Investigating the role of OTULIN and the regulation of linear ubiquitination in the pathogenesis of Activated B-Cell-like Diffuse Large B-Cell Lymphoma

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Dysregulation of NF-ĸB signalling underlies many forms of cancer. Activated B-cell Diffuse Large B-Cell Lymphoma (ABC-DLBCL), a heterogeneous form of non-Hodgkin lymphoma, is characterised by chronic active NF-ĸB signalling, rendering ABC-DLBCL addictive to NF-ĸB. This dependency often originates from gain-of-function mutations in critical regulators of B-cell receptor (BCR)-mediated NF-ĸB signalling, like BCR subunits, MALT1, MyD88, CARD11 and LUBAC, the linear ubiquitin-specific E3 ligase. Chronic BCR and NF-ĸB activation trigger non-degradative linear ubiquitination of several substrates, among them LUBAC itself affecting LUBAC function in a context-dependent manner. Inhibition of LUBAC selectively kills ABC-DLBCL cells, suggesting that LUBAC and linear ubiquitin are central oncogenic nodes in ABC-DLBCL and that modulating the linear ubiquitin axis could potentially serve as therapeutic target. This is of special interest, since ABC-DLBCL is generally associated with a poor prognosis and the success of standard chemotherapy strongly depends on the mutational background. Although linear ubiquitination and LUBAC function are counteracted by two deubiquitinating enzymes, CYLD and OTULIN, CYLD is proteolytically inactivated by MALT1 in ABC-DLBCL, supporting LUBAC-mediated linear ubiquitination as oncogenic driver. In contrast, the role of OTULIN in regulating linear ubiquitination, LUBAC function, oncogenic NF-ĸB signalling and tumorigenesis in ABC-DLBCL remains unknown and elusive. Therefore, this proposal aims to unravel the role of OTULIN in controlling LUBAC E3 ligase function, cellular linear ubiquitination, chronic NF-ĸB signalling and ABC-DLBCL tumorigenesis. Using systematic multidisciplinary and state-of-the-art approaches, including CRISPR/Cas9-mediated genetic knock-out and knock-in, mass-spectrometry combined with classical biochemistry and in vivo evaluation of ABC-DLBCL tumorigenesis in preclinical mouse models, this proposal will reveal the precise roles of OTULIN in oncogenic linear ubiquitination and NF-ĸB in ABC-DLBCL tumour cell fate control.The outcomes of this proposal are expected to provide crucial novel insights in how OTULIN controls linear ubiquitin and LUBAC in ABC-DLBCL tumorigenesis and will contribute to novel therapeutic approaches to selectively target ABC-DLBCL. Understanding OTULIN function will also lead to a better understanding of other tumour entities, like certain subtypes of acute myeloid leukaemia, chronic myeloid leukaemia and multiple myeloma, that rely on linear ubiquitination and LUBAC. Finally, the proposed research project will also have fundamental implications for OTULIN function in innate immunity and inflammation.