Selinexor Potentiates The Cytotoxic Effect Of Natural Killer Cell On Acute Myeloid Leukemia Possibly Via The HLA-E/NKG2A Pathway

Objective:The proportion and number of natural killer cells(NK cells)from peripheral blood were amplified by the anti-CD52 monoclonal antibody combined with the NK cell amplification kit to explore a new method of NK cell amplification.By comparing the ability of NK cells to kill small molecule drug selinexor pretreated and untreated acute myeloid leukemia(AML)cells,to explore whether selinexor can enhance the efficacy of NK cells in the treatment of AML,and further study the possible mechanism of selinexor affecting the killing activity of NK cells,providing new ideas for the treatment of patients with AML in clinic.Methods:Peripheral blood mononuclear cells(PBMCs)from healthy donors were co-stimulated with anti-CD52 monoclonal antibodies(anti-CD52 mAb)and cultured for 14 days in NK Cell Induction Culture Kit,and percentages of NK cells were measured by flow cytometry.Then,NK cells,AML cell lines and primary AML cells were pretreated with selinexor(0nmol/L、50nmol/L、100nmol/L and 400nmol/L)or DMSO control for 24 h.Next,AML cells were treated with the indicated concentrations of Selinexor(100nmol/L)for 24 h and co-cultured with NK cells for 4 h.Cells proliferation was determined by carboxy fluorescein succinimidyl ester(CFSE)staining,and cell apoptosis was analyzed by Annexin V/7-AAD staining.Surface expression of NKG2A,NKG2D,MICA/MICB and HLA-E was detected using flow cytometry.Results:Using anti-CD52 mAb plus NK Cell Induction Culture Kit,the number of NK cells(CD3-CD56+)was increased about 1000-fold,and the purity of NK cells increased from 1.39±2.01(%)to 86.65±5.03(%)after amplification.Low concentration of selinexor did not affect the apoptosis and proliferation of AML cells and NK cells compared with DMSO control(P>0.05).Selinexor-pretreated AML cells increased the susceptibility of AML cells to NK mediated cytotoxicity,characterized by decreased proliferation and induced apoptosis of AML cells compared with DMSO control(P<0.05).Moreover,selinexor down regulated the expression of HLA-E ligands on AML cells and the inhibitory NK cell receptors NKG2A(P<0.05),while the expression changes of MICA,MICB and NKG2D were not observed(P>0.05).Conclusion:A large number of NK cells with high purity could be amplified from PBMC by the combination of anti-CD52 mAb and cytokines.When the concentration of selinexor was low,it did not show significant killing activity against AML cells.Selinexor-pretreated AML cells increased the susceptibility of AML cells to NK mediated cytotoxicity,characterized by decreased proliferation and induced apoptosis of AML cells.Low concentration of selinexor may enhance the killing effect of NK cells through the HLA-E/NKG2A pathway.