Project Summary Wnt signaling regulates cell replication and proliferation of most adult stem cells, and its abnormal activation has been linked to many cancers. Both small-molecular inhibitors that inhibit Wnt production and blocking antibodies against the Wnt receptor Frizzled (FZD) have been developed, and some are currently in clinical trials. Humans express 19 Wnt and 10 Wnt receptor Frizzed proteins (FZDs), and each tissue type may have its own profile of Wnt-FZD pairs. As Wnt is critical for maintaining almost all adult stem cells, a major challenge is to minimize the side effects to healthy tissues. One approach to protecting these tissues is to develop blocking agents that specifically target the particular set of Wnt-FZD pair(s) expressed in the cancer tissues being targeted. However, this has proven difficult, as both Wnt and FZD family members are highly conserved. In particular, a specific and effective blocking agent targeting FZD7, which is upregulated in triple-negative breast cancer, has yet to be established. Here we propose to develop a novel class of therapeutic proteins that are highly effective in blocking Wnt signaling mediated by FZD1/2/7 (these three share almost-identical Wnt-binding domains). This is based on our previous finding that a bacterial toxin, C. difficile toxin B (TcdB), binds to FZD1/2/7 as its high-affinity receptor, and its binding to FZD1/2/7 potently inhibits Wnt signaling. Our preliminary studies showed that the FZD-binding domain of TcdB (TcdBFBD) inhibits growth in a mouse triple-negative breast cancer organoid in vitro and xenograft model in vivo, without damaging Wnt-sensitive tissues. We propose to use protein engineering to improve the biochemical and pharmacological properties of TcdBFBD, and to further evaluate the therapeutic potential of TcdBFBD using xenograft models and human breast cancer organoids. These studies will further establish Wnt signaling as an important therapeutic target, and develop a new class of therapeutic proteins for labeling and treating Wnt-dependent tumors.