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Introduction

Fms-like tyrosine kinase 3 (FLT3) is a member of the class III receptor tyrosine kinase receptor family (1) and is the most frequently mutated gene (20–30%) in acute myelogenous leukemia (AML) (2–4). Activating mutations, as well as overexpression of FLT3, are prevalent in AML, with a role in leukemogenesis (3,5–8). In adult AML, ~24% of such mutations are internal tandem duplications (ITD) in the juxtamembrane domain (2,3), which result in ligand-independent dimerization and tyrosine phosphorylation of the receptor (9). In addition, activating point mutations in the FLT3 tyrosine kinase domain (TKD; FLT3-TKD), mainly at aspartic acid 835, are identified in ~7% of AML patients (10).

ITD in FLT3 (FLT3-ITD) is associated with a higher leukocyte count, increased relapse risk, decreased disease-free survival (DFS) and decreased overall survival (OS) (11). Furthermore, multivariate analyses have shown that FLT3-ITD is the most significant factor for predicting an adverse outcome in AML (11–14). By contrast, FLT3-TKD mutations have a smaller effect compared with FLT3-ITD, but tend to worsen the DFS and OS (10), with the differences being statistically significant for OS in patients aged ≤60 years (15).

FLT3-ITD alters chemotherapy responses in vitro and in vivo and confers resistance to doxorubicin, which depends on p53 (16). Additionally, DNA repair contributes to the FLT3-ITD drug-resistant phenotype of primary AML (17). Several studies demonstrate drug resistance conferred by FLT3-ITD (16,18,19); however, thus far, no studies have demonstrated the effect of mutations in the FLT3 TKD on anticancer drug resistance.

The present study examined the effects of FLT3-ITD and FLT3-TKD on cytotoxic drugs by employing an IL-3 dependent cell line, Ba/F3. In this cell line, interleukin-3 (IL-3)-independent cell growth occurs in response to stably transduced oncogenic signaling, such as via FLT3-ITD and -TKD (20). This system was used to evaluate the effect of FLT3-ITD and -TKD oncogenic signals on the cytotoxicity of daunorubicin (DNR) and cytarabine (Ara-C). As a result, FLT3-ITD and -TKD signals were observed to alter the response to DNR.

Materials and methods

Generation of Ba/F3 cells expressing FLT3-ITD and FLT3-TKD

Our previous studies established the Ba/F3 FLT3-WT and-ITD cells (6,21). To generate stably expressing Ba/F3-FLT3-TKD cells, pcDNA FLT3-TKD (D835Y) was generated using pcDNAFLT3-WT (6) and the Quick change site directed mutagenesis kit XL (Stratagene, La Jolla, CA, USA) with the following primers: Sense, 5′-CTTTGGATTGGCTCGATATATCATGAGTGATTC-3′ and anti-sense, 5′-GAATCACTCATGATATATCGAGCCAATCCAAAG-3′. The construct was confirmed by sequence analysis and transfected into Ba/F3 cells using a CLB-Transfection device (Lonza, Basel, Switzerland). Stably transfected Ba/F3 clones were isolated by limiting dilution and selection with 400 µg/ml neomycin in RPMI (Gibco BRL, Thermo Fisher Scientific, Rockville, MD, USA) containing 10% heat-inactivated fetal bovine serum, 100 ng/ml recombinant mouse IL-3 (R&D Systems, Minneapolis, MN, USA) and 50 µmol/l 2-mercaptoethanol. Cells were cultured at 37°C in a humidified 5% CO2 atmosphere.

mRNA analysis

cDNA was prepared from cells using reverse transcriptase (Transcriptor First Strand cDNA Synthesis kit; Roche, Indianapolis, IN, USA). Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed using the Quantitect SYBR-Green PCR reagent (Qiagen, Miami, FL, USA) according to the manufacturer's protocol and using an Opticon Mini Real-time PCR Instrument (Bio-Rad, Hercules, CA, USA), as described previously (22). The primer sequences were: FLT3 forward, 5′-TCAAGTGCTGTGCATACAATTCCC-3′ and reverse, 5′-CACCTGTACCATCTGTAGCTGGCT-3′; and GAPDH forward, 5′-GAAGGTGAAGGTCGGAGT-3′ and reverse; 5′-GAAGATGGTGATGGGATTTC-3′. The thermal cycling conditions for FLT3 and GAPDH were incubation at 95°C for 15 min, followed by 35 cycles of 95°C for 30 sec, 55°C for 30 sec and 72°C for 45 sec. The copy number of each sample was calculated as previously described (21).

