Somatic mutation of the FLT3 gene, in which the juxtamembrane domain has an internal tandem duplication, is found in 20% of human acute myeloid leukemias and causes constitutive tyrosine phosphorylation of the products. In this study, we observed that the transfection of mutant FLT3 gene into an IL3-dependent murine cell line, 32D, abrogated the IL3-dependency. Subcutaneous injection of the transformed 32D cells caused leukemia in addition to subcutaneous tumors in C3H/HeJ mice. To develop a FLT3-targeted therapy, we examined tyrosine kinase inhibitors for in vitro growth suppression of the transformed 32D cells. A tyrosine kinase inhibitor, herbimycin A, remarkably inhibited the growth of the transformed 32D cells at 0.1 μM, at which concentration it was ineffective in parental 32D cells. Herbimycin A suppressed the constitutive tyrosine phosphorylation of the mutant FLT3 but not the phosphorylation of the ligand-stimulated wild-type FLT3. In mice transplanted with the transformed 32D cells, the administration of herbimycin A prolonged the latency of disease or completely prevented leukemia, depending on the number of cells inoculated and schedule of drug administration. These results suggest that mutant FLT3 is a promising target for tyrosine kinase inhibitors in the treatment of leukemia.
Mutations of receptor tyrosine kinases (RTK), including c-KIT, PDGFRβ and FLT3, have been found in human leukemia.123 An internal tandem duplication (ITD) of the juxtamembrane (JM) domain-coding sequence of the FLT3 gene is the most frequent mutation among them.456 In the mutated FLT3 gene with ITD, the juxtamembrane (JM) domain-coding sequence, which primarily consists of exon 11 but sometimes includes intron 11 and exon 12, is arranged in a direct head-to-tail succession. Although its location and length vary from sample to sample, the mutated FLT3 gene is always readable in frame, and the transcripts actually code mutant FLT3 with a long JM domain without affecting other domains. The FLT3 gene mutation is found in 20% of acute myeloid leukemia (AML) and in 3% of myelodysplastic syndrome (MDS) cases, whereas it is very rare in chronic myeloid leukemia and lymphoid malignancies.4 The presence of the FLT3 gene mutation was significantly related to high peripheral white blood cell (WBC) counts and a poor prognosis.56 The ITD of the FLT3 gene sometimes emerged during progression of MDS or at relapse of AML which had no ITD at the first diagnosis.78 These findings strongly suggest that the FLT3 gene mutation promotes leukemia progression.
FLT3 products preferentially expresses on hematopoietic progenitor cells and its ligand (FL) on bone marrow stroma, suggesting an important role in their survival, proliferation, and differentiation.910 Clinical samples from AML frequently express functional FLT3, suggesting the FLT3-signal pathway is also important to proliferation and/or inhibition of apoptosis in leukemia cells.1112 The mutant FLT3 is ligand-independently dimerized and phosphorylated in a dominant manner.13 Similarly, in mast cell leukemia, the c-KIT gene reportedly has a mutation of the JM domain and the products are dimerized and phosphorylated.14 The JM domain of RTK might be thus associated with regulation of RTK dimerization.
Recently the inhibition of the tyrosine kinase activities and the following signal pathways has been recognized as new therapeutic strategy for leukemia.15 Using murine models, several experiments presented promising results the Bcr-Abl products-directed tyrosine kinase inhibitors could treat the Bcr-Abl+ leukemia in vivo, as well as in vitro.16171819 In this study, we transformed IL3-dependent murine cell lines with mutant. Furthermore, we examined tyrosine kinase inhibitors for in vitro and in vivo growth suppression of the transfectants to develop a model for FLT3-targeted therapy for leukemia.
Materials and methods
Herbimycin A, CGP 52411, genistein, tyrphostin A9, and erbstatin were purchased from Sigma-Aldrich (St Louis, MO, USA). They were dissolved in DMSO at appropriate concentrations and stored at −20°C until use. Recombinant murine IL3 was a generous gift of Kirin Brewery (Tokyo, Japan). Recombinant human FLT3 ligand (FL) was purchased from R&D Systems (Minneapolis, MN, USA).
