FIG 4.
Tnks inhibitors induce telomeric stress in human cells independently of Wnt/β-catenin pathway inhibition. (A) Porcn inhibitors afford a Tnks-independent approach to disrupting Wnt/β-catenin signaling. Chemicals targeting the Wnt acyltransferase Porcn abrogate Wnt palmitoylation, which in turn results in loss of activity of the Frizzled (Fzd) family of Wnt receptors. Fzd receptors directly induce activation of the cytoplasmic signaling molecule Dishevelled (Dvl) in a ligand-dependent manner, which in turn results in activation of β-catenin and transcription mediated by the TCF family of DNA binding proteins. IWP-2 and the clinical candidate LGK-974 represent two structurally distinct Porcn inhibitors. (B) Tnks, but not Porcn, inhibitors induce DNA damage at telomeres. The appearance of colocalized Terf2 and pH2A.X (an indicator of DNA damage at the telomere ends, TIF) was scored using indirect immunofluorescence in HeLa cells (expressing wild-type APC protein). (C) Quantification of results represented in panel B. Cells with >5 TIF were scored as positive for telomere damage. The mean total phosphorylated H2A.X (gamma H2A.X) was used to measure general DNA damage responses. (D) Tnks and Porcn inhibitors disrupt β-catenin-dependent transcription in HeLa cells. qPCR analysis of Axin2, a target gene of Wnt/β-catenin signaling, reveals that both classes exert influence on β-catenin activity. The error bars indicate standard deviations. (E) IWP-2 and LGK-974, but not Tnks, inhibitors block ligand-dependent phosphorylation of Dvl2, a cytoplasmic signaling molecule regulated by the Frizzled family of Wnt receptors. (F) IWR compounds do not inhibit telomerase. Digital droplet-based telomerase extension analysis of genomic DNA isolated from HeLa cells treated for 24 h with the indicated compounds.