BACKGROUND: Osteosarcoma (OS) is highly heterogeneous and often exhibits an immunosuppressive tumor microenvironment, contributing to poor outcomes. The cell-type-resolved role of the ATF/CREB stress-response network, particularly ATF4, remains unclear in OS. METHODS: We integrated public OS single-cell RNA-seq and bulk transcriptome cohorts. Single-cell analyses included malignant program discovery (cNMF), regulon inference (SCENIC), and cell-cell communication (CellChat). Bulk cohorts (TARGET and GEO) were used to build and validate a prognostic model. ATF4-associated immune features were assessed using ESTIMATE, immune deconvolution, and TIDE-related metrics. WNT/β-catenin activity was evaluated by gene-set scoring. ATF4-related drugs were screened using DSigDB/Enrichr and assessed by docking. Sorafenib was selected for molecular dynamics simulation. ATF4 function was validated by siRNA knockdown (MG63, U2OS) and overexpression (HOS). RESULTS: A stress-associated malignant program showed enriched ATF/CREB activity and prominent signaling interactions. Intersecting its markers with ATF/CREB genes identified ATF3/ATF4/CREB5, which were used to construct an ATF/CREB-associated risk score (ACS) that stratified survival in TARGET and external cohorts. ATF4 showed the most consistent adverse prognostic association and remained an independent factor in multivariate analyses. ATF4-high tumors were associated with a more immunosuppressive profile and stronger WNT/β-catenin signaling, and these features tracked with the ACS. Among the compounds tested in silico, sorafenib ranked highest in docking, and subsequent molecular dynamics simulations suggested that the ATF4-sorafenib interaction can remain stable. In cell-based assays, silencing ATF4 curtailed proliferation, colony formation, and migration, accompanied by reduced β-catenin, CyclinD1, and c-MYC. By contrast, ATF4 overexpression led to the opposite pattern. CONCLUSION: Our results place ATF4 at the intersection of stress-related tumor programs, immune suppression, and WNT/β-catenin activation in OS, and this pattern is associated with poorer clinical outcomes. The ACS offers a practical way to stratify risk, and sorafenib warrants further evaluation as a potential ATF4-oriented therapeutic lead.
Ding et al. (Wed,) studied this question.