinib on growth of CML cells. Ba/F3p210wt cells (A), Ba/F3p210Y253F cells (B), and Ba/F3p210E255K cells (C) were incubated with various concentrations of rapamycin and imatinib at a constant ratio of 1:1000 for 48 h. Thereafter, cell growth was determined by [3H]thymidine incorporation assay. Results are means ± SD of triplicates. D) Imatinib-resistant K562 cells were grown in the absence of imatinib for 24 h and then incubated with various concentrations of imatinib (100 nM–1 µM) in the absence (imatinib) or presence (imatinib+Rapamycin) of rapamycin (10 nM). Thereafter, proliferation was measured by [3H]thymidine incorporation assay. Results are means ± SD of triplicates.


Identification of mTOR as a novel bifunctional target in chronic myeloid leukemia: dissection of growth-inhibitory and VEGF-suppressive effects of rapamycin in leukemic cells

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