Assessment of viable cells

The proportion of viable cells was determined using a dye reduction assay involving a tetrazolium salt, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitro- phenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8; Dojindo, Tokyo, Japan), which is a modification of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolim assay. The effective dose (ED)50 values were calculated from the data obtained from the cell growth assays. Exponentially growing cells were seeded at 1×104 cells per well in flat-bottomed 96-well plates, with or without IL-3 in the medium. Seven different doses were selected for DNR (Sigma, St. Louis, MO, USA) (1.5, 3.1, 6.2, 12.5, 25, 50 and 100 nM) and Ara-C (Sigma) (0.15, 0.31, 0.62, 1.25. 2.5, 5 and 10 µM). No DNR or Ara-C was added to the controls cells. Assays were performed 2 days after the addition of the drugs. For IL-3(−) cells, exponentially growing cells were washed twice with phosphate-buffered saline and seeded without IL-3. Viable cells (%) were calculated as the ratio of the absorbance (490 nm) of DNR or Ara-C-treated cells to the absorbance of untreated cells. At least three independent experiments were performed. The calculated ratios were analyzed and the ED50 values were obtained using tools at http://www.vector.co.jp/soft/win95/edu/se248471.html.

Statistical analysis

Data are expressed as the mean ± standard error of the mean and P<0.05 (denoted by one asterisk) was considered to indicate a statistically significant difference. Comparison of the means was performed using Student's t-test (http://www.physics.csbsju.edu/stats/t-test_bulk_form.html).

Results

Generation of Ba/F3-FLT3-TKD cells

Our previous study established the generation of Ba/F3-FLT3-ITD cells (21), and for the present study, to clarify whether FLT3-ITD or FLT3-TKD mediate any specific anticancer drug effects, Ba/F3-FLT3-TKD cells were also generated. The pcDNA FLT3-TKD (D835Y) vector was electroporated into Ba/F3 cells and stably transfected lines were isolated by limiting dilution with medium containing neomycin. Among >20 lines obtained, two clones, Ba/F3-FLT3-TKD16 and Ba/F3-FLT3-TKD22, exhibited increased levels of FLT3 expression compared with the parental vector (pcDNA3.1) transfected Ba/F3-vec cells (Fig. 1). To confirm that the FLT3-TKD transgene was functionally active, Ba/F3-FLT3-TKD16 and Ba/F3-FLT3-TKD22 cells were deprived of IL-3. These two lines showed factor-independent growth, as previously reported (21,23). Similar growth was observed for Ba/F3-FLT3-ITD (positive control), but not for Ba/F3-FLT3-WT (negative control) (Fig. 2).

Figure 1. Expression of FLT3 transgene in Ba/F3 cells. Reverse-transcription quantitative polymerase chain reaction (PCR) analyses of FLT3 using newly established Ba/F3-FLT3-TKD16 and Ba/F3-FLT3-TKD22 lines and the previously generated Ba/F3-vec, Ba/F3-FLT3-WT (6) and Ba/F3-FLT3-ITD27 (21) lines. FLT3 transcript levels were adjusted relative to the expression of GAPDH. The data presented were obtained from three independent PCR amplifications.

Figure 2. Effect of the FLT3-TKD transgene on the growth of Ba/F3 cells. To measure cell proliferation, Ba/F3-FLT3-TKD (TKD16, TKD22) cells together with Ba/F3-FLT3-WT and Ba/F3-FLT3-ITD (ITD27) cells were counted at days 3 and 6 following IL-3 deprivation, using a hemocytometer and the trypan blue exclusion method. The plotted values were obtained from an average of four counts, and the result is a representative of two independent experiments. IL-3, interleukin-3.

