SU11652 Inhibits tyrosine kinase activity of FLT3 and growth of MV-4-11 cells
- Yao Guo†1, 2,
- Yun Chen†2,
- Xuesong Xu1, 3,
- Xueqi Fu1Email author and
- Zhizhuang Joe Zhao1, 2Email author
© Guo et al.; licensee BioMed Central Ltd. 2012
Received: 24 October 2012
Accepted: 26 November 2012
Published: 6 December 2012
FLT3-ITD and FLT3-TKD mutations are frequently found in acute myeloid leukemia (AML). This makes tyrosine kinase FLT3 a highly attractive target for therapeutic drug development. However, effective drugs have not yet emerged. This study is intended to identify and to characterize new FLT3 inhibitors.
By using the protein substrate GST-FLT3S to analyze kinase activity of recombinant proteins carrying the catalytic domain of wild type and mutant forms of FLT3, we screened a chemical library containing 80 known protein kinase inhibitors. We identified SU11652 as a potent FLT3 inhibitor and further employed FLT3-ITD-positive MV- 4–11 cells to study its effects on cell growth, apoptosis, cell cycles, and cell signaling.
SU11652 strongly inhibited the activity of wild type, D835Y, and D835H mutant forms of FLT3 with IC50 values of 1.5, 16, and 32 nM, respectively. It effectively blocked the growth of FLT3-ITD -positive MV-4-11 cells at nanomolar concentrations but exhibited much less effects on several other cells which do not carry mutations of FLT3. SU11652 inhibited growth of MV-4-11 cells by inducing apoptosis, causing cell cycle arrest, and blocking activation of the ERK, Akt, and STAT signaling pathways.
SU11652 is a potent FLT3 inhibitor which selectively targets FLT3-ITD-positive cells. It should serve as a good candidate for development of therapeutic drugs to treat AML.
KeywordsTyrosine kinase FLT3 Inhibitor screening SU11652 Acute myeloid leukemia
Acute myeloid leukemia (AML) is the most common malignant myeloid disorder in adults and is featured by abnormal differentiation and proliferation of hematopoietic progenitor cells of the myeloid lineage . The median age of AML patients at diagnosis is 67 years, and it is estimated that 13,780 (7,350 men and 6,430 women) will be diagnosed with and 10,200 will die of the disease in 2012 in USA . Currently, there is no effective cure for the disease. With standard chemotherapy, the 3-year event-free survival rate of patients with favorable cytogenetics is 60%; however, the median survival of patients with unfavorable cytogenetics is only 6 months [1, 3]. Therefore, novel therapies with higher efficacy and fewer side effects are badly needed. With the increased understanding of the pathogenesis of AML, several molecular lesions have been identified, which allows development of targeted drugs . Among them, FMS-like tyrosine kinase 3 (FLT3) is one of the most attractive targets, and related drug therapies are currently under development [5, 6].
FLT3 is a receptor tyrosine kinase mainly expressed in early myeloid and lymphoid progenitors, and it plays a role in the development of hematopoietic progenitor cells . Mutations of FLT3 have been found in about 30% of AML patients . Among these mutations, the most common type is FLT3-ITD caused by internal tandem duplication (ITD) in the juxtamembrane domain, and the less common FLT3-TKD type involves point mutations in the tyrosine kinase domain (TKD). These mutations lead to constitutive activation of FLT3 and result in uncontrolled proliferation of leukemia cells , and they are strongly associated with poor prognosis in AML patients . Since mutations found in AML cause activation of FLT3, FLT3 has become an obvious target for anticancer drugs. Up to date, several small molecule inhibitors and monoclonal antibodies against FLT3 have been developed [11–14]. Clinical trials have shown the efficacy of some FLT3 inhibitors in treatment of AML, but the responses are incomplete and often limited by the acquired resistance during the treatment [15–17]. For off-label use, sorafenib, which was initially identified as an inhibitor of VEGFR/PDGFR tyrosine kinases and Raf kinases, is the only FDA-approved anticancer drug targeting FLT3 . Needless to say, identification of novel FLT3 inhibitors with higher potency is required.
In this study, by screening a protein kinase inhibitor library using a protein substrate we recently developed , we identified SU11652 as a potent FLT3 inhibitor. We further performed cell-based assays to demonstrate its inhibitory selectivity and potency for FLT3-ITD-positive cells and the cellular and molecular mechanism underlying its action. We believe that SU11652 should serve as a good candidate for development of targeted drugs to treat AML.