Transformation of 32D cells
Murine IL3-dependent myeloid cell line, 32D, was obtained from the RIKEN cell bank (Tsukuba, Japan) and maintained in RPMI1640 containing 10% fetal calf serum and 1 ng/ml murine IL3. Human full-length mutant FLT3 cDNAs were cloned into the pMKIT-NEO vector and transfected into the cell lines by using TransFast (Promega, Madison, WI, USA) according to the manufacturer's instructions. Mt 4 clone13 was used as the mutant FLT3 cDNA. Two days after transfection, cells were selected by the culture with G418 (Gibco BRL, Gaithersburg, MD, USA) at a concentration of 800 μg/ml. Expression of FLT3 products was examined by flow cytometer (EPICS ELITE; Coulter, Hialeah, FL, USA) using an anti-human FLT3 monoclonal antibody (SF1.340; Immunotech, Marseille, France). The mutant FLT3-transfected cells were washed three times with the medium without IL3, thereafter maintained without IL3.
In vitro screening assay of tyrosine kinase inhibitors
Each cell line (2 × 105/ml) was seeded in 24-well culture dishes. After overnight culture, tyrosine kinase inhibitors were added at various concentrations in triplicate. Viable cells were counted using the trypan blue exclusion assay. IC50 was defined as the concentration of compound, which decreased the cell proliferation to 50%, compared with each untreated control.
Immunoprecipitation and immunoblot analysis
Transfection of wild-type and mutant FLT3 cDNA into Cos7 cells was described previously.13 Transfected cells were treated with herbimycin A at 0.1, 0.5 and 1.0 nM in combination with or without FL at 50 ng/ml for 3 h. After the treatment of herbimycin A, cells were washed twice with ice-cold phosphate buffered saline (PBS), and analyzed for the phosphorylation status of FLT3 as described previously.13
In vivo administration of herbimycin A
Eight-week-old female C3H/HeJ mice purchased from Japan SLC (Hamamatsu, Japan) were kept under standard laboratory conditions according to the guidelines of the Institute for Laboratory Animal Research, Nagoya University School of Medicine. This study was approved by the Institutional Ethics Committee for Laboratory Animals Used in Experimental Research. Mutant or wild-type FLT3-transfected 32D cells were subcutaneously inoculated in the right back. Treatment was started 1 day after the inoculation. A stock solution of herbimycin A (1 mM) prepared in DMSO was diluted with phosphate buffered saline (PBS) before each administration. Control mice were given an equal volume of PBS. Tumor size was monitored every day. Tumor weight was evaluated by the following formula: TW (mg) = (d2 × D)/2, where d (mm) and D (mm) are the shortest and longest diameters of the tumor, respectively.
The statistical difference of tumor weight was calculated using Mann–Whitney U test. Survival time was analyzed by Kaplan–Meier curves and compared by the log-rank (Mantel–Cox) test. Statistical analyses were performed using the program StatView (SAS Institute, Cary, NC, USA).
Transformation of 32D cells and effects of tyrosine kinase inhibitors
In the absence of IL3, 32D cells transfected with mutant FLT3 cDNA (mFLT3/32D) proliferated without either IL3 or FL. The growth speed of mFLT3/32D was more rapid than that of parental 32D stimulated with IL3 (Figure 1a). The expression of FLT3 was confirmed by flow cytometer (data not shown). In an additional two independent experiments, IL3-independent clones were obtained by the transfection with mutant FLT3.
Several tyrosine kinase inhibitors were examined for growth inhibition using mFLT3/32D and parental 32D cells. Herbimycin A suppressed the proliferation of mFLT3/32D cells more significantly than 32D cells stimulated with IL3 at low concentrations from 0.1 to 0.3 μM (Figure 1b). The viable cell number after 3-day culture was reduced by 50% (IC50) at 0.06 and 0.2 μM in mFLT3/32D cells and 32D cells, respectively (Figure 1b). In the other tyrosine kinase inhibitors (CGP52411, genistein, tyrphostin A9, and erbstatin), the IC50 to mFLT3/32D cells were 8, 3, 0.5 and 13 μM, respectively. However, the selective cytotoxicity as in herbimycin A was not observed. Accordingly, mFLT3/32D cells and herbimycin A were used in the murine model below.
