A ubiquitin specific protease (USP) network regulates wnt/β-catenin activity in myeloid leukemia cells

Abstract

Abstract Canonical Wnt signalling is an important regulator of haematopoietic stem/progenitor cells (HSPC) and frequently dysregulated in myeloid neoplasms. Overexpression, overactivity or mislocalisation of the central mediator β-catenin has been reported in multiple acute myeloid leukaemia (AML) subtypes where it's associated with poor prognosis and the emergence, maintenance, and drug resistance of leukaemia stem cells (LSC). Despite being an attractive therapeutic target, efforts to pharmacologically target β-catenin have been hampered by a poor understanding of its molecular interactions in leukaemia cells. Following a detailed exploration of β-catenin's interaction network in myeloid leukemia cells, we revealed the significant enrichment of several de-ubiquitinating enzymes (DUB) called ubiquitin-specific proteases (USP). USPs stabilise target proteins through removal of ubiquitin chains, thus resisting ubiquitin-proteasome mediated degradation. USP dysregulation has been frequently observed in human cancer, but how it impacts Wnt signalling in a leukaemia context has not been sufficiently examined, despite abundant evidence from solid tissues showing several critical Wnt signalling components are subject to ubiquitin/USP regulation. In this study we confirm that several USPs including USP5, 39 and 48 co-immunoprecipitate with β-catenin from both myeloid cell lines and primary AML patient samples. We found these USPs Co-IP together in myeloid cells indicating they may regulate target substrates collectively. Consistent with this lentiviral mediated suppression of individual USPs via shRNA, did not impact β-catenin stability or nuclear translocation but pan inhibitors of DUB activity (DUBi) including WP1130, P22077, and PR619 all significantly reduced Wnt signalling output (TCF/LEF activity) suggesting alternative mechanisms of Wnt signalling regulation. Specifically, further investigation into USP48 found it to be a predominantly nuclear localised USP and although shRNA mediated depletion did not impact overall β-catenin level, we observed accelerated nuclear β-catenin exit dynamics upon withdrawal of the Wnt agonist CHIR99021 in myeloid cell lines, suggesting a role for USPs in the nuclear retention/stability of β-catenin. These findings highlight a novel and complex role of USPs in regulating Wnt signalling activity in leukaemia cells and may provide new therapeutic strategies for limiting aberrant nuclear β-catenin via USP48 targeting in myeloid leukaemia.