Analysis Of Common Targets And Exploration Of New Targets In The Immunotherapy Of Acute Myeloid Leukemia

Part 1 Analysis of common target antigens in immunotherapy of acute myeloid leukemiaObjectiveAcute myeloid leukemia(AML)is a highly heterogeneous tumor,and it is difficult to select a universal immunotherapy target.Only through in-depth analysis of the target antigens of AML subsets with different characteristics,it is possible to find suitable targets and achieve safe and efficient personalized immunotherapy.Therefore,the first part of this study aims to analyze the expression of common and potential target antigens of AML with different gene mutations,de nove or secondary,newly diagnosed or relapsed/refractory(R/R)AML,to find more suitable targets and potential new targets with different characteristics of AML subsets.MethodsPatients newly diagnosed with AML(non-acute promyelocytic leukemia)in Tongji Hospital affiliated to Huazhong University of Science and Technology from January 2013 to December 2021 were enrolled.The clinical and laboratory data of these patients were collected.The patients were divided into groups according to risk stratifications,gene mutation status,de nove or secondary,newly diagnosed or R/R AML,and the differences of target antigen expressions between groups were compared.ResultsA total of 1024 patients with AML were included in this part of the study.Flow cytometry immunotyping analysis showed that among the target antigens,stem/progenitor cell antigen CD117 had the highest positive rate of 97.2%,followed by myeloid antigen CD33,whose positive rate was 93.7%.However,the expression of lymphoid antigen was relatively low,and the highest expression was CD9,with positive rates of 49.5%.The positive rates of CD38 and CD96 in patients with favorite risk category were significantly higher than those in patients with poor risk category,while the positive rates of CD4 and CD9 in patients with favorite risk category were significantly lower than those in patients with poor risk category(P<0.05).There was no significant difference in the expression of CD117,CD123,and CD33 among the three groups(P>0.05).Compared with AML patients with FLT3wt/NPM1wt,the positive rate of CD123 was significantly increased in patients with FLT3wt/NPM1mut,the positive rates of CD 123,CD4,and CD9 were significantly increased in patients with FLT3-ITDmut/NPM1wt and the positive rates of CD 123 and CD9 were significantly increased in patients with FLT3-ITDmut/NPM1mut(P<0.05).The expression of CD38,CD7,and CD96 in patients with CEBPA double mutation was significantly higher than that in patients without CEBPA double mutation(P<0.05).There was no significant difference in the expression of these target antigens between patients with DNMT3A mutation and wild-type patients(P>0.05).The positive rates of CD4,CD7,and CD96 in patients with IDH mutation were significantly lower than those in patients without IDH mutation(P<0.05).The positive rate of CD38 in patients with secondary AML was lower than that in patients with de nove AML.There was no significant difference in the expression of other antigens between the two groups.The positive rates of CD38,CD123,CD4,CD7,and CD9 in R/R AML were significantly higher than those in newly diagnosed patients(P<0.05).A paired test showed that CD38,CD 123,and CD9 were obtained with high frequency at R/R,making the expression of these antigens higher at R/R than at initial diagnosis(P<0.05).Meanwhile,the CD4 and CD9 positive rates of R/R AML at initial diagnosis were significantly higher than those of non-R/R AML patients during the same period(P<0.05).The multivariate Logistic regression analysis showed that CD9 was an independent risk factor for R/R in AML patients(P=0.010).In terms of prognosis,the overall survival(OS)was significantly lower in CD123 positive patients than that in CD123 negative patients(median OS 65.4 months vs not yet achieved,P=0.011).The OS of CD9 positive and CD9 negative patients also showed the same trend(median OS 38.8 months vs not yet achieved,P=0.012).Other target antigens had no significant effect on the OS of AML(P>0.05).ConclusionThere was significant heterogeneity in the expression of target antigens in AML patients.CD9 is associated with FLT3-ITD mutation,R/R,and poor prognosis in AML patients,and may be a new immunotherapy target for AML.In addition,patients with FLT3-ITD mutations have high expression of CD 123 and CD9,and may be more likely to benefit from immunotherapy targeting CD 123 and CD9.Except that CD38 is more suitable as the target of de nove AML,other targets have similar applicability to de nove and secondary AML.CD38,CD9,and CD123 are highly