The role of deubiquitylating enzymes (DUBs) in maintaining β-Catenin stability in intestinal stem cells and colorectal cancer (CRC) is not well characterized. Using an unbiased screening approach, we identified the DUB USP10 as a stabilizer of β-Catenin specifically in APC-truncated CRC models, both in vitro and in vivo. Mechanistic analyses, including in vitro binding assays and computational modelling, indicated that USP10 interacts with β-Catenin through its unstructured N-terminal region, an interaction that is competitively inhibited by full-length APC, promoting β-Catenin degradation. In contrast, in APC-truncated CRC cells, USP10 binds β-Catenin and enhances its stability, which is critical for preserving an undifferentiated tumor phenotype. Loss of USP10 decreased WNT and stem cell signature gene expression while inducing differentiation markers. Notably, silencing USP10 in both murine and patient-derived CRC organoids demonstrated its necessity for NOTUM signaling and the APC super-competitor phenotype, reducing the tumorigenicity of APC-truncated CRC. Clinically, USP10 dependence in patient-derived organoids correlates with worse CRC prognosis. These results reveal USP10 as a key regulator of CRC cell identity, stemness, and tumor growth through β-Catenin stabilization, resulting in aberrant WNT activity and degradation-resistant tumors, highlighting USP10 as a promising therapeutic target in APC-truncated CRC.