Inhibition of tyrosine phosphorylation of FLT3 by herbimycin A
To study whether at the above concentrations, herbimycin A directly suppresses the phosphorylation of FLT3, FLT3 cDNA was transfected into Cos7 cells and the phosphorylation status of FLT3 was analyzed (Figure 2). Phosphorylation of FLT3 was augmented by the addition of the FLT3 ligand (FL), which was poorly inhibited by the treatment with herbimycin A. In contrast, mutant FLT3 was significantly phosphorylated in a FL-independent manner. Herbimycin A remarkably suppressed the phosphorylation of mutant FLT3. The expressed level of FLT3 did not change during the treatment with herbimycin A. These results indicated that herbimycin A inhibits the autophosphorylation of mutant FLT3 more significantly than the ligand-dependent phosphorylation of wild-type FLT3.
Establishment of an in vivo model
Inoculation of mFLT3/32D at 1 × 106 cells or more, produced subcutaneous tumors in non-irradiated C3H/HeJ mice with 100% efficiency. The latency time was from 2 to 4 weeks, depending on the inoculated cell counts. However, parental 32D cells did not produce tumors even when inoculated at 2 × 107 cells. After observation of the subcutaneous tumors, mice died within 5 weeks. Histopathologic examination of the subcutaneous tumors revealed features of myeloid leukemia, including intermediate-sized myeloblasts partly differentiated. All lymph nodes were affected, as was the periportal region of the liver, with diffuse leukemic infiltration of the lung, spleen and bone marrow (Figure 3).
In vivo treatment with herbimycin A
The effect of herbimycin A was examined in this murine model 1 × 107 cells of 32D/mFLT were injected subcutaneously into eight mice (Figure 4a). Half of the mice (A5 to A8) were administered by intraperitoneal (i.p.) injection of 2.5 μg herbimycin A from days 1 to 12. Tumor growth inhibition was statistically significant from day 22 (P = 0.02, by Mann–Whitney U test), which continued during the observation. On day 28, tumor weight was 475 ± 561 and 2125 ± 122 μg in treated and non-treated groups, respectively. However, two of four treated mice (A5 and A6) gradually developed subcutaneous tumors. Another two mice (A7 and A8) had a long latency of tumors and died in the 10th week. Thus survival time was elongated by the treatment (P = 0.006, by the log-rank test). One of the treated mice (A5) showed oral bleeding, whereas the other mice displayed no obvious sign of toxicity. This experiment indicates that the treatment for 12 consecutive days could not eradicate leukemia but caused some side-effects.
In the second experiment, we tried a longer treatment schedule, in which mice were administered by i.p. injection 3 days per week for 4 weeks (Figure 4b). In two of five mice (B6 and B7) treated with this schedule, tumor latency was prolonged by approximately the same duration as herbimycin A therapy. In the other three mice (B8 to B10), tumors did not develop. No sign of side-effects was observed. This experiment suggests that the longer schedule with intermittent administration might be better than the short one with daily administration.
In the third experiment, the inoculated cell number was reduced to 1 × 106 (Figure 4c). All of the non-treated mice developed subcutaneous tumors within 6 weeks after injection, and tumor weight was 3380 ± 2550 μg on day 42. However, none of the treated mice developed tumors. Histopathologic examination of the survivors did not reveal any signs of leukemia infiltration. These results suggested that herbimycin A inhibited the growth of tumor or prevented tumor progression, but complete eradication depended on the initial tumor cell number.
In this study, we showed that (1) herbimycin A inhibited the in vitro growth of mutant FLT3-transformed 32D cells but not IL-3 dependent 32D cells at the concentration of 0.1 μM; (2) herbimycin A inhibited the phosphorylation of mutant FLT3 but not that of ligand-stimulated wild-type FLT3; and (3) in vivo administration of herbimycin A prolonged the latency of disease or prevented the tumor progression, depending on the number of cells inoculated and the schedule of drug administration.
