Background: Dysregulated Wnt/?-catenin signaling has been implicated in the pathogenesis of many common human cancers, making this an attractive clinical target. However, this has proven challenging because i) ?- catenin plays critical roles in normal tissue homeostasis, ii) its signaling pathways form part of a complex network of intersecting pathways, and iii) ?-catenin's mode of interaction with active sites on its partner proteins makes it difficult to identify molecular probes that specifically and selectively disrupt its oncogenic activity. Preliminary data: i) Development of peptidomimetics of the BCL9-HD2 domain that selectively suppress oncogenic Wnt activity by blocking interaction of ?-catenin with its transcriptional co-activator BCL9; ii) A high-throughput screening (HTS) AlphaScreen assay allowing identification of several small-molecule inhibitors of ?-catenin/BCL9 interaction has been implemented; iii) An in vivo model using BCL9 transgenic mice that develop lymphomas as well as lung and gastric adenocarcinomas has been put into place. These studies offer compelling proof-of-concept for i) a therapeutic strategy addressing a known oncogenic role of the Wnt/?-catenin/BCL9 transcriptional complex, and ii) a pharmacologic intervention via targeted disruption of this complex. Working hypothesis: i) The Wnt/?-catenin/BCL9 transcriptional complex performs critical roles in cancer pathogenesis, and targeted disruption of this complex represents a promising pharmacologic strategy for blocking oncogenic Wnt activity in cancer; ii) Potent, specific, competitive inhibition of the ?-catenin/BCL9 interaction can be accomplished by using small organic molecules that bind tightly to the BCL9-HD2 binding domain on ?-catenin. Goals: i) to characterize HTS ?hits? biophysically via rapid calorimetry-based assay; ii) to test inhibitors for functional activity in Wnt-relevant cell-based assays to ensure that the inhibitors have the desired molecular specificity. Experimental tools: i) Homogeneous biophysical assays (i.e. enthalpy change measurement and Isothermal titration calorimetry) that will robustly monitor disruption of ?-catenin/BCL9 binding in vitro, ii) X-ray crystallography to unequivocally establish the structural basis of this interaction and will lay groundwork for possible future structure-guided synthetic chemistry; iii) Cellular assays that will enable us to monitor the consequences of disrupting the ?-catenin/BCL9 complex in intact cells; and iv) Established xenograft and transgenic mouse models of cancers with a dysregulated Wnt/?-catenin/BCL9 complex will allow evaluation of the possible clinical usefulness of small-molecule inhibitors of BCL9/?-catenin interaction. Expected results: Structurally validated small-molecule chemical probes that i) target ?-catenin, ii) dissociate native ?-catenin/BCL9 complexes, iii) selectively suppress Wnt transcriptional activity, and iv) possess mechanism-based antitumor activity in vitro, and in vivo with minimal toxicity, will be identified. Implications to Medicine: Our work in this project is expected to afford highly selective clinical probes of oncogenic Wnt activity and innovative targeted therapies against Wnt/?-catenin/BCL9 dependent human cancers.