expressed in patients with R/R AML,which can be used as more suitable targets for immunotherapy in patients with R/R AML.Part 2 Effect of decitabine on immunotherapy targets of AML cellsObjectiveIn the first part of this study,it was found that there was significant heterogeneity in the expression of target antigens in different AML subsets,and the expression density of target antigens seriously affected the effect of AML immunotherapy.So,how to improve the expression of target antigens to enhance the effect of immunotherapy is particularly important.Therefore,the second part of this study aimed to analyze the effects of hypomethylating drug decitabine on the expression of common and potential immunotherapeutic targets in AML cells and the related molecular mechanisms.MethodsData were collected from patients who were newly diagnosed with AML(non-acute promyelocytic leukemia)and received a decitabine-containing regimen in the first course of treatment in Tongji Hospital affiliated to Huazhong University of Science and Technology from January 2013 to December 2020.The expressions of common target antigens were compared before and after treatment.Flow cytometry was used to verify the changes of target antigens of AML cells treated with decitabine in 9 AML cell lines:THP-1,SKM-1,KG-1,OCI-AML3,MV4-11,Kasumi-6,Molm13,HL-60,and U937.RNA-seq was performed on SKM-1,MOLM-13,and U937 AML cell lines treated with decitabine to compare the expression differences of 15 common and 6 potential immunotherapy targets of AML between the decitabine-treated group and the control group.GO,KEGG,and GSEA enrichment analyses were performed for the differentially expressed genes(DEGs)in these three cell lines to explore possible involved signaling pathways.ResultsA total of 95 AML patients who received a decitabine-containing regimen were enrolled in this part of the study.The expression of target antigen in AML patients before and after treatment was compared,and it was found that the positive rates of CD38,CD123,and CD9 were significantly up-regulated after decitabine treatment(P<0.05).A paired analysis showed that CD38,CD123,and CD9 changed from negative to positive in 18.4%,48.3%,and 60.0%of AML patients,respectively,after decitabine treatment.Flow cytometry was used to detect the expression of CD9,CD38,and CD 123 in 9 AML cell lines.It was found that the positive rate and intensity of expression of these three surface antigens were significantly different in AML cell lines.It was found that the positive rate and expression intensity of the three target antigens in the above AML cell lines were significantly different,with the positive rate ranging from 1%to 100%.Compared with the control group,CD9 and CD3 8 were significantly upregulated in the above 9 AML cell lines after decitabine treatment,and CD 123 was significantly upregulated in the 7 AML cell lines except for Molm13 and MV4-11 after decitabine treatment.Among the three antigens,CD9 was the most up-regulated,and its average fluorescence intensity in the U93 7 cell line was about 10 times that of the control group.Transcriptome sequencing showed that among the 15 common and 6 potential AML immunotherapy targets,in addition to CD9,CD38,and CD123,5 targets(CCR1,CLL-1,CD70,SIGLEC6,CD 117)were significantly up-regulated in 2 cell lines after treatment with decitabine,while LILRB2,a member of leukocyte immunoglobulin-like receptor subfamily B,was significantly up-regulated in all 3 cell lines after treatment with decitabine.GO and KEGG enrichment analysis showed that the common DEGs of the three cell lines were mainly enriched in immune-related biological processes and signaling pathways.Further GSEA enrichment analysis of each cell line showed that signaling pathways such as cytokine-cytokine receptor interaction,interleukin,inflammatory response,and immunomodulatory interaction between lymphoid and non-lymphoid cells were significantly enriched and up-regulated in these three cell lines.These results suggest that the intervention of decitabine on AML involves the mechanism of immune regulation.ConclusionDecitabine can up-regulate many kinds of AML targets such as CD9,CD38,CD 123,and LILRB2,suggesting that there is a potential synergistic effect between immunotherapy for these targets and decitabine.Gene enrichment analysis indicated that decitabine may enhance the immune response through the immunomodulatory signaling pathway and inhibit the proliferation of AML cells through the immune microenvironment.Part 3 Exploration of LILRBs as targets for immunotherapy of AMLObjectiveThe main problem of AML immunotherapy is still finding suitable targets.The second part of this study found