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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 22, 2005 as doi:10.1096/fj.04-1973fje. |
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* Department of Internal Medicine I, Division of Hematology and Hemostaseology and
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, USA;
¶ Department of Adult Oncology, Dana Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA; and
# Howard Hughes Medical Institute, Portland, Oregon, USA
1Correspondence: Department of Internal Medicine I, Division of Hematology and Hemostaseology, Medical University of Vienna, Waehringer Guertel 18-20, Vienna A-1097, Austria. E-mail: matthias.mayerhofer{at}univie.ac.at
SPECIFIC AIMS
Chronic myeloid leukemia (CML) is a myeloproliferative disease associated with the Bcr-Abl oncogene. Recent data suggest that the mammalian target of rapamycin (mTOR) is activated by Bcr-Abl and that inhibition of mTOR by rapamycin results in reduced growth of Bcr-Abl-transformed cell lines. In the present study, we analyzed growth-inhibitory effects of rapamycin on primary CML cells in vitro and in vivo, and asked whether rapamycin-induced suppression of VEGF in leukemic cells is related to growth inhibition. Moreover, we investigated effects of rapamycin on CML cells resistant to the Bcr-Abl tyrosine kinase inhibitor imatinib and determined potential cooperative growth inhibitory effects of rapamycin and imatinib.
PRINCIPAL FINDINGS
1. Rapamycin inhibits growth of primary CML cells
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. Even when grown in the presence of GM-CSF, the proliferation of primary CML cells was significantly inhibited by rapamycin at pharmacologic concentrations. Thus, rapamycin (10 nM) decreased 3H-thymidine uptake of GM-CSF-treated CML cells to 50 ± 17% of control (P<0.05). Growth inhibition was also seen when the CML-derived cell lines K562 and KU812 were incubated with rapamycin. In these experiments, K562 cells were less sensitive to rapamycin compared with KU812 cells.
2. Rapamycin inhibits growth of imatinib-resistant leukemic cells
Although imatinib is a most effective drug in patients with CML, some patients develop resistance to imatinib, which can represent a serious clinical problem. Therefore, current research is focusing on antineoplastic drugs capable of inhibiting the growth of leukemic cells in these patients. In the current study, we asked whether rapamycin would inhibit the growth of imatinib-resistant CML cells. Rapamycin was found to inhibit the proliferation of primary CML cells obtained from patients with imatinib-resistant disease (IC50: 1–10 pM) (Fig. 1
). Rapamycin produced a dose-dependent inhibition in proliferation of imatinib-resistant K562 cells. Moreover, rapamycin inhibited growth of Ba/F3 cells expressing imatinib-resistant mutants of Bcr-Abl. These data show that rapamycin inhibits growth of imatinib-resistant CML cells.
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3. 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. In this study, rapamycin was found to induce a G1 cell cycle arrest in K562 cells. Moreover, rapamycin (20 nM) decreased the viability of primary CML cells in a time-dependent manner. The number of apoptotic cells was found to be substantially higher in rapamycin-treated cultures compared with cells kept in control medium. These data suggest that rapamycin suppresses the growth of CML cells through induction of cell cycle arrest and by increasing the rate of apoptosis.
4. 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, K562 cells as well as Ba/F3p210 cells were exposed to various combinations of these drugs. 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 greater than 1.0 indicates antagonism, whereas a CI less than 1.0 indicates synergism. We found that rapamycin and imatinib exert synergistic inhibitory effects on growth of Ba/F3p210WT cells as well as on Ba/F3p210 clones containing the imatinib-resistant Bcr-Abl mutants E255K and Y253F. Synergistic growth inhibitory effects of rapamycin and imatinib were also obtained with the imatinib-resistant subclone of K562.
5. In vivo effects of rapamycin in a patient with imatinib-resistant CML
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 p.o. daily for 17 consecutive days. To determine the in vivo inhibitory effects of the drug, the numbers of peripheral blood leukocytes and blast cells 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. 2
). After discontinuation of rapamycin (day 17), no increase in blast cells or LDH was seen during the ensuing 4 wk. Thereafter, blast cells again increased. The patient then received imatinib again, and experienced a short-lived response. These data show that rapamycin can reduce growth of CML cells in vivo.
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6. Rapamycin does not inhibit growth of CML cells through down-regulation of expression of VEGF
We have previously shown that VEGF is expressed in primary CML cells, and that rapamycin counteracts Bcr-Abl-dependent production of VEGF in these cells. In the current study, we found that rapamycin down-regulates expression of VEGF in imatinib-resistant leukemic cells. In addition, a substantial decrease in expression of VEGF mRNA was recorded in ex vivo-analyzed blood leukocytes 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. We found that VEGF slightly up-regulates 3H-thymidine incorporation in K562 cells. However, exogenously added VEGF did not counteract rapamycin-induced inhibition of proliferation of K562 cells or KU812 cells. These data suggest that the rapamycin-induced growth inhibition of CML cells was not caused by suppression of an autocrine loop involving production and secretion of VEGF in CML cells.
CONCLUSIONS AND SIGNIFICANCE
A number of recent data suggest that rapamycin, apart from its immunosuppressive activities, exhibits potent antineoplastic effects in various tumors. We have recently shown that rapamycin suppresses expression of VEGF in neoplastic cells in patients with CML. In the current study, we report that rapamycin inhibits the growth and survival of primary CML cells in vitro and in vivo. Our data also 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 novel antileukemic drug (Fig. 3
).
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Recent data have shown that rapamycin is a highly active and potent antineoplastic drug. In studies analyzing the actual potency of rapamycin in human tumor cell lines, sufficient inhibition occurred at very low concentrations with IC50 values of less than 10–8 M. In line with these data, rapamycin produced significant inhibitory effects on primary CML cells at low concentrations in the present study. 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 for rapamycin effects 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 antileukemic effects in GM-CSF-treated cells at pharmacological concentrations. Thus, our in vitro data show that antileukemic 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 p.o. for 17 consecutive days. In this particular patient, rapamycin was found to decrease the leukocyte count and the numbers of myeloblasts in the peripheral blood. The effect of rapamycin was sustained and lasted for more than 4 wk after cessation of rapamycin (without further cytoreductive therapy).
Apart from direct inhibition of tumor cell growth, rapamycin may inhibit tumor formation by suppressing the production (or release) of autocrine or paracrine growth regulators. Likewise, recent data have shown that rapamycin has antineoplastic effects in vivo in xenotransplanted mice by inhibiting the production of VEGF in tumor cells and hence tumor-associated angiogenesis. Since we have recently shown that rapamycin inhibits VEGF production in CML cells, 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, and favors two independent drug effects (Fig. 3)
. However, 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.
Together, 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 (alone or in combination with imatinib) shows beneficial in vivo effects in other patients with CML, remains to be determined.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1973fje; doi: 10.1096/fj.04-1973fje
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