Abstract TEAD transcription factors bind to the transcription co-activators YAP/TAZ and control the transcriptional output of the Hippo pathway. Deregulation of Hippo-YAP signaling is implicated in diverse human cancers; however, it remains difficult to directly target TEAD-YAP complex with small molecules. We previously reported that TEADs possess intrinsic “enzyme-like” activities and undergo autopalmitoylation. Palmitate binds into a deep hydrophobic pocket and is essential for TEAD protein stability and transcriptional activation. Here we report MGH-CP1 as a small-molecule inhibitor of TEAD autopalmitoylation. We determined the crystal structure of inhibitor-bound TEAD2 and showed that MGH-CP1 binds to the conserved lipid-binding pocket in TEAD2 and displaces palmitate binding. In cells, MGH-CP1 maintains TEAD stability and TEAD-Vgll4 repressor complex and allosterically inhibits TEAD-YAP association and YAP-induced transcriptional activation. Through a large-scale profiling of cancer cell line growth inhibition, followed by intersecting with the Cancer Dependence Map analysis, we demonstrate that the sensitivity to MGH-CP1 treatment correlates significantly with YAP-dependency in a broad range of human cancer cells. Furthermore, we show that MGH-CP1 blocks YAP-dependent liver overgrowth induced by liver-specific Lats1/2 deletion in mice and inhibits xenograft tumor growth of human uveal melanoma cells in vivo. Together, our work suggests pharmacologic inhibition of TEAD palmitoylation as a promising therapeutic strategy for diseases associated with deregulated Hippo signaling, and opens a door to target “autopalmitoylation” of proteins. Citation Format: Xu Wu. Targeting autopalmitoylation of TEAD transcription factors [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr IA12.