In vitro activity: Super-TDU (SVDDHFAKSLGDTWLQIGGSGNPKTANVPQTVPMRLRKLPDSFFKPPE) is a rationally designed inhibitor peptide that targets the YAP-TEADs interaction by mimicking the VGLL. VGLL4 is a previously identified YAP antagonist which also functions as a regulator of Wnt/β-catenin signalling. Super-TDU demonstrated therapeutic potentials for treating human cancer with YAP-activation. It reduced endogenous interaction between YAP and TEADs as shown by coIP experiment. Super-TDU inhibited cell viability and colony formation of GC cell lines MGC-803, BGC-823, and HGC27, but not MKN-45 as predicted. Moreover, the Super-TDU downregulated expression of YAP-TEADs target genes CTGF, CYR61, and CDX2. Further chromatin immunoprecipitation (ChIP) assays also showed that the Super-TDU dose-dependently reduced the amount of CTGF and CDX2 promoter cDNA ChIPed by YAP antibody. To verify the specificity of the Super-TDU, two Super-TDU mutants were created which harbor mutations that disable TEADs binding. As expected, the mutant Super-TDUs failed to interact with TEADs and inhibit cell proliferation. Thus the Super-TDU appears to have a broad but specific antitumor activity toward YAP-driven human cancers in which YAP/VGLL4 ratio should be considered as an important prognostic marker for personalized treatment.

Kinase Assay: proteins used in this study were expressed in Escherichia coli using pET28a-derived vectors and purified by affinity chromatography and size-exclusive chromatography. Diffraction data were collected at beamline BL17U, Shanghai Synchrotron Radiation Facility (SSRF) of China.

Cell Assay: Cells were transfected with the indicated plasmids of pcDNA3.1 or Flag-VGLL4 wild-type or mutant and/or HA-tagged YAP. ATP cell viability assay was used for detecting cell proliferation. ATP content was measured in accordance with the protocol of the CellTiter-Glo luminescent cell viability assay kit (Promega). Briefly, 100 μl of assay reagent was added to the wells and mixed for 2 min at room temperature. After 10 min, intracellular ATP content was measured using a luminescence multilabel counter.