Reactivating PP2A By FTY720 As A Novel Therapy For AML With C-KIT Tyrosine Kinase Domain Mutation

PART I Detection of C-KIT/D816V mutation in Acute Myeloid Leukemia with Different Subtypes in ChinaObjective To investigate and analyze the incidence of C-KIT/D816V mutation in AML patients in China.Methods Blood samples from 286 AML patients with various French-American-British (FAB) classifications were collected from Center for Stem Cell Research and Application between 2008 and 2011. The diagnosis of AML and the assignment of FAB classification were based on morphology and immunophenotype. Then the quantitative realtime-PCR was used to examine the C-KIT/D816V mutation.Results Of the 286 AML samples studied,8 were shown to have a point mutation at D816V, including 6 male cases and 2 female cases. Its incidence in male and female was 3.9% and 1.5% respectively, thus it was more inclined to occur in male than female (P< 0.05). This mutation was typically located within the M2 (5 cases) and M4 (1 cases) subclasses, with the other 2 mutation AML cases not definitely diagnosed. In the present work, the D816V mutation occurred in 6.8% of M2,4.3% of M4 subclasses, and the overall incidence of C-KIT/D816V was 2.8% in AML.Conclution C-KIT/D816V most frequently occurs in M2 subutype, then M4 in AML, and it is more inclined to occur in male than female. Our research provided significant evidence for the incidence of C-KIT/D816V mutation in AML and its subtypes in China. partⅡAnalysis of Changes of the Activity of PP2A in Acute Myeloid LeukemiaObjective To analyze the changes of PP2A activity in AML patients with C-KIT/WT and C-KIT/D816V and compare the PP2A activity in HL60 cell harboring C-KIT/WT and kasumi-1 cell harboring C-KIT/TKD.Methods Eight AML patients harboring C-KIT/D816V mutation, twelve AML patients harboring wide-type C-KIT (C-KIT/WT) and twenty healthy controls were recruited and informed consent at Union hospital of Wuhan in this study. Real-time quantitative PCR was used to identify C-KIT/WT and C-KIT/D816V. Peripheral blood mononuclear cells (PBMCs) of patients with AML and normal controls were collected. In addition, HL60 and kasumi-1 cells were cultivated in vitro. The activity of PP2A in the PBMCs of patients and the cell lines was assayed with a PP2A Immunoprecipitation Phosphatase Assay Kit, the data was analyzed by SPSS 16.0.Results We detected PP2A activity of all samples with PP2A Immunoprecipitation Phosphatase Assay Kit. The results indicated that the PP2A activity in AML patients harboring C-KIT/WT(565.69±122.56 pmol) was obviously lower than the normal controls(706.02±154.22 pmol), and the PP2A activity in AML patients harboring C-KIT/D816V(419.01±96.97 pmol) was the lowest. There was significant difference between the three groups (P<0.05). In addition, PP2A activity in Kasumi-1 cell(886.420±152.187 pmol) was obviously lower than HL60 cell(1196.442±219.483 pmol). There was significant difference between them (P<0.05).Conclution It is concluded that from the standpoint of prevalence, PP2A activity is decreased in patients with C-KIT/TKD AML. In addition, compared with AML cells lines harboring C-KIT/WT, PP2A activity of AML cells harboring C-KIT/TKD is obviously lower. PP2A protein plays a key role in the genesis and development of AML, and may imply a diagnosis and treatment of AML, especially the refractory C-KIT/TKD AML. PARTⅢCytotoxic Effect of FTY720 on AML Cell Lines harboring C-KIT/TKD and C-KIT/WT and Its MechanismObjective To observe the cytotoxicity of FTY720 in C-KIT/TKD AML and develop FTY720 as a novel therapy for C-KIT/TKD AMLMethods Cell lines were treated with FTY720 alone or combined with specific PP2A inhibitor okadaic acid (OA) in culture. Cell growth was assessed using the CCK8 Kit. Cell apoptosis was analyzed by flow cytometry after dual staining with annexinⅤ/ propidium iodide. PP2A activity was assayed with a PP2A Immunoprecipitation Phosphatase Assay Kit. Western blotting was used to analyze the protein changes. All experiments were repeated three times. Statistical analysis was performed using SPSS 16.0.Results FTY720 induced toxicity in all AML cells, including Kasumi-1 and HL60 in a time-and dose-dependent manner. PP2A inhibitor OA rescued these cells from FTY720-induced apoptosis. Furthermore, Increased PP2A activity was detected after FTY720 treatment. When cells were pretreated with OA, PP2A activity was partially rescued. Finally, PP2A expression remained unchanged after FTY720 treatment. Our results strongly suggested that FTY720-induced cytotoxicity was mediated by PP2A activation without altering its expression. Interestingly, it was observed that cells harboring C-KIT/TKD mutation were more sensitive to this agentConclusions Our study provides evidence for the PP2A-dependent toxicity of FTY720 in AML cells, and these effects of FTY720 appear to be most marked in C-KIT/TKD AML where conventional chemotherapeutic approaches are most likely to fail. The significant preclinical efficacy of FTY720 indicates that it may be valuable therapeutic agents for refractory C-KIT/TKD AML.