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

项目总结 霍奇金淋巴瘤(HL)是年轻人中最常见的(每年新增6,000至7,000例)淋巴瘤亚型 成人期。尽管在过去的几十年里取得了很大的进步，但患者的存活率 晚期或复发/难治性疾病的诊断率仍然较低。对当前的理解 这种疾病的生物学已经转化为治疗药物的开发和批准 靶向HL特异性抗原或免疫检查点途径。然而，完全治愈的患者数量 缓解率低，复发频繁，导致预后不佳。因此，一个更清晰的 了解HL的分子病理学对于开发新的治疗策略是必要的。HL是 以少数恶性霍奇金和Reed/Sternberg(HRS)细胞为特征 致密的炎性细胞。然而，HRS细胞已经失去了他们的B细胞表型，并逃脱了BCR- 介导细胞凋亡和免疫消除。因此，仍然存在两个主要问题：(A)HRS细胞如何 逃脱免疫系统的控制；(B)在没有bcr表达的情况下，它们如何存活？至 为了解决这些知识差距，我们应用了无偏见的高通量CRISPR筛查，并确定了 线性泛素链组装复合体(LUBAC)在HL发病中的重要作用。我们的预赛 研究表明，LUBAC活性促进HRS细胞存活和免疫逃逸，这显著地 与HL，A20中最常见的基因突变状态重叠。临床上，LUBAC活动 在大多数原发HL病例中持续升高，这与A20的低表达有关。此外， 利用rna-seq分析，我们鉴定了一组在HL中受LUBAC调控的基因，这些基因与 反映核因子κB和JAK-STAT活性以及TH2型细胞因子和细胞表面的信号 免疫抑制分子。出乎意料的是，我们的BioID蛋白质组筛选显示了CD30-LUBAC复合体 在HL中，提示LUBAC-A20轴在CD30介导的NF-κB信号、TH2型细胞因子产生、 和STAT6激活。最后，一种高度特异的LUBAC小分子抑制剂显示出良好的抗肿瘤活性。 HL在体外和小鼠异种移植模型中。总之，这些发现为我们的假设提供了强有力的支持 LUBAC-A20轴调节HL的发病机制，靶向LUBAC可能是一种新的治疗方法 治疗这种疾病的策略。在本研究中，我们将：1)研究LUBAC-A20 Axis支持HRS的存活和增殖；2)评估线性泛素依赖的信号如何调节 驱动HL肿瘤免疫逃逸的分子回路；3)开发靶向治疗的潜力 LUBAC为HL的靶向和免疫治疗提供新的干预策略。这些研究 承诺揭示驱动这种淋巴癌的分子电路的关键见解，以及提供 开发靶向和免疫疗法治疗HL的新策略的独特机会。

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.