Abstract
Hippo signaling is one of the top pathways altered in human cancer, and intensive focus has been devoted to developing therapies targeting Hippo-dependent transcription mediated by YAP1 and TAZ interaction with TEAD proteins. However, a significant challenge in evaluating the efficacy of these approaches is the lack of models that can precisely characterize the consequences of TEAD inhibition. To address this gap, our laboratory developed a strategy that utilizes a fluorescently traceable, dominant-negative protein named TEADi. TEADi specifically blocks the nuclear interactions of TEAD with YAP1 and TAZ, enabling precise dissection of Hippo TEAD-dependent and independent effects on cell fate. In this study, we aimed to enhance TEADi effectiveness by altering post-transcriptional modification sites within its TEAD-binding domains (TBDs). We demonstrate that a D93E mutation in the YAP1 TBD significantly increases TEADi inhibitory capacity. Additionally, we find that TBDs derived from VGLL4 and YAP1 are insufficient to block TAZ-induced TEAD activity, revealing crucial differences in YAP1 and TAZ displacement mechanisms by dominant-negative TBDs. Structural differences in YAP1 and TAZ TBDs were also identified, which may contribute to the distinct binding of these proteins to TEAD. Our findings expand our understanding of TEAD regulation and highlight the potential of an optimized TEADi as a more potent, specific, and versatile tool for studying TEAD-transcriptional activity.