E2F1 signatures stratify immunotherapy response and reveal YWHAQ vulnerability in esophageal squamous cell carcinoma

To improve biological interpretation of immune checkpoint inhibitor (ICI) response in esophageal squamous cell carcinoma (ESCC), we developed an E2F1 transcriptional regulation-related framework integrating spatial multi-omics and experimental validation. Single-cell and post-treatment spatial analyses localized E2F1-associated activity predominantly to malignant epithelial states. In ten ESCC Xenium specimens, paired patient-level analyses linked E2F1-high epithelial neighborhoods to lower CD8 effector T-cell and B-cell densities, a lower immune-active niche score, and higher epithelial CD274 -positive fractions. In harmonized public ICI cohorts, E2F1 .Sig functioned as an exploratory response-stratification signature, achieving the highest mean area under the curve (AUC) among evaluated immune signatures across validation and external testing cohorts. Separately, TCGA analyses supported prognostic associations of a 20-gene Hub- E2F1 .Sig risk score. In ESCC, Hub- E2F1 .Sig-based subtyping defined three immune microenvironmental states with distinct survival patterns. Cluster1 showed a more favorable immune contexture and better prognosis, suggesting its association with immune responsiveness. YWHAQ , a key Hub- E2F1 .Sig gene, was associated with poor prognosis, and YWHAQ depletion reduced malignant phenotypes, increased cisplatin-associated apoptosis, and attenuated PD-1/PD-L1-associated immunosuppressive features in vitro and in vivo. Virtual screening identified 6’-Sialyllactose as an in silico candidate YWHAQ-binding compound. These findings support an exploratory E2F1-centered framework and nominate YWHAQ as an ESCC-focused therapeutic hypothesis.