Activation of non-classical Wnt signaling pathway effectively enhances HLA-A presentation in acute myeloid leukemia

Abstract

Objective: The escape from T cell-mediated immune surveillance is an important cause of death for patients with acute myeloid leukemia (AML). This study aims to identify clonal heterogeneity in leukemia progenitor cells and explore molecular or signaling pathways associated with AML immune escape.

Methods: Single-cell RNA sequencing (scRNA-seq) was performed to identified AML-related cellular subsets, and intercellular communication was analyzed to investigate molecular mechanisms associated with AML immune escape. Bulk RNA sequencing (RNA-seq) was performed to screen differentially expressed genes (DEGs) related to hematopoietic stem cell progenitors (HSC-Prog) in AML, and critical ore signaling pathways and hub genes were found by Gene Set Enrichment Analysis (GSEA), Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. The mRNA level of the hub gene was verified using quantitative real-time PCR (qRT-PCR) and the protein level of human leukocyte antigen A (HLA-A) using enzyme-linked immuno sorbent assay (ELISA).

Results: scRNA-seq analysis revealed a large heterogeneity of HSC-Prog across samples, and the intercellular communication analysis indicated a strong association between HSC-Prog and CD8⁺-T cells, and HSC-Prog also had an association with HLA-A. Transcriptome analysis identified 1748 DEGs, enrichment analysis results showed that non-classical wnt signaling pathway was associated with AML, and 4 pathway-related genes (RHOA, RYK, CSNK1D, NLK) were obtained. After qRT-PCR and ELISA validation, hub genes and HLA-A were found to be down-regulated in AML and up-regulated after activation of the non-classical Wnt signaling pathway.

Conclusion: In this study, clonal heterogeneity of HSC-Prog cells in AML was identified, non-classical wnt signaling pathways associated with AML were identified, and it was verified that HLA-A could be upregulated by activation of non-classical wnt signaling, thereby increasing antigen presentation.

Keywords: HLA-A; acute myeloid leukemia; immune escape; non-classical Wnt signaling pathway; scRNA-seq.

Figure 1

Cell quality control and classification. (A) Identification of 91772 cells and 22032 genes after quality control analysis of scRNA-seq for AML; (B) Scatterplots of the relationship between the number of sequences and the proportion of mitochondria and the number of genes before quality control, respectively; (C) Characteristic variance plot of gene expression profiles in the sample; (D) PCA results.

Figure 2

Identification of 19 cellular subsets and their intercellular communication based on scRNA-seq data. (A) Classification of cellular subsets in each sample; (B) Bubble plot of the expression level of the markers used for cell annotation; (C) The UMAP plot of the cell annotation results; (D) Stacked plot of the proportion of different cells in AML samples or healthy samples.

Figure 3

Cellular communication between HSC-Prog and other cellular subsets. (A) Interactions probabilities of ligand-receptor signaling associated with intercellular communication; (B) Cellular communication diagram.

Figure 4

Results of HSC-Prog-based differential gene expression analysis and enrichment analysis. (A) Results of differential gene expression analysis of AML1 and other samples; (B) Enriched signaling pathways by GSEA; the top 10 hub genes; (C) Biological processes, (D) Molecular functions, and (E) Cellular components; (F) The top 10 KEGG pathways for hub genes.

Figure 5

Identification of hub gene expression. (A–D) The relative expression levels of RHOA, RYK, CSNK1D and NLK in HL group, AML group, Foxy-5+AML group and Box5+AML group were detected by qRT-PCR; (E) HLA-A protein levels in HL group, AML group, Foxy-5+AML group and Box5+AML group were detected by ELISA; (F) Protein levels of HLA-A in various cells from bone marrow. Notes: Compared with HL, ** means P<0.01, *** means P<0.001; Compared with AML group, # means P<0.05, ## means P<0.01 and ### means P<0.001.