ETS1 Orchestrates a Hybrid EMT Program Driving Metastasis and Immune Evasion.

Transcriptional intratumoral heterogeneity (ITH) is a hallmark of aggressive cancers. Investigation into the ITH programs that drive tumor metastasis and immune evasion could help identify potential treatment and prevention approaches. Through single-cell RNA sequencing analysis of upper aerodigestive squamous cell carcinoma (UASCC) cells and patient tumors, we uncovered a hybrid epithelial-mesenchymal transition (hEMT) ITH program linked to metastatic dissemination. The transcription factor ETS1 was identified as a master regulator of the hEMT program, directly activating prometastatic genes and promoting distant spread in vivo. Unexpectedly, ETS1 also orchestrated an immune-cold tumor microenvironment by transcriptionally activating STAT1 and CD274 (PD-L1), suppressing T lymphocyte infiltration, and elevating immune checkpoint molecules. Clinically, high ETS1 expression in tumors strongly correlated with poor survival and resistance to immune checkpoint blockade across multiple cohorts. Drug screening demonstrated that ETS1-high cancers were vulnerable to HSP90 inhibitors (e.g., alvespimycin), which suppress ETS1 by disrupting hypoxia-inducible factor 1 alpha-mediated transcriptional activation. Together, this work reveals ETS1 as a dual driver of tumor distal metastasis and immune evasion in UASCC, while nominating HSP90 inhibition as a tailored treatment strategy for ETS1-driven tumors. These findings provide a roadmap for targeting aggressive ITH subsets and overcoming immunotherapy resistance. SIGNIFICANCE: Transcriptional intratumoral heterogeneity drives metastasis and immune evasion via an ETS1-regulated hybrid EMT program, which can be targeted with HSP90 inhibitors to treat aggressive, immunotherapy-resistant tumors.