that LILRB2 may be a potential target for AML,while it is unclear whether other LILRB members can be used as immunotherapy targets.Therefore,the third part of this study aims to analyze the role of the LILRBs gene family in the expression,prognosis,progression,and immunosuppression of AML,and explore their possibility as potential immunotherapeutic targets of AML.MethodsThe mRNA data of the LAML dataset from the TCGA database and corresponding normal samples from the GTEx database were downloaded using the UCSC Xena database.The differential expression levels of LILRBs between AML and normal samples were compared.The clinical information of TCGA-LAML patients was downloaded from the TCGA database,and the survival differences between LILRBs high and low expression groups were compared.The DEGs were obtained by comparing the gene expression of LILRB 1-4 high and low expression groups(<50%vs>50%)in the TCGA-LAML dataset.The functional enrichment of DEGs such as GO,KEGG,and GSEA was performed.The ssGSEA algorithm was used to evaluate the enrichment levels of 24 immune cells in the LAML dataset.The relationships between LILRBs and the expression levels of 23 common immunosuppressive genes were analyzed by the spearman’s test.Genes that were significantly positively correlated with LILRB 1-4 were screened and intersected to obtain genes that co-expressed with LILRB 1-4.The genes significantly related to the prognosis of AML were screened from these genes by univariate Cox regression analysis.Lasso regression was used to screen genes from co-expressed prognostic genes to establish a prognostic risk score model for AML.Univariate and multivariate Cox regression were used to evaluate the independent prognostic value of the risk score model and common risk factors.A prognostic Nomogram diagram was established using prognostic independent risk factors to predict the 1-year,2-year,and 3-year survival probability of AML patients.The Calibration curve was used to judge the prediction efficiency of the Nomogram diagram.Statistical analysis was performed and plotted using R software(v.3.6.3).ResultsThe expression levels of LILRB1-5 in AML were significantly higher than those in normal control samples.The overexpression of LILRB1-4 was significantly correlated with poor prognosis.The GO and KEGG enrichment analysis showed that DEGs were mainly enriched in immune-related biological processes and signaling pathways.The GSEA enrichment analysis showed that chemokine,IL-10,and other immune signal pathways,MAPK,PI3K-AKT-mTOR,and other carcinogenic signal pathways,cell adhesion molecules,leukocyte transendothelial migration,vascular endothelial growth factor receptor,and other drug resistance signal pathways were significantly enriched in LILRB1-4 high expression groups.Immune cell infiltration analysis showed that compared with the low expression groups of LILRB1-4,the enrichment levels of immunosuppressive cells such as immature DC,tumor-associated macrophages,and Treg cells were significantly increased,while the enrichment level of NK cells was significantly decreased in the high expression groups of LILRB1-4(P<0.05).The correlation analysis also showed that the expression levels of LILRB1-4 were positively correlated with the enrichment level of immunosuppressive cells mentioned above,but negatively correlated with the enrichment level of NK cells(P<0.05).In addition,LILRB1-4 were positively correlated with the expression of most immunosuppressive molecules such as TIM-3,CTLA-4,IDO1,IL-10,IL10-R,and TGFβ1(P<0.05).Six genes(DESI1,HTR7,GNGT2,S100A4,TFEB,and STIM2)were screened out to construct the prognostic risk score model by co-expression analysis,Cox regression analysis,and Lasso regression analysis.The time-dependent ROC curve showed that the prognostic risk score model had high accuracy,and the area under the curve for 1-year,3-year,and 5-year OS was 0.766,0.762,and 0.802,respectively.The multivariate Cox analysis showed that age,poor risk stratification,and risk score were independent risk factors for OS in patients with AML.The prognostic Nomogram diagram was constructed using the above independent risk factors.The Calibration curve confirmed that this Nomogram has strong predictive power for 1-year,2-year,and 3-year survival probability of AML patients.ConclusionLILRB1,LILRB2,LILRB3,and LILRB4 were overexpression in AML,play important roles in the progression and the formation of an immunosuppressive microenvironment of AML,and have high prognostic value,so they are likely to be potential immunotherapy targets for AML.Immunotherapy against these targets can not only kill AML cells but also inhibit the immune escape of AML cells.