Analysis and Therapeutic Targeting of the Linear-Ubiquitination Pathway in Hodgkin Lymphoma

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PROJECT SUMMARY Hodgkin lymphoma (HL) is the most common (6,000 to 7,000 new cases per year) lymphoma subtype in young adulthood. Although there has been great progress during the last few decades, the survival rate for patients diagnosed at an advanced stage or with relapsed/refractory disease remains low. The current understanding of the biology of the disease has been translated into the development and approval of therapeutic agents that target HL-specific antigens or immune checkpoint pathways. However, the number of patients in complete remission has been low, and relapse frequently develops, leading to poor outcomes. Thus, a clearer understanding of the molecular pathology of HL is necessary to develop new treatment strategies. HL is characterized by a minority population of malignant Hodgkin and Reed/Sternberg (HRS) cells in a background of dense inflammatory cells. HRS cells have lost their B cell phenotype, however, and escaped from BCR- mediated apoptosis and immune elimination. Therefore, two major questions remain: (a), how do HRS cells escape the control of the immune system; (b), how do they survive despite the absence of BCR expression? To address these gaps in knowledge, we applied an unbiased high throughput CRISPR screening, and identified an essential role for the linear ubiquitin chain assembly complex (LUBAC) in HL pathogenesis. Our preliminary studies demonstrated that LUBAC activity promotes HRS cell survival and immune escape, which significantly overlaps with mutation status of the most recurrent genetically altered gene in HL, A20. Clinically, LUBAC activity is consistently elevated in most primary HL cases, and this is correlated with low A20 expression. Moreover, using RNA-seq analysis, we identified a set of LUBAC-regulated genes in HL that overlapped significantly with signatures reflecting NF-κB and JAK-STAT activities, as well as TH2 cytokines and cell surface immunosuppressive molecules. Unexpectedly, our BioID proteomic screening reveals a CD30-LUBAC complex in HL, suggesting the role of LUBAC-A20 axis in the CD30 mediated NF-κB signaling, TH2 cytokine production, and STAT6 activation. Finally, a highly specific LUBAC small molecular inhibitor shows promising activity against HL in vitro and in a mouse xenograft model. Altogether, these findings provide strong support for our hypothesis that the LUBAC-A20 axis regulates HL pathogenesis, and that targeting LUBAC could be a novel therapeutic strategy in this disease. In this study, we will: 1) investigate the mechanistic basis by which the LUBAC-A20 axis supports HRS survival and proliferation; 2) evaluate how linear-ubiquitin-dependent signaling regulates the molecular circuitry that drives tumor immune escape of HL; and 3) exploit the therapeutic potential of targeting LUBAC to provide novel intervention strategies for both targeted and immune therapies in HL. These studies promise to reveal critical insights into the molecular circuitry that drives this lymphoid cancer, as well as provide unique opportunities for the development of novel strategies for both targeted and immune therapies to treat HL.