Tim-3 As A Prognostic Candidate And Therapeutic Target In Acute Myeloid Leukemia

BackgroundAcute myeloid leukemia is a polyclonal malignant tumor in which a large number of primitive myeloid cells proliferate and destroy the hematopoietic function of the bone marrow,accounting for about 90%of adult leukemia.After induction therapy,70%-80%of adult patients(<60 years old)and 50%of elderly patients(>60 years old)can achieve complete remission.However,approximately 70%to 80%of patients relapse after achieving complete remission.Therefore,in order to overcome chemotherapy resistance,researchers need to find new AML targets that can predict prognosis and provide new treatment options.Tim-3,the full name of T cell immunoglobulin mucin 3,was first discovered in Thl cells,and later found to be expressed in dendritic cells,NK cells,monocytes and macrophages and lymphocytes,and negatively regulates T cell function.,In addition,Tim-3 is also expressed in tumor cells.Kikushige et al.found that Tim-3 is highly expressed in leukemia stem cells,which may be a new marker of LSC,by targeting Tim-3 to eliminate residual leukemia cells in patients.Caixia Li et al.found that Tim-3 was highly expressed in CD8+T cells in AML in NCCN high-risk group,suggesting that Tim-3 may be associated with poor prognosis of patients.On the other hand,Tim-3 can inhibit T cell proliferation by affecting the expression of TCF1,and blocking immune checkpoints can restore its function.At present,there is no report on the expression and clinical significance of Tim-3 in primary cells and T cells of newly treated acute leukemia.This study explores the expression of Tim-3 on promyelocytes and CD3+T cells in acute leukemia patients and its relationship with prognosis in order to find new targets for AML therapy.Objective(1)To explore the expression of Tim-3 in bone marrow cells of newly diagnosed AML patients and its correlation with prognosis.(2)To explore the relationship between Tim-3 and acute myeloid leukemia LSC and study the relationship between Tim-3 expression and prognosis in CD34+leukemia stem progenitor cells.(3)To study the expression of Tim-3 in CD3+T cells of AML patients and its relationship with prognosis.Research objects and methodsCase data:173 cases of TCGA-AML RNA-seq data were downloaded from the cBioportal website,GSE37642-GPL570,GSE71014 and GSE12417 three AML data sets with complete prognostic information were downloaded from GEO to verify the conclusions of the TCGA cohort;GSE14924 was downloaded from GEO to study AML patients T Differences in gene expression between cells and healthy human T cells.Bone marrow cells of newly diagnosed AML who were hospitalized in the Department of Hematology in Nanfang Hospital from March 2019 to December 2019 were collected,and the expression of Tim-3 in CD34+cells and Tim-3 in CD3+T cells were detected by flow cytometry.Research method:(1)Using the GEPIA website to analyze the difference in the expression of Tim-3 in bone marro w cells of AML patients and healthy people online;(2)TCGA-AML patients were divided into Tim-3 high and Tim-3 low groups according to the expression of Tim-3,and the overall survival time(OS)and diseasefree survival status(DFS)of the two groups were compared.The GSE37642-GPL570,GSE71014 and GSE12417 datasets were combined to validate the results from the TCGA-AML cohort.Gene difference analysis was performed between the two groups of Tim-3 high and Tim-3 low,and enrichment analysis was performed to understand the differences in cell signaling pathways between the two groups,and to explore the relationship between Tim-3 expression and stem cell pathways.(3)Flow cytometry was used to detect cell membrane molecules,and to detect the expression of Tim-3 in AML patients in our center.(4)Western blot was used to detect the expression level of Tim-3 in AML cell lines and primary cells from AML patients before and after treatment.(5)To compare the differences in CD3+T-cell Tim-3 and related immune checkpoint expression between AML patients and healthy individuals according to the GSE14924 cohort,and to perform GO and KEGG analysis of the differential genes between the two groups.(6)We divided CD3+T-cell Tim-3 expression into two groups,CD3 Tim-3 high and CD3 Tim-3 low,according to our center,and compared the survival.Results(1)The expression of Tim-3 in bone marrow cells of TCGA-AML patients was significantly higher than that of healthy people,and the OS and DFS in the Tim-3 high group were significantly worse than those in the Tim-3 low group.Multivariate analysis showed that Tim-3 was an independent prognostic factor.The prognostic significance of Tim-3 was confirmed in the GEO-AML dataset and the Southern Hospital Hematology AML single-center cohort.(2)The expression of Tim-3 in CD3+T cells of GEE14924-AML patients was significantly higher than that of healthy people.The single-center cohort of Nanfang Hospital was divided into two groups:CD3 Tim-3 high and CD3 Tim-3 low according to Tim-3 expression.Survival analysis showed that the OS and DFS in the CD3 Tim-3 high group were significantly worse than those in the CD3 Tim-3 low group,and CD3+T cells’ Tim-3 was an independent prognostic factor.Conclusion(1)The higher expression of Tim-3 in leukemia cells in AML patients indicates that the stem cell signaling pathway is more abundant,the expression of Tim-3 may be positively correlated with LSC,and the prognosis of patients with high expression of Tim-3 in leukemia cells is worse,and Tim-3 is an independent prognostic factor in AML patients,targeting Tim-3 may clear LSC.(2)Tim-3 of CD3+T cells in AML patients is significantly higher than that in healthy people.The increase of Tim-3 and PD1 may mediate T cell function exhaustion,and patients with high Tim-3 expression of CD3+T cells have a worse prognosis.Targeting Tim-3 of CD3+T cells may restore T cell function and enhance anti-tumor effect.