Heterogeneity and regulatory mechanisms of ALK-mutant and wild-type epithelial cells in non-small-cell lung cancer: Single-cell transcriptomics and chromatin accessibility.

[BACKGROUND] ALK mutations are key oncogenic drivers in non-small-cell lung cancer (NSCLC). Although ALK tyrosine kinase inhibitors (TKIs) elicit high initial response rates, relapse due to acquired resistance is common. Conventional approaches fail to resolve tumor heterogeneity and epigenetic control, necessitating multi-omic integration to dissect molecular differences between ALK-mutant and wild-type (WT) tumors.

[METHODS] Single-cell RNA sequencing (scRNA-seq) and matched chromatin accessibility (scATAC-seq) data of 5 WT and 4 ALK-mutant NSCLC samples from GEO (GSE274934) were integrated. "Seurat" and "Harmony" packages in R were used for data preprocessing and batch correction. "Monocle" package reconstructed epithelial differentiation trajectories. "CellChat" package delineated cell-cell communication. "Signac" package integrated multiple omics data and analyzed the differentially accessible chromatin regions. Motif enrichment based on the JASPAR database identified key transcription factors. The role of CEACAM6 in the malignant progression of ALK-mutant-mutant NSCLC was assessed by qRT-PCR, CCK-8, colony formation, and wound-healing assays.

[RESULTS] ALK-mutant epithelial cells exhibit disrupted differentiation trajectories, characterized by loss of SPINK1 expression and pan-expression of MARCKSL1. ALK-mutant cells relied on CEACAM5/6 signaling for communication; WT cells primarily engaged in multi-directional MIF interactions. ALK-mutant cells showed increased global chromatin accessibility. Motif analysis revealed enrichment of PRC pathway components and EGR1 binding sites (survival/drug resistance-associated) in ALK-mutant cells, whereas WT cells specifically enriched KLF5, ZNF148, and GLIS3 motifs. In vitro, CEACAM6 expression was significantly upregulated in ALK-mutant compared to WT cells. CEACAM6 knockdown significantly inhibited cell proliferation and migration, and reduced p-AKT and EGR1 protein levels, indicating that CEACAM6 promotes malignant progression of ALK-mutant NSCLC through the AKT-EGR1 signaling axis.

[CONCLUSION] ALK mutations drive tumor stemness and drug resistance by blocking epithelial cell differentiation, activating CEACAM6-mediated signaling, enhancing chromatin accessibility, and remodeling the EGR1/PRC regulatory network. Targeting CEACAM6 and its downstream effector EGR1 may represent an effective strategy to overcome ALK-TKI resistance.