Mapping the B-Catenin Interactome: CRISPR-Based functional proteomics, proximity labelling, and small molecule screening for wnt-targeted cancer therapy

Abstract

The Wnt signaling pathway is critical for embryonic development and the homeostasis of adult tissues, with β-catenin acting as a key transcriptional co-activator in canonical Wnt signaling. Dysregulated β-catenin activity, often due to mutations or stabilization, is a hallmark of Wnt-driven cancers, particularly colorectal cancer (CRC). Despite its central role, direct therapeutic targeting of β-catenin has proven challenging, with limited success in clinical applications. In this study, we sought to further characterize the β-catenin interactome and identify novel protein-protein interactions (PPIs) and small-molecule modulators. First, we generated a CRISPR/Cas9-mediated CTNNB1 knockout (KO) in U2OS cells to examine proteomic changes associated with β-catenin loss, confirming its pivotal role in cancer-associated signaling networks. Next, we employed a proximity-dependent biotinylation approach (MiniTurbo) fused to β-catenin in U2OS cells, coupled with affinity purification mass spectrometry (AP-MS) to map the β-catenin interactome. This approach identified 112 high-confidence β-catenin interactors, verified by BIOGRID and INTACT databases, and revealed 39 novel candidate interactors, suggesting previously uncharacterized roles in β-catenin signaling regulation. To explore potential therapeutic interventions, we screened small-molecule inhibitors and identified a carnosic acid derivative (MAM4-141) as a Wnt signaling modulator. Whole-cell proteomic profiling in SW480 colorectal cancer cells, combined with interactome analysis using the MiniTurbo system, demonstrated cell toxicity and revealed potential effects on βcatenin-associated pathways, providing initial insights into its mechanism of action. These findings establish a robust β-catenin interactome platform and highlight MAM4-141 as a promising candidate for further investigation in Wnt-targeted therapies.

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Identifiers

ORCID for Steve Shaju John: orcid.org/0009-0005-9463-2491

ORCID for Matthias Baud: orcid.org/0000-0003-3714-4350

ORCID for Graham Packham: orcid.org/0000-0002-9232-5691

ORCID for Paul Skipp: orcid.org/0000-0002-2995-2959

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