Artificial modulation of Epstein-Barr virus latent-lytic switch in EBV-associated epithelial cancers

Ph.D.Epstein Barr virus (EBV) is a human gamma-herpesvirus associated with multiple malignant diseases, including nasopharyngeal carcinomas (NPC), a subgroup of gastric carcinomas (GC) and several types of lymphomas (e.g.: Burkitt’s lymphoma, Hodgkin lymphomas, NK-T lymphomas, post-transplant and AIDS-associated lymphomas).This study aims to develop novel strategies for the efficient induction of viral lytic replication in EBV-infected epithelial cells, which may serve as novel therapies for EBV-associated epithelial cancers. Although a number of chemical inducers have been reported for reactivating the lytic cycle in NPC or EBV-associated gastric cancer (EBVaGC) cells, their effects were found to be limited and varied among different EBV-positive cancer cell lines. Here, the efficiency of different classes of reported lytic inducers (HDACi, PKC activator, conventional chemotherapeutic agents) in reactivation of the immediate early genes (BZLF1 and BRLF1) was examined in four native EBV-positive cancer cell lines (C666-1, NPC43, C17, and SNU719). By treatment with different lytic inducing agents, BZLF1-expressing cells detected in these EBV-positive cancer cell lines were varied from 0 to 10%. Nevertheless, none of the reported lytic inducers was able to consistently activate BZLF1 expression in all of these EBV positive cancer cell lines. The findings imply that the switch of latency to the lytic cycle is cell-context specific, depending on the cellular features and genetic background of these tumors.To develop an efficient oncolytic therapeutic strategy for EBV-associated cancers, a novel approach directly reactivating lytic genes by CRISPR activating system was explored in this study. Through transfection or transduction of a dCas9/CRISPR transactivator system and specifically designed single-guide RNAs (sgRNAs) targeting the promoter site of immediately early lytic gene BZLF1, endogenous BZLF1 expression was artificially activated in a panel of EBV positive epithelial cancer cell lines. Significant induction of BZLF1 and downstream lytic genes in the cancer cells were detected by both qRT-PCR and Western blot. By flow cytometry, RNAscope, and immunofluorescence staining, BZLF1-positive cells were detected in up to 60% of tumor cells. By the inducible dCas9/CRSIPR system targeting BZLF1, potent in vitro and in vivo cytotoxic activity was demonstrated in the tumor cells treated by artificial BZLF1 activation and an antiviral drug, Ganciclovir (GCV). As a prodrug, GCV can be converted to a cytotoxic drug by an EBV early lytic protein BGLF4 during reactivation of the lytic cycle. The findings indicate that artificial activation of EBV lytic genes by the CRISPR system is a potential approach for oncolytic therapy in EBV-associated epithelial cancers.The current study firstly provides convincing evidence for the treatment of EBV-positive epithelial cancers via artificial activation of the EBV lytic cycle. Further study on the development of in vivo delivery of this dCas9/CRSIPR-EBV targeting system and elucidation of the synergic effect of multiple lytic gene induction may help to develop efficient clinical interventions for these viral-associated cancers.EB病毒 又名 人類孢疹病毒第四型，是一種常見的 雙鏈 DNA病毒。 EB病毒與多種惡性 腫瘤 有關，這些 腫瘤 包括鼻咽癌， 部分胃癌和 多種淋巴腫瘤（伯奇氏淋巴瘤，霍奇金淋巴瘤， 自然杀伤 /T细胞淋巴瘤 ，艾滋病相關淋巴瘤）。本研究希望通過有效地誘導EB病毒感染的上皮細胞的病毒裂解循環，從而為EB病毒相關上皮惡性腫瘤治療提供新的方案。儘管有報道表示部分化學誘導劑可以重新激活鼻咽癌或者部分 EB病毒相關胃癌細胞內的病毒裂解循環，但是在不同的 EB病毒陽性腫瘤細胞系中出現明顯的效率差異性。在本研究中，我們在 4種原生 EB病毒陽性上皮惡性腫瘤細胞系（ C666-1, NPC43, C17和 SNU719中測試多種化學誘導劑激活 EB病毒裂解循環基因 BZLF1 和 BRLF1表達的效率。這些化學誘導劑包括 織蛋白去乙醯酶抑制劑，蛋白激酶 C活化劑以及化療藥物。結果所示，經過不同的裂解循環誘導劑的處理後出現 BZLF1表達的細胞在這些細胞系中所占比例在 0和 10%之間。然而沒有發現任何一種裂解循環誘導劑可以在所有 EB病毒陽性腫瘤細胞系中誘導 BZLF1的表達。這些結果說明病毒潛伏期到病毒裂解循環的轉變依賴這些腫瘤細胞的細胞特性以及遺傳背景並具有細胞專一性。為了研發一種高效的通過病毒裂解治療EB病毒相關癌症的方案，本研究中嘗試使用 CRISPR技術直接激活病毒裂解複製相關基因作為激活病毒裂解循環的新方法。多種 EB病毒相關腫瘤細胞係中的 BZLF1在轉染 和轉導 CRISPR/dCas9激活體系以及相關的靶 EB病毒裂解基因 BZLF1啟動子的引導核糖核酸（ sgRNA下被人工激活。 Western免疫印迹法還有熒光定量 PCR實驗表明 BZLF1還有病毒裂解循環相關的下游基因在這些癌症細胞內顯著表達。流式細胞術 以及 免疫熒光等方法 檢測發現在這些腫瘤細胞中 BZLF1陽性細 胞高達 60%。通過在 EB病毒陽性腫瘤細胞系構建誘導性 CRISPR/dCas9 BZLF1活化 系統，利用抗病毒藥物 更昔洛 韋 在體外腫瘤細胞和小鼠體內 證明了其具有 細胞毒性。抗病毒藥物更昔洛 韋 是一種前體藥物，通過 EB病毒相關基因 BGLF4表達的蛋白激酶催化的磷酸化反應可以激活更昔洛 韋 產生細胞毒性。實驗表明通過 dCas9/CRISPR技術可以人工誘導激活 EB病毒陽性腫瘤中的病毒裂解循環相關基因 ，這個技術未來有可能作為一種新的 EB病毒相關腫瘤的治療方案。這個實驗第一次展示了直接人工誘導EB病毒 裂解複製可以殺死腫瘤細胞進一步 治療 EB病毒相關上皮腫瘤。之後的實驗將進一步利用非病毒類體系傳遞相關的特異性激活系統進入體內提高傳遞效率，完善其相關治療效果。這個治療方式將有助於發展 EB病毒相關腫瘤的治療，提高治療效果。Wu, Man.Thesis Ph.D. Chinese University of Hong Kong 2020.Includes bibliographical references (leaves 159-171).Abstracts also in Chinese.Title from PDF title page (viewed on February 8, 2022)