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 multi-omics analyses of RNA sequencing (RNA-seq) and ChIP sequencing (ChIP-seq) for YAP and H3K27Ac in primary EACSCC and noncancerous¬ ear skin samples. RNA-seq indicated hyperactivation of YAP/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 PITX TFs in EACSCC, and presence of EACSCC-specific super enhancers (SEs). Importantly, YAP-bound SEs were involved in oncogenic transcription including EGFR signaling. For further validations, functional experiments were performed in vitro and in vivo. Small molecule TEAD inhibitor (smTEADi) VT104 significantly suppressed proliferation and clonogenicity of EACSCC-derived cells. Interestingly, smTEADi not only inhibited YAP-TEAD interaction but also induced YAP-PITX2 binding, suggesting that PITX2 could represent an alternative partner TF of YAP under TEAD-inhibited condition 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 co-expressed in primary EACSCC tissues, and significantly correlated with poor prognosis of EACSCC patients. Together, this study highlighted hyperactivated YAP-driven transcriptional program and its potential as a therapeutic target in EACSCC.