Single-Cell Lineage Trajectory Defines Cyclin-Dependent Kinase Inhibitor-Sensitive Cells-of-Origin in Esophageal Squamous Cell Carcinoma.

Background and Aims: Understanding the cells of origin is essential for overcoming therapy resistance and improving early intervention strategies in esophageal squamous cell carcinoma (ESCC). Despite recent advances in genomic profiling, the precise cellular hierarchies and molecular programs driving ESCC initiation remain poorly defined. Methods: We utilized machine learning-based single-cell trajectory analysis on 4-nitroquinoline 1-oxide-induced murine models and genetically engineered organoids to identify cellular lineages during tumorigenesis. Combined with gene regulatory network analysis, we identified transcriptional drivers of tumor initiation and employed transcriptome-based drug repurposing to predict compounds targeting these initiating populations. Results: Our analyses revealed multiple distinct epithelial clusters that function as cellular origins of ESCC, exhibiting diverse stem and progenitor signatures. Gene regulatory network analysis of these populations indicated activation of stem/progenitor cell regulators, including CEBPβ and TFAP2A/C. Translating these findings, a transcriptome-based drug repurposing screen identified 5 chemical candidates, 4 of which are potent cyclin-dependent kinase inhibitors, aligning with the frequent loss-of-function mutations in Conclusion: This research delineates the potential cellular origins of ESCC and their key regulons, thereby pioneering a single-cell-derived therapeutic strategy that exposes vulnerabilities in tumor-initiating cells.