A large-scale screening strategy using induced protein proximity to identify regulators of Wnt signalling in colorectal cancer

• Applicant: Dr. Lucie Wolf
• Subject Area: General Genetics and Functional Genome Biology ; Cell Biology
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 533617596

Colorectal cancer is a major contributor to global disease burden and nearly all cases present with activating mutations in the beta-catenin dependent Wnt signaling pathway. Nevertheless, no treatment for diseases with increased Wnt target gene transcription is currently in clinical practice, emphasizing the need for novel therapeutic strategies. Recent advancements in targeted protein degradation (TPD) provided such new approaches with the development of small molecule degraders that induce physical proximity between a Protein Of Interest (POI) and a component of the proteasomal degradation machinery (e.g. an E3 Ubiquitin Ligase), resulting in POI degradation. However, the scope of application of these degraders is limited. This is because only a small number of E3s are being used for protein regulation and a systematic approach to find further candidates, possibly even including alternative protein classes and working mechanisms, is missing. Here, I propose a systematic approach to find candidates promoting beta-catenin degradation, and thus Wnt signalling regulation, in colorectal cancer cells. To this end, I conducted two screens, one for beta-catenin function and one for beta-catenin protein abundance. This near proteome-wide screening strategy relies on targeted recruitment of endogenous beta-catenin into proximity of over 15 000 open reading frames and allowed me to query E3 ubiquitin ligases as well as alternative protein classes and working mechanisms. Success of this induced-proximity screening strategy was confirmed by many E3 ubiquitin ligases among the screen hits, including the known beta-catenin regulator SIAH2. Surprisingly, the screen uncovered that inducing proximity to members of the Casein kinase I (CSNK1) family strongly regulated beta-catenin. While CSNK1alpha is a member of the beta-catenin-degrading destruction complex in healthy cells, the potential of CSNK1 as TPD effector has not been explored. So far, I could show that beta-catenin degradation by CSNK1 is proximity-, kinase activity-, and proteasome-dependent and requires activity of the E3 ubiquitin ligase beta-TrCP2. Thus, I propose the formation of a phospho-degron on beta-catenin for this kinase-dependent degradation mechanism. In my upcoming experiments, I aim to expand on these results by further characterizing the molecular mechanism of action, focusing on the involved E3 ubiquitin ligases and ubiquitination status of beta-catenin. As proof of principle experiments for kinase-dependent degradation of target proteins, I plan to generate a set of cell lines which allow the inducible regulation of beta-catenin abundance and function via small molecules and to transfer the phospho-degron approach to alternative protein targets. This project contributes to a more comprehensive understanding of how the induced proximity concept can be leveraged in the context of cancer and how to expand the toolbox of targeted protein degradation by kinase-dependent effectors.

• DFG Programme: WBP Fellowship
• International Connection: Canada
• Host: Professor Dr. Stéphane Angers