Aberrant Hippo-YAP/TEAD Signaling Drives Malignant Transcriptional Reprogramming in External Auditory Canal Squamous Cell Carcinoma.

External auditory canal squamous cell carcinoma (EACSCC) is an exceptionally rare malignancy related to chronic tissue damage and inflammation. The molecular underpinnings of EACSCC are poorly understood, and evidence-based therapeutic strategies are not fully developed. In this study, we performed integrated multiomics analyses of RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) for Yes-associated protein (YAP) and histone 3 lysine 27 acetylation (H3K27Ac) in primary EACSCC and noncancerous ear skin samples. RNA-seq indicated hyperactivation of the YAP/TEA domain family members (TEAD)-mediated transcriptional program in EACSCC, which was significantly correlated with poor clinical outcomes. ChIP-seq suggested gained accessibility for transcription factor (TF) binding sites for TEAD, AP-1, and paired-like homeodomain (PITX) TFs in EACSCC and the presence of EACSCC-specific super-enhancers (SE). Importantly, YAP-bound SEs were involved in oncogenic transcription, including EGFR signaling. For further validations, functional experiments were performed in vitro and in vivo. The small-molecule TEAD inhibitor (smTEADi) VT104 significantly suppressed the proliferation and clonogenicity of EACSCC-derived cells. Interestingly, smTEADi not only inhibited the YAP-TEAD interaction but also induced YAP-PITX2 binding, suggesting that PITX2 could represent an alternative partner TF of YAP under TEAD-inhibited conditions in EACSCC. Knockdown of PITX2 enhanced sensitivity to VT104, inhibiting cell growth and migration of EACSCC and head and neck squamous cell carcinoma cells, whereas overexpression of PITX2 induced oncogenic gene expression programs, as well as YAP/TEAD target genes, promoting tumor growth in vivo. Of note, nuclear YAP and PITX2 were coexpressed in primary EACSCC tissues and significantly correlated with the poor prognosis of patients with EACSCC. Together, this study highlighted the hyperactivated YAP-driven transcriptional program and its potential as a therapeutic target in EACSCC. SIGNIFICANCE: This study provides evidence for the hyperactivation of YAP/TEAD-driven transcriptional programs in EACSCC, an exceptionally rare malignancy related to chronic tissue damage and inflammation. A comprehensive multiomics approach, including YAP and H3K27Ac ChIP-seq in clinical samples, not only suggested hyperactivation of YAP/TEAD but also identified YAP-PITX2 as a potential oncogenic transcriptional machinery under TEAD-inhibited conditions. Our results may provide a better understanding of EACSCC and contribute to the future development of therapeutic strategies.