Exposing synthetic lethal vulnerabilities in EBV-positive AIDS-NHL through novel replication dependency factors

项目摘要 病毒相关淋巴瘤在HIV感染者中引起显著的发病率和死亡率--事实上， 口腔病原体EB病毒（EBV）导致高达90%的弥漫性大B细胞淋巴瘤（DLBCL）， 40%的伯基特淋巴瘤（BL）。虽然联合抗逆转录病毒治疗（cART）和化疗， 尽管艾滋病淋巴瘤的预后得到改善，但挑战仍然存在，特别是与病毒相关的艾滋病淋巴瘤， 促使人们努力更好地了解病毒相关因素和途径。特别是，EBV驱动的细胞 对淋巴瘤增殖至关重要的基因组复制仍然未被探索。 在感染B细胞后，EBV驱动宿主DNA复制，这对于建立病毒潜伏期是必需的 以及癌细胞的增殖。然而，这种病毒癌蛋白驱动的DNA复制受到以下因素的困扰： 物理和功能障碍，导致复制压力。这种复制压力是癌症的障碍。 然而，EBV癌细胞如何克服复制叉的这种压力以成功增殖还不清楚。 明白为了解决这一知识缺口，我们将新生DNA上的蛋白质分离（iPOND）和 质谱分析来发现新的叉蛋白。这揭示了ZC 3 H18（或ZC 3）作为一种生物活性剂的关键作用。 EBV上调以确保宿主基因组复制和淋巴瘤细胞复制依赖因子 增殖;值得注意的是，ZC 3以前没有与DNA复制相关。事实上，来自艾滋病的EBV+ DLBCL 与EBV-淋巴瘤相比，患者具有升高的ZC 3表达。一种内在无序的蛋白质，ZC 3 具有在复制叉处集中多种蛋白质的潜力。我们发现， ZC 3和MCM 7（复制解旋酶复合物的核心组分），进一步指出ZC 3的影响作用 EBV转化细胞的增殖。重要的是，ZC 3与其他复制依赖因子的伙伴关系 暴露EBV-淋巴瘤细胞的合成致命性-这种疗法利用的性质，癌细胞容忍 单个基因的干扰，但屈服于多个遗传事件的共同破坏。 在本申请中，我们将测试EBV调节DNA复制机制的假设， 转化细胞在面对复制应激时的增殖，并增强对 合成杀伤力我们将使用体外模型执行以下目标，并将我们的结果转化为患者- 来源于AIDS和癌症标本资源（ACSR）的EBV+ AIDS淋巴瘤。 目标1。研究新的依赖性因素如何揭示EBV中的合成致命漏洞- 转化细胞和Aim 2.研究ZC 3上调，复制机器重新布线， 以及复制依赖因子对EBV+ AIDS淋巴瘤的作用。 这些研究将确定机制并产生新的范式，揭示机会性病毒如何 调节宿主复制机制以维持转化状态。我们的长期目标是确定 新的药物靶点，显示出对HIV/AIDS患者的EBV+淋巴瘤的合成致死性。

PROJECT SUMMARY Virus-associated lymphomas cause significant morbidity and mortality in HIV-infected individuals – indeed, the oral pathogen Epstein-Barr virus (EBV) contributes to up to 90% of diffuse large B-cell lymphomas (DLBCL) and 40% of Burkitt lymphomas (BL). Although combined antiretroviral therapy (cART) and chemotherapy have improved outcomes for AIDS lymphomas, challenges remain particularly with virus-associated AIDS lymphomas, prompting efforts to better understand virus-associated factors and pathways. In particular, EBV-driven cellular genome replication which is essential to lymphoma proliferation remains underexplored. Upon infection of B cells, EBV drives host DNA replication which is essential for establishment of viral latency as well as proliferation of cancer cells. However, such viral oncoprotein-driven DNA replication is plagued with physical and functional obstacles, resulting in replication stress. Such replication stress is a barrier to cancer. And yet, how EBV-cancer cells overcome such stress at replication forks to successfully proliferate is not well understood. In addressing this knowledge gap, we combined isolation of proteins on nascent DNA (iPOND) and mass spectrometry to discover novel fork proteins. This revealed a critical role for ZC3H18 (or ZC3) as a replication dependency factor that EBV upregulates to ensure host genome replication and lymphoma cell proliferation; notably, ZC3 had not been previously linked to DNA replication. Indeed, EBV+ DLBCL from AIDS patients have elevated ZC3 expression compared to EBV- lymphomas. An intrinsically disordered protein, ZC3 has the potential to concentrate a variety of proteins at replication forks. We find a direct interaction between ZC3 and MCM7 (a core component of the replicative helicase complex), further pointing to ZC3’s influential role in proliferation of EBV transformed cells. Importantly, ZC3’s partnership with other replication dependency factors exposes EBV-lymphoma cells to synthetic lethality – such therapies exploit the property that cancer cells tolerate perturbation of a single gene but succumb to co-disruption of multiple genetic events. In this application, we will test the hypothesis that EBV modulates the DNA replication machinery, ensuring proliferation of transformed cells in the face of replication stress and enhancing the potential for susceptibility to synthetic lethality. We will perform the following aims using ex vivo models and translate our results to patient- derived EBV+ AIDS lymphomas obtained from the AIDS and Cancer Specimen Resource (ACSR). Aim 1. Investigate how novel dependency factors unmask synthetic lethal vulnerabilities in EBV- transformed cells & Aim 2. Investigate mechanisms of ZC3 upregulation, replication machinery rewiring, and contribution of replication dependency factors to EBV+ AIDS lymphomas. These studies will identify mechanisms and generate new paradigms that reveal how an opportunistic virus modulates the host replication machinery to maintain the transformed state. Our long-term goal is to identify novel druggable targets that demonstrate synthetic lethality against EBV+ lymphomas in persons with HIV/AIDS.