ED50 of DNR increases in IL-3-deprived Ba/F3-FLT3-ITD27, -ITD29 and -TKD22 cells

Cell viability and ED50 values were examined in the Ba/F3-FLT3-ITD and Ba/F3-FLT3-TKD cells. In Ba/F3-FLT3-ITD27, -ITD29 and -TKD22 cells, the ED50 value was significantly increased in the absence of IL-3, compared with the controls (Fig. 3A). Additionally, in Ba/F3-FLT3-TKD16 cells, the median value was higher [IL-3(−), 17.7 nM; IL-3(+), 14.8 nM], although this difference was not statistically significant (Fig. 3A). By contrast, there were no differences in the ED50 value for Ara-C between IL-3(−) and IL-3(+) cells (Fig. 3B). These results indicate that FLT3-ITD, as well as the mutation of the FLT3-TKD, may confer DNR resistance to Ba/F3 cells.

Figure 3. ED50 values for (A) DNR and (B) Ara-C. The ED50 values were calculated from the data obtained from the cell growth assays. WST-8 assays were performed on cells grown with or without IL-3 in the medium and 2 days after the addition of drugs: DNR (1.5, 3.1, 6.2, 12.5, 25, 50 or 100 nM) and Ara-C (0.15, 0.31, 0.62, 1.25. 2.5, 5 or 10 µM). Control cells were grown without the addition of DNR or Ara-C. Viable cells (%) were determined by the ratio of the absorbance (490 nm) of DNR or Ara-C-treated cells relative to the absorbance of untreated cells. The ratio of viable treated cells to untreated cells was used to calculate ED50 values. At least three independent experiments were performed. These data are shown as boxplots representing the 25 and 75 percentiles, median and 5–95 range. Data are expressed as the mean ± standard error of the mean and *P<0.05 was considered to indicate a statistically significant difference. ED, effective dose; DNR, daunorubicin; IL-3, interleukin-3; n.s., not significant.

Discussion

The present data suggest that in AML patients with FLT3-ITD or FLT3-TKD mutations, DNR is not efficacious and only causes toxicity. This is consistent with the findings of a clinical trial showing that while the majority of AML patients benefit from intensified anthracycline dosing regimens, high-dose DNR did not provide a significant survival benefit in patients who had the FLT3-ITD mutation (24). Lee et al (25) also support this notion that FLT3-ITD causes resistance to doxorubicin; dual treatment of PML-RARα FLT3-ITD transgenic mice with FLT3 inhibitor SU11657 and doxorubicin increased sensitivity. Pardee et al (16) recently reported that FLT3-ITD confers resistance to doxorubicin in a p53-dependent manner. In addition to the well-known role of p53 in apoptosis induction, it has also been shown to induce multiple prosurvival and DNA repair genes (26). Consistent with this, FLT3-ITD-expressing AML cell lines and primary patient samples have increased the levels of reactive oxygen species, double-strand DNA breaks and increased DNA repair capacity (17,27).

The present findings are consistent with and support the above. However, using murine myeloid HF6 and human myeloid K562 cells, our previous study found that FLT3-ITD induced Ara-C resistance through repression of equilibrative nucleoside transporter 1 expression (19), which was not observed in the present study. This discrepancy may be due to the use of different cell lines. The present study employed Ba/F3 cells, a murine B-lymphoid cell line, which is distinct from HF6 and K562 cells. The Ba/F3 cells were used due to their IL-3 deprivation characteristic, which can reveal the effect of FLT3 oncogenic signals. This enabled a specific DNR response to be identified.

The study also highlights the FLT3-TKD mutations, which may confer resistance to anthracycline. Although the data are from a cell line model and are somewhat preliminary, these results may indicate the benefit of therapy combining anthracycline and FLT3 inhibitors for patients carrying FLT3-ITD and FLT3-TKD mutations.

Acknowledgements

The authors would like to thank Mr. Tatsuya Osaka and Mr. Wataru Nomura for establishing the cell lines and Ms. Hiroko Nakano for her technical assistance. The study was supported in part by Grants-in-Aid for Scientific Research (grant no. 26460685) from the Ministry of Education, Science and Culture (Japan), and the Takeda Sceince Foundation, a foundation from Kitasato University School of Allied Health Sciences (Grant-in-Aid for Research Project, no. 2015-1003). Edanz Group Ltd. (www.edanzediting.com) provided editorial assistance.

References