Abstract 1666: Novel peptides target the β-catenin/TBL1 complex to competitively inhibit dysregulated Wnt signaling in cancer

Abstract

Abstract Purpose: The canonical Wnt pathway is a stem cell pathway essential during embryogenesis, and functions to direct stem cell fate, renewal, and proliferation. Mutations in this pathway induce overexpression of Wnt genes resulting in a variety of human cancers including breast, colorectal, and ovarian. Canonical Wnt signaling is β-catenin-dependent and when active, β-catenin forms a complex with transducin β-like protein 1 (TBL1) and transducin β-like related protein 1 (TBLR1) to promote gene TCF/LEF-dependent expression downstream the Wnt pathway. Previously we developed a small-molecule inhibitor that disrupts this protein complex to inhibit Wnt signaling and promote a cytotoxic effect in tumor cells. While this approach is effective, our current efforts are focused to develop a peptide-based therapy that targets the β-catenin/TBL1 complex which will result in increased target specificity and less overall toxicity compared to small-molecule counterparts. We have generated a library of peptides corresponding to the binding interface of β-catenin and TBL1 with the aim of developing a specific drug that disrupts the complex. Methods: Selected peptides were tested for their ability to affect the aberrant Wnt pathway on the Wnt-activated colon cancer cell line SW480. Displacement of β-catenin from TBL1 was assessed using a heterologous competition ELISA screen in cell lysate. Hit peptides were identified then tested for specific binding to recombinant TBL1 using an additional ELISA. Further validation to determine associated levels of β-catenin with and without peptide treatment was performed with a co-immunoprecipitation. In situ detection of β-catenin/TBL1 complex dissociation in response to peptide treatment was investigated with a proximity ligation assay (PLA). A ubiquitin enrichment kit was used to measure induced β-catenin degradation. Hit peptides were tested with a viability assay for efficacy in their ability to promote a cytotoxic effect on tumor cells. Transcriptional TCF/LEF activation, which is promoted by β-catenin, was evaluated with TOPFlash in the Wnt activated TCF/LEF reporter HEK293 cell line. Results: We identified a series of peptides that selectively bind TBL1 and displace β-catenin from the complex in Wnt-driven colon cancer cell line SW480 in a dose-dependent manner. Further, we found that treatment with 5 μM peptides in TCF/LEF reporter cells significantly blocks the activity of TCF/LEF, inhibiting downstream transcription of Wnt genes. The disruption of the β-catenin/TBL1 complex results in the ubiquitination of β-catenin. Our findings confirmed this data showing that peptide treatment depletes ubiquitinated β-catenin over time. When these peptides are used to treat Wnt-driven tumor cells, they display strong on-target cytotoxic effects indicating a potential clinical application for the use of these specific peptides as a novel therapeutic. Citation Format: Shelby Rheinschmidt, Trason Thode, Samuel Sampson, Alexis Weston, Tithi Ghosh Halder, Serina Ng, Sydney Adamson, Kate Gutowsky, Mohan Kaadige, Raffaella Soldi, Sunil Sharma. Novel peptides target the β-catenin/TBL1 complex to competitively inhibit dysregulated Wnt signaling in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1666.