Defining deregulated ubiquitylation events in B-NHL

• Applicant: Professor Dr. Florian Bassermann
• Subject Area: Hematology, Oncology
• Term: from 2014 to 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 258522452

B-Cell Non Hodgkins Lymphomas (B-NHL) are characterized by high and cumulative levels of genomic instability. Recently, the central role of the ubiquitin proteasome system (UPS) in the cellular DNA damage response machinery has been appreciated, thus suggesting roles in both B-NHL development and as a target structures. Indeed, proteasomal inhibition has been successfully introduced into the therapy of B-NHL, yet considerable variations exist between different entities, indicating the presence of disease-specific deregulated ubiquitylation events. Their identity in B-NHL has however remained largely unknown. Starting from systematic analyses of genomewide aCGH and expression studies, we identified the previously orphan SCF-type E3 ubiquitin Fbxo25 and the USP-type deubiquitylase (DUB) USP9X as promising candidates predicted to be deleted and overexpressed in B-NHL, respectively. Using unbiased mass-spectrometric screens, we subsequently found that Fbxo25 targets the pro-survival protein Hax-1 for proteasomal degradation in response to apoptotic stimuli. Further studies in B-NHL cell lines and a murine B-NHL in vivo model suggest that deletions of Fbxo25 contribute to lymphomagenesis through Hax-1 stabilization. Indeed, we find monoallelic deletion and low expression of Fbxo25 in different B-NHL patient samples with a particular enrichment in mantle cell lymphoma (MCL) samples. These findings distinguish Fbxo25 as a potential novel haploinsufficient tumorsuppressor in B-NHL. With regard to USP9X, we identified the mitotic phosphatase Cdc14B as an upstream regulator of USP9X, and XIAP (X-linked inhibitor of apoptosis) as a novel ubiquitylation substrate. Our data suggest that the Cdc14B-USP9X-XIAP axis contributes to the regulation of mitotic cell death. Initial IHC studies in patient samples revealed an enrichment of USP9X overexpression particularly in DLBCL. Based on these results, we propose to further functionally investigate how deregulation of Fbxo25 and USP9X contributes to B-NHL development and progression. These approaches will include studies in amenable cell culture models to functionally characterize the relevant ligase/DUB-substrate pairs on the biochemical and cell biological level. Subsequently, these mechanistic findings will be further investigated in different human B-NHL tissue culture models, B-NHL mouse models and B-NHL patient cohorts with available clinical follow up data. Moreover, as a complimentary approach, we propose to generate a conditional Fbxo25 knock-out mouse to further investigate the role of Fbxo25 as a novel tumorsuppressor. In summary, our interdisciplinary approach comprising functional proteomics, cell biology, mouse models, and analyses of defined patient cohorts is anticipated to gain further mechanistic insight into the pathophysiology of B-NHL, and identify relevant drugable target structures of the ubiquitin proteasome system for the specific treatment of these diseases.

• DFG Programme: Research Grants