Exploring the Role of Cuproptosis-related Genes in Acute Myeloid Leukemia Through WGCNA, Single-cell Sequencing and Experiments

Background: Cuproptosis, a newly discovered form of programmed cell death, has potential implications for tumorigenesis and cancer progression. This study investigates the role of cuproptosis in Acute Myeloid Leukemia (AML) and identifies associated biomarkers using bulk and single-cell RNA sequencing. Despite recent advances, the mechanisms of cuproptosis in AML remain unclear, and its relationship with immune cell infiltration could reveal novel therapeutic targets. Methods: RNA-seq data from 151 AML patients and 70 healthy controls were obtained from TCGA and GTEx databases, and single-cell RNA-seq data from 10 AML patients (GEO) were used for validation. Differential expression of Cuproptosis-Related Genes (CRGs) was analyzed via RCircos and correlation analysis. Immune cell infiltration was assessed using CIBERSORT and ssGSEA. WGCNA identified key genes for AML and cuproptosis subtypes, which were validated with single-cell data. Intercellular communication was analyzed through ligand-receptor interactions. RNA interference experiments validated TLR4 and NCF2, with gene expression measured through RT-qPCR. Apoptosis and CCK-8 assays assessed cell viability. Results: We identified 18 CRGs with differential expression between AML subtypes linked to immune cell infiltration. Subtype analysis classified AML patients into C1 and C2 subgroups enriched in biosynthesis and metabolism pathways. WGCNA identified 2701 genes associated with AML and 92 with cuproptosis, leading to 15 intersecting genes. RETN was highlighted as key in intercellular communication. Experimental validation showed that elesclomol-induced cell death in THP-1 cells is reversible by TTM. Knockout of TLR4 and NCF2 promoted cuproptosis. Conclusion: These findings offer new insights into the role of cuproptosis in AML, highlighting novel biomarkers, such as TLR4 and NCF2, which may provide promising targets for the development of future therapeutic strategies in AML treatment. result: We obtained 18 CRGs that were differentially expressed between the two populations and found that these genes were closely related to the infiltration of immune cells. Through subtype analysis, we divided AML patients into C1 subtype and C2 subtype based on CRGs. The differential genes between these subtypes are mainly enriched in biosynthesis and metabolism-related pathways. Through WGCNA analysis, we obtained 2701 key genes related to AML and 92 key genes related to cuproptosis classification, culminating in the identification of 15 intersectional key genes. Single-cell transcriptome data from the GEO database revealed that RETN may be a crucial pathway affecting intercellular communication in AML through these intersecting key genes. After verifying the expression of these genes through the single-cell transcriptome of the peripheral blood of a patient with congenital AML in our hospital, we screened out two key genes, TLR4 and NCF2, for subsequent experimental verification. We demonstrated with experiments that the copper ion carrier elesclomol can induce death in the THP-1 cell line, and cell death can be reversed with the copper chelator TTM. For the first time, we found that knocking out TLR4 and NCF2 can promote cuproptosis in THP-1 cells.