Results and discussion
SU11652 is a potent FLT3 inhibitor
SU11652 inhibits the growth of FLT3-ITD-positive cells
SU11652 induces apoptosis and cell cycle arrest in MV-4-11 cells
SU11652 inhibits FLT3 downstream signaling in MV-4-11 cells
By screening a chemical library with an effective FLT3 kinase assay system, we have identified SU11652 as a potent inhibitor of FLT3 with an IC50 value of 1.5 nM. More importantly, our in vitro cell-based assays demonstrated that SU11652 selectively inhibited the growth of FLT3-ITD-positive MV-4-11cells with equivalent potency. Furthermore, we showed that SU11652 induced apoptosis, caused cell cycle arrest, and blocked FLT3 downstream signaling transduction. FLT3 is an obvious target for therapeutic drugs to AML, but no effective drug has emerged. Our study provides a new candidate. Considering the potency and selectivity of SU11652 according to biochemical and cell-based assays, further preclinical study with animal models and clinical studies with FLT3-ITD -positive AML patients appears to be well warranted.
InhibitorSelect Protein Kinase Library I containing 80 protein kinase inhibitors including SU11652 was purchased from Calbiochem (CA, USA). Monoclonal anti-phosphotyrosine antibody PY20 was from BD Biosciences (CA, USA), while antibodies against pFLT3 (pY591), pERK1/2(pT202/pY204), pAKT(pS473), and pSTAT5(pY694) were from Cell Signaling Technology (MA, USA). MV-4–11, HL-60, and Jurkat cell lines were obtained from ATCC (VA, USA). Karpas 299 cells were kindly provided by Yi Zhao (University of Southern California, ). MV-4-11 cells were cultured in Iscove’s Modified Dulbecco’s Medium containing 10% fetal bovine serum, and the rest of cells were maintained in RPMI medium supplemented with 10% fetal bovine serum.
FLT3 kinase activity assays and inhibitor screening
Protein kinase activity assays and inhibitor screening were performed as previously described [19, 25]. The FLT3 substrate GST fusion protein GST-FLT3S was purified from E. coli cells by using a glutathione-Sepharose column, and recombinant proteins containing the catalytic domain of wild type FLT3 and its D835H and D835Y mutant forms were isolated from recombinant baculovirus-infected Sf9 insect cells by using the NTA-Ni resin . Phosphorylation of GST-FLT3S by isolated FLT3 tyrosine kinases was carried out in a reaction buffer containing 25 mM Tris–HCl (pH 7.5), 10 mM MgCl2, 0.2 mM adenosine 5′-triphosphate, and 2 mM dithiothreitol in the presence of various concentrations of protein kinase inhibitors. The level of GST-FLT3S tyrosine phosphorylation was determined by immunoblotting with anti-phosphotyrosine antibody PY20 followed by horseradish peroxidase-conjugated secondary antibody. Detection and quantification of enhanced chemiluminescence signals were done by using FluorChem SP imaging system from Alpha Innotech .
Cell viability assays
MV-4-11, HL-60, Karpas 299, and Jurkat cells were incubated with various concentrations of SU11652 for 48 hours. To measure the viability of cells, 0.5 mg/ml MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was added into the medium. After incubation at 37°C for 3 hours, the medium was removed by centrifugation and the precipitated dye was dissolved in 1 ml isopropanol containing 0.04 M HCl. Absorbance at 570 nm was then measured with a spectrophotometer.
Apoptosis and cell cycle analyses
For apoptosis analysis, the cells were stained with Annexin V-Cy5 and propidium iodide (Biovision, CA, USA). To assess cell cycle arrest, the cells were fixed with ethanol overnight and then stained with propidium iodide in the presence of RNAse. Flow cytometric assays were performed by using a FACSCalibur flow cytometer (BD Biosciences) at the Flow and Image Cytometry Laboratory of University of Oklahoma Health Sciences Center.
Cell signaling assays
Cells treated with SU11652 or the control solvent were extracted with a whole-cell extraction buffer containing 25 mM β-glycerophosphate (pH 7.3), 5 mM EDTA, 2 mM EGTA, 5 mM β-mercaptoethanol, 1% Triton X-100, 0.1 M NaCl, 1 mM sodium vanadate, and a protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN, USA). Cell lysates were cleared by centrifugation in a microfuge at 13,000 g, and clear cell extracts containing equal amounts of total proteins were separated on SDS gels for western blot analyses with antibodies against pFLT3, pERK, pAKT, and pSTAT5.
Acute myeloid leukemia
This work was supported by grants HL079441 and HL094591 from the National Institutes of Health, and a grant from Oklahoma Center for the Advancement of Science & Technology (to ZJ Zhao).
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