The first finding indicates that 32D and mFLT/32D cells are useful for screening compounds for mutant FLT3-target therapy. Since the specific inhibitor of FLT3 kinase is unknown, herbimycin A, CGP 52411, genistein, tyrphostin A9, and erbstatin were examined in this study. Erbstatin, genistein and CGP 52411 were isolated or synthesized by the screening for the inhibition of EGFR kinase.2021 Tyrphostin A9 is an inhibitor of the kinase activity of the platelet-derived growth factor (PDGF) receptor.22 Herbimycin A was isolated from the culture broth of a strain of Streptomyces on the basis of its ability to revert rat kidney cells transformed by v-src to a normal morphology.23 Further study of this compound revealed that it inhibited the kinase activity of p60v-src.24 Now it is known that herbimycin A inactivates various non-receptor tyrosine kinases and reverts cultured cells transformed by oncogenes including src, yes, fps, ros and abl.25 Direct binding to the Cys residue of the kinase reportedly causes inhibition of the kinase activity. Herbimycin A was also reported to selectively down-regulate RTK,26 although the expression level of FLT3 did not change in this study. Preferential growth suppression of Ph1-positive human leukemia cell lines has been achieved using low concentrations of herbimycin A.1718
The second finding is particularly notable, in that the ITD of FLT3 does not affect the kinase domain. Research into signal transduction has revealed that several SH2-containing proteins, such as PLCγ, SHC, SHP-2 and Grb2, recognize the phophorylated Tyr residues of FLT3.2728 Although the functional role of the JM domain of FLT3 remains unclear, it is possible that like PDGFR, src-family kinase(s) is physically associated with the JM domain.29 In general, herbimycin A suppresses non-receptor kinases rather than RTK.25 Accordingly, one reason why herbimycin A inhibited mutant FLT3 more than the wild-type is that since the elongated JM domain might aberrantly recruit some tyrosine kinase(s) which augments phosphorylation of mutant FLT3, herbimycin A inhibits the tyrosine kinase(s) in addition to FLT3 kinase. A cell-free kinase assay is necessary to clarify this issue.
The third finding is important to the development of tyrosine kinase inhibitors as targeted therapy for cancer. In murine models of Bcr-Abl+ leukemia, administration of herbimycin A prolonged the latency periods of leukemia and/or suppressed the tumor growth, although it could not eradicate leukemia.18 We showed that the treatment schedule was significantly associated with the therapeutic response. The longer schedule (three times a week for 4 weeks) was more effective than consecutive 12-day administration, even if the total dose was the same. Moreover, the inoculated cell number was a critical factor for in vivo response, as pointed out previously.17 Accordingly, tyrosine kinase inhibitors should not be used solely in vivo. Since herbimycin A reportedly accelerates the apoptosis of Bcr-Abl+ leukemia cells following etoposide treatment or γ-irradiation,30 the treatment using tyrosine kinase inhibitors might be further improved by the combination with other cytotoxic drugs.
The importance of tyrosine kinase signaling as a potent target for novel cancer treatment has recently been noticed. A synthetic tyrosine kinase inhibitor, CGP57148B, inhibited the phosphorylation of Bcr-Abl and eradicated a Bcr-Abl+ human leukemia cell line transplanted in nude mice.19 Although CGP57148B is not specific for Bcr-Abl, in vivo the use of the compound produced a significant therapeutic response. The investigators noticed that not a single but a continuous block of Bcr-Abl kinase was needed to produce important biological effects in vivo. Further analysis of tyrosine kinase inhibitors in the animal model should identify novel therapies to treat human leukemias. Herbimycin A might serve as a starting compound for the development of derivatives.
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We thank Chisato Kamiya, Yoko Kudo and Yoko Tagawa for their technical assistance, and Kirin Brewery Company Ltd for providing murine IL3.
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