ABSTRACT Dysregulation of Wnt/-catenin signaling is found in more than 20% of all cancers, including 50% of breast can- cers, and contributes to cancer initiation, angiogenesis, tumor migration and metastasis. However, attempts at developing a drug to inhibit -catenin have been thwarted by the inability of small molecule inhibitors to separate -catenin’s oncogenic from homeostatic functions, producing a toxic safety profile. BCL9 is a transcriptional co-activator that regulates oncogenic β-catenin-mediated gene transactivation. Disrup- tion of BCL9 interaction with β-catenin results in potent inhibition of -catenin-mediated oncogenic gene trans- activation without the GI toxicity associated with loss of signaling via APC or Axin. As BCL9 is highly expressed in tumors, but generally not normal cells, the β-catenin interaction with BCL9 represents an attractive, novel therapeutic target. Based on the above, we hypothesize that a peptide antagonist of β-catenin interaction with BCL9 will prove a safe and effective strategy for Wnt-driven cancers. Sapience has designed and synthesized a panel of β-catenin antagonist peptides (BCAPs) to disrupt the inter- action of β-catenin with BCL9, in which we introduced amino acid substitutions to the native BCL9 HD2 domain to enhance target affinity, included a non-toxic cell penetrating domain (CPD) for intracellular entry and D-amino acids to protect from proteolysis and confer chemical stability. Through structure-activity relationship (SAR)- based design, we developed a lead candidate, BCAP-58, with improved potency in vitro and in vivo. Through this SBIR program, we propose to develop an innovative inhibitor of -catenin/BCL9 interaction as a therapeutic for Wnt/-catenin-driven cancers, starting with breast cancer. We propose to characterize the in vitro (Specific Aim #1) and in vivo (Specific Aim #2) activity of BCAP-58. Initial experiments will evaluate the binding affinity of BCAP-58 with -catenin (Aim 1.1). Subsequently, the impact of BCAP-58 on Wnt/β-catenin activity will be determined by TOPFlash reporter, β-catenin phosphorylation and β-catenin subcellular localization (Aim 1.2). Finally, we will test the impact and target specificity of BCAP-58 on β-catenin-target gene expression by qPCR (Aim 1.3), and on cell proliferation and viability (Aim 1.4), against a panel of Wnt-dependent or -independent cell lines of various breast cancer subtypes and patient-derived tumoroids. To establish proof of concept for BCAP- 58 as a therapeutic for Wnt-dependent breast cancer, we will assess its repeat-dose toxicity and in vivo efficacy. First, we will establish a no adverse effect level (NOAEL) and PK profile in mice, to define the upper dosing limit (Aim 2.1). Next, we will investigate the dose-dependent activity of BCAP-58 in mouse models of the subtypes that demonstrate susceptibility to BCAP-58 in Aim 1 (Aim 2.2). Finally, we will characterize the impact of BCAP- 58 on β-catenin-mediated gene transactiv...