The Wnt/wingless (wg) pathway is one of a core set of evolutionarily conserved signaling pathways that regulate many aspects of metazoan development. Inappropriate activation of the Wnt pathway has been associated with tumorigenesis of the liver, colon, breast and skin. One of the most important effectors of the Wnt pathway is encoded by the transcription factor, beta-catenin (-cat)/armadillo (arm). Since -catenin Responsive Transcription (CRT) has been implicated in the genesis of many cancers, it makes an ideal target for developing therapeutics that could be utilized to modulate the nuclear activity of -cat. We employed a novel cell-based chemical genetic high-throughput screening (HTS) methodology, where we screened for small molecule “modifiers” of RNAi-induced loss-of-function phenotypes of genes, such as Axin and APC, that are commonly mutated in cancers associated with enhanced Wnt/-cat activity. We identified 3 potent inhibitors of CRT, called iCRT3, iCRT5 and iCRT14. We demonstrate that iCRTs specifically and potently inhibit Wnt/-cat-induced phenotypes in a variety of Wnt-responsive cell lines. Mechanistic studies suggest that the iCRTs directly bind to -cat and disrupt its interaction with its transcriptional partner TCF4. Importantly, the iCRTs inhibit cell growth/proliferation of pathologically relevant and oncogene/-cat-addicted colon cancer cells (such HCT116, HT29, SW480), both in vitro and in xenograft models in vivo. Notably, iCRT3 displayed potent cytotoxic effect on patient-derived primary tumor microspheroids from a variety of cancers at concentrations that are comparable to known FDA-approved chemotherapeutics that are currently in use for treatment. These studies have now enabled us to investigate the molecular mechanisms by which dysregulated Wnt signaling may influence tumor progression and relapse in a variety of cancers, including castrate-resistant prostate cancer, where the role for oncogenic -cat activity remains underexplored. Finally, we are also in the process of identifying FN3-monobodies (FNDYs) against oncogenic -catenin that can be developed as novel therapeutics or biosensors to modulate and monitor activity of cell signaling pathways in cancer cells in vivo.