Analysis of the E7-Mediated Mechanism of MHC Class I Repression in HNSCC

摘要 头颈部鳞状细胞癌（HNSCC）是世界上第六大最常见的癌症类型， 美国每年有超过8,000人死于这种疾病由以下原因引起的HNSCC病例数 人乳头瘤病毒（HPV）感染呈上升趋势，尤其是在40-65岁的患者中。HPV目前与 80%的口咽癌和5-10%