Decitabine Treatment Induces CD48 Expression In AML Cells And Stimulates NK Cell Responses

Background and purpose:Decitabine(DAC),5-aza-2'-deoxycytidine,has a cytotoxic effect at high concentrations and a demethylation effect at low concentrations.DAC has been used to treat myelodysplastic syndrome(MDS),acute myeloid leukemia(AML)and other malignancies.CD48,a costimulatory molecule that mediates the killing effect of natural killer(NK)cells,may be regulated by DNA methylation and affect the prognosis of AML.Our previous study showed that the expression level of CD48 affects the prognosis of AML by methyl C-capture sequencing(MCC-Seq)and bioinformatics techniques.Therefore,the effects of DAC on CD48 and its effects on the treatment and prognosis of AML deserve further exploration.Methods:The regulation of DNA methyltransferase(DNMTs)and CD48 at messenger RNA(mRNA)and protein levels by DAC was detected by Real-time Quantitative PCR Detecting System(QPCR),Flow Cytometry(FCM)and Western Blotting.The killing activity of NK cells to DAC-treated WEHI-3 cells at different concentrations was measured by FCM.A mouse leukemia model was constructed and divided into AML group,AML+DAC group and AML+DAC+anti-CD48 group.All groups were infused with NK cells.The survival of three groups was compared.Pathological analysis of liver and spleen paraffin sections was performed using hematoxylin-eosin staining.Peripheral blood mononuclear cells were obtained by Ficoll separation solution.APC anti-mouse CD49b antibody and BV421 anti-mouse CD 107a were stained.And CD 107a positive cell ratio was detected by FCM to monitor NK cells killing activity in vivo.Results:MCC-seq results showed that CD48 methylation levels were higher in AML patients than in normal subjects,and CD48 methylation levels were down-regulated after DAC treatment.Bioinformatics analysis showed the expression of CD48 mRNA in AML patients was significantly lower than that of healthy people(P<0.01).The DNA methylation level was significantly decreased after 10 days of treatment with DAC(P<0.0001),with increasing expression of CD48 mRNA.Online database analysis showed that overall survival(OS)and event-free survival(EFS)were significantly longer in patients with CD48high AML than that in patients with CD48low AML(P<0.01).DAC could up-regulate the mRNA expression levels of CD48 in human SKNO-1,KASUMI-1 cell lines and mouse EL-4 and WEHI-3 cell lines,which were statistically significant(P<0.01).FMC showed that DAC significantly up-regulated the expression of CD48 on cell surface in the former four cell lines;WB results showed that CD48 protein expression was increased in WEHI-3 and SKNO-1 cells stimulated by DAC.While the expression levels of DNA methyltransferase(DNMTs)proteins(including DNMT1,DNMT3A,DNMT3B)decreased.DNMTs and CD48 expression levels were negatively correlated.The results of NK cell killing experiments showed that the killing activity after DAC treatment was higher than that of the control group(P<0.001).After the anti-mouse CD48 blocking antibody was added,the percentage of cells in the control group and the DAC-treated group was changed from 52.7%and 44.1%to 48.0%and 48.4%,indicating that anti-mouse CD48 antibody could reduce the killing effect of NK cells to DAC-treated WEHI-3 in mice(P<0.001).The spleen weight of AML+DAC group was lighter than that of AML group(P<0.05),and the spleen weight of AML+DAC+anti-CD48 group was heavier than that of AML+DAC group(P<0.05).Compared with the AML group,the quantity of nodules caused by extramedullary infiltration of liver was significantly smaller in the AML+DAC group,and the coverage area was significantly more limited.Compared with the DAC group,the AML+DAC+anti-CD48 group had a larger number of nodules and a larger coverage area.However,when compared with the AML group,the AML+DAC+anti-CD48 group had a smaller number of nodules and more limited coverage area.The results of HE staining of spleen paraffin sections showed disordered structure,abnormal deeply stained red pulp,increased number of high nuclear-plasma ratio cells and fatty necrotic cells in the AML group.Germinal centers were decreased or even disappeared in this group.The aberration degree was as listed:AML group>AML+DAC+anti-CD48 group>AML+DAC group.HE staining results of liver paraffin sections showed that all the three groups had deep-stained tumor cells with high nuclear-plasma ratio.The infiltration range was as listed:AML group>AML+DAC+anti-CD48 group>AML+DAC group.The proportion of CD 107a positive cells in the AML+DAC group and the AML group were 11.4%and 2.48%(P<0.05).The proportion of CD 107a positive cells in the AML+DAC+anti-CD48 group was 5.76%,which was lower than that in the AML+DAC group(P<0.05).The survival time of AML+DAC group was significantly longer than that of AML group(P<0.0001).After adding anti-mouse CD48 antibody,the survival time of AML+DAC+anti-CD48 group was significantly shortened than that of AML+DAC group(P=0.0019).Conclusion:DAC up-regulated the expression of CD48 by down-regulating its DNA methylation level,leading to enhanced affinity of NK cells and AML cells and increased cytotoxicity of NK cells.Up-regulation of CD48 by DAC in combination with NK cell infusion can reduce tumor burden and prolong survival of AML mice.Demethylating drugs followed by NK cell infusion provides a new therapeutic strategy for AML.