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Background: KDM6A, encoding a histone demethylase, is one of the top ten mutated epigenetic cancer genes. The effect of mutations on its structure and function are however poorly characterized. Methods: Database search identified nonsense and missense mutations in the N-terminal TPR motifs and the C-terminal, catalytic JmjC domain, but also in the intrinsically disordered region connecting both these two well-structured domains. KDM6A variants with cancer-derived mutations were generated using site directed mutagenesis and fused to eGFP serving as an all-in-one affinity and fluorescence tag to study demethylase activity by an ELISA-based assay in vitro, apoptosis by FACS, complex assembly by Co-immunoprecipitation and localization by microscopy in urothelial cells and apoptosis by FACS. Results: Independent of the mutation and demethylase activity, all KDM6A variants were detectable in the nucleus. Truncated KDM6A variants displayed changes in complex assemblies affecting (1) known interactions with the COMPASS complex component RBBP5 and (2) KDM6A-DNA associated assemblies with the nuclear protein Nucleophosmin. Some KDM6A variants induced a severe cellular phenotype characterized by multiple acute effects on nuclear integrity, namely, release of nuclear DNA into the cytoplasm, increased level of DNA damage indicators RAD51 and p-γH2A.X, and mitosis defects. These damaging effects were correlated with increased cell death. Conclusion: These observations reveal novel effects of pathogenic variants pointing at new specific functions of KDM6A variants. The underlying mechanisms and affected pathways have to be investigated in future research to understand how tumor cells cope with and benefit from KDM6A truncations. [ABSTRACT FROM AUTHOR]