Cytoplasmic Mutated NPM1 Interacted With Akt And Regulating The Proliferation And Apoptosis Of Leukemia Cells Through AKT/FOXO3A Pathway

Objective Nucleophosmin (NPM) gene mutations are the most common genetic alteration in acute myeloid leukemia (AML), NPM1 mutation A (NPM1 mA) occurs in about 75%-80% of cases, which have an important impact on the pathogenesis, treatment and prognosis of leukemia. However, the potential role of cytoplasmic NPM1 mA to the molecular mechanism in leukemogenesis is far from elucidated. This paper aim to observe the interaction between NPM1 mA and the master regulator AKT, and investigate the potential effect of NPM 1 mA to the activation of the AKT/FoxO3a signaling pathway in leukemia, then discussed the impact of NPM1 mA on proliferation and apopotosis through AKT activation in leukemia cells.Method Co-immunoprecipitation assay was used to explore the interaction between NPM1 mA and AKT. The subcellular location of NPM 1 mA and AKT was tested by immunofluorescence. The expression of p-AKT and its downstream factor FoxO3a after NPM1 mA disturbance and overexpression was determined by Western blot. The protein level of p-AKT was observed after treated with different concentration of AKT inhibitor TV (AKT IV) in leukemia cells. The influence of activated AKT which mediated by NPM1 mA to cell proliferation ability was assayed through CCK-8 and colony formation. Flow cytometry and Hoechst 33258 staining were used to test the effect of activated AKT which mediated by NPM1 mA to cell apoptosis.Results NPM1 mA interacted with AKT in OCI/AML3 cells and co-located in cytoplasm. After NPM1mA knockdown in OCI/AML3, the expression of p-AKT(S473)、p-AKT(T308) and its downstream factor p-FoxO3a was downregulated. Overexprssion of NPM1 mA in HEK293T and K562 cells promoted the expression of p-AKT(S473、p-AKT(T308) and p-Fox03a. The AKT inhibitor (AKT IV) could reduce the expressiion of p-AKT(S473) and p-AKT(T308) in dose dependent manner while not influence the level of AKT. CCK-8 assay found that the proliferation of mutation group cell (K562 mA) was significantly increased compared to the wild group (K562 wt) and mock group (K562 c1) (P<0.001). After treated with AKT IV for 48 h, the growth of K562 wt and K562 cl group was depressed, while the K562 mA group was still in proliferation. Colony formation assay displayed that the colony forming rate of K562 mA was (1.2±0.4)% after treated with AKT IV for 2 weeks, while the K562 wt and K562 cl group haven’t colony forming. The difference was statistically significant (P<0.001). Flow cytometry showed that the rate of apoptotic cells was (8.33±0.85)% in K562 mA group after serum starvation for 4 h, which was significant lower than K562 wt group (15.01±2.24)%(P<0.05). After treated with AKT IV, the apoptosis rate of K562 mA cells was (42.95±3.35)%, which was higher than K562 wt group (30.97±1.3)%. The difference was obvious (P<0.05). In addition, Hoechst 33258 staining showed that the apoptotic cells in K562 mA group was lower than K562 wt and K562 cl group, after treated with AKT IV, the apoptotic cells in K562 mA group was increased, while there was no obvious difference in K562 wt group.Conclusion Our data indicate that cytoplasmic NPM1 mA interacted with AKT and regulating the proliferation and apoptosis of leukemia cells through AKT/FoxO3a pathway.