FLT3: Kinase Assay Development And Inhibitor Screening

Acute myeloid leukemia (AML) is the most common malignant myeloid disorderin adults and is featured by abnormal differentiation and proliferation ofhematopoietic progenitor cells of the myeloid lineage. Currently, chemotherapy isthe mainstay of AML treatment. However, due to the complexity of AML,chemotherapy is only effective in a selected group of patients. Besides, the sideeffects of chemotherapy is too severe to be tolerated by some patients. Bone marrowtransplant is an effective way to treat AML, but it is always hard to find a matcheddonors. With the development of modern molecular medicine and betterunderstanding of pathogenesis of AML in recent years, several molecular lesionshave been identified in AML patients. This sets a foundation for development oftarget drug therapy in AML.FMS-Like Tyrosine Kinase3(FLT3) is mutanted in about30%of AML patients,representing the most frequent mutation found in the disease. The high frequency ofFLT3mutations makes it an attractive target in AML therapy. Among thesemutations, the most common type is FLT3-ITD caused by internal tandemduplication in the juxtamembrane domain, and another mutation FLT3-TKD typeinvolves point mutations in the tyrosine kinase domain (TKD), usually occurring atD835. In addition, there are several less commonly found deletions and insertions injuxtamembrane domain of FLT3. The presence of FLT3-ITD interrupt the structureof juxtamembrane domain, which is believed to negatively regulate kinase activity ofFLT3, while FLT3-TKD mutations can change kinase activity of FLT3directly.Therefore, these two types of FLT3mutations make FLT3constitutively active, andlead to the activation of downstream signaling pathways thereby resulting inuncontrolled proliferation of leukemia cells. Usually, FLT3mutations is related toadverse prognosis in AML patients.FLT3mutations are important prognosis markers and therapeutic targets. And FLT3related targeted therapies are under development. Currently, several smallmolecule FLT3inhibitors have been identified. However, the clinical effects of theseFLT3inhibitors are still limited by their potency and specificity. Needless to say,more potent and specific FLT3inhibitors are needed.In our study, in order to screen novel FLT3inhibitors, we developed a FLT3kinase assay by using substrate GST-FLT3S.Firstly, we designed the substrate GST-FLT3S, which is a GST phusion proteincontaining the autophosphorylation site of FLT3. It was expressed in E. coli cells andpurified by using a glutathione-Sepharose column. After purification, the purity ofGST-FLT3S can achieve99%. In order to perform FLT3kinase assay, we expressedHis-tagged recombinant protein containing FLT3wild type and mutant catalyticdomain by using the baculovirus expression system. These recombinant proteinswere purified by using Ni-NTA columns. To identify their kinase activity, theautophosphorylation of these recombinant protein was detected by western blot withanti-PY antibody PY20.With GST-FLT3S and recombinant proteins containing FLT3wild type andmutant catalytic domain, we established a new FLT3kinase assay system. Byperforming kinase assays with different amounts of the substrate and kinases forvarious periods of reaction time, we found that the two TKD mutant forms of FLT3had much higher kinase activities than wild type FLT3.To avoid the tediousness of western blot, we performed kinase assaysGST-FLT3S immobilized on glutathione-Sepharose beads and detected thephosphorylation of the immobilized GST-FLT3S by using a fluorescent microscope.Although this method is an easier and fast, it is hard to quantify the signal. Therefore,we employed the western blot method for most of our studies.To validante that our FLT3kinase assay can be used for inhibitor screening, wetried several well-studied kinase inhibitors in the FLT3kinase assay. As expected,known FLT3inhibitors can inhibit the phosphorylation of GST-FLT3S effectively,but other kinase inhibitors failed to inhibit this phosphorylation. Therefore, the GST-FLT3S-based FLT3kinase assay is effective in FLT3inhibitor screening.In order to screen novel FLT3inhibitors, we apply a kinase inhibitor library fromCalbiochem. This library contains80ATP competitive protein kinase inhibitorsincluding several known FLT3inhibitors. The screening led to identification of acompound named SU11652, which is a novel FLT3inhibitor.SU11652is a known kinase inhibitor, but we found that it inhibits FLT3withhigher potency. We measured IC50of SU11652to wild type of FLT3as well as twomutant forms of FLT3. The results demonstrate that SU11652can inhibit mutantforms of FLT3effectively with a little higher IC50compared with wild type FLT3.In cell-based assays, we applied FLT3-ITD positive leukemia cell line MV-4-11to study the effectiveness of SU11652. We found that SU11652can inhibit theproliferation of MV-4-11effectively as revealed by MTT assay.In addition, themorphology of MV-4-11changed significantly when the cells were treated bySU11652. However, the growth and morphology of control HL60, Jurkat and Karpas299cells were not affected by SU11652. Therefore, SU11652inhibits the growth ofMV-4-11effectively and selectively.To reveal further how SU11652inhibits the growth of MV-4–11cells, weconducted apoptosis assays, cell cycle analyses, and signaling pathway studies.Apoptosis was demonstrated by staining cells with Annexin V and propidium iodide.The results showed that the percentage of Annexin V-positive and propidiumiodide-negative MV-4-11cells was increased following SU11652treatment,indicating induction of apoptotic cell death. For cell cycle analysis, we applied coldethanol to fix SU11652-treated cells and stained these cells by propidium iodide..Flow cytometric analysis revealed that the percentages of S and G phase cells inSU11652-treated cells were reduced significantly. Together, the data indicate thatSU11652induces both apoptosis and cell cycle arrest of MV-4-11cells.Finally, we applied western blot to detect the phosphorylation of ERK1/2, Akt,and STAT5in SU11652treated MV-4-11cells. GAPDH was also blotted as theloading control. As expected, we found a dose-dependent reduction in the phosphorylation levels of FLT3, ERK1/2, Akt, and STAT5in MV-4-11cells treatedwith SU11652. This indicates that the activation of these downstream signalingproteins in MV-4–11is blocked by SU11652, thereby providing a molecularmechanism for the inhibition of cell growth by the inhibitor.In summary, we developed an FLT3kinase inhibitor screening system by usingthe unique substrate GST-FLT3S. The inhibitor screening led to identification of anovel small molecule FLT3inhibitor SU11652. It is more potent and specificcompared with FLT3inhibitors currently available. It is also effective and specific ingrowth inhibition of FLT3mutant cell lines. Considering the potency and selectivityof SU11652according to biochemical and cell-based assays, our study provides anew drug candidate for AML therapy in the future.