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

项目总结 病毒相关淋巴瘤在艾滋病毒感染者中会导致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)和 40%的Burkitt淋巴瘤(BL)。尽管联合抗逆转录病毒疗法(CART)和化疗已经 改善艾滋病淋巴瘤的预后，挑战依然存在，尤其是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未得到充分研究。 在感染B细胞后，EBV驱动宿主DNA复制，这是建立病毒潜伏期的关键 以及癌细胞的增殖。然而，这种由病毒癌蛋白驱动的DNA复制受到 生理和功能障碍，导致复制应激。这种复制压力是癌症的屏障。 然而，EBV-癌细胞如何克服复制分叉的压力而成功增殖并不是很好。 明白了。为了解决这一知识差距，我们将新生DNA上蛋白质的分离(IPOND)和 质谱学发现新的叉蛋白。这揭示了ZC3H18(或ZC3)作为 EBV上调以确保宿主基因组复制和淋巴瘤细胞的复制依赖因子 特别是，ZC3以前与DNA复制没有关联。事实上，EBV+DLBCL来自艾滋病 与EBV淋巴瘤相比，患者ZC3表达升高。一种天生无序的蛋白质，ZC3 有可能在复制叉子上集中各种蛋白质。我们发现了一种直接的相互作用 ZC3和MCM7(复制解旋酶复合体的核心成分)，进一步指出ZC3的S的影响作用 在EBV转化细胞的增殖中。重要的是，ZC3的S与其他复制依赖因素建立了伙伴关系 使EBV-淋巴瘤细胞暴露于人工致死--这种疗法利用了癌细胞耐受的特性 单个基因的扰动，但屈服于多个遗传事件的共同干扰。 在这个应用中，我们将检验EBV调节DNA复制机制的假设，确保 面对复制应激时转化细胞的增殖，并增加对 合成杀伤力。我们将使用体外模型实现以下目标，并将我们的结果转化为患者- 来源于艾滋病和癌症标本资源(ACSR)的EBV+艾滋病淋巴瘤。 目的1.研究新的依赖因素如何揭示EBV中的合成致命漏洞 转化细胞&目的2.研究ZC3上调、复制机制重排、 复制依赖因子在EBV+AIDS淋巴瘤中的作用。 这些研究将确定机制并产生新的范例，揭示机会性病毒如何 调节宿主复制机制以维持转换状态。我们的长期目标是确定 新的可药物靶点，展示了对艾滋病毒/艾滋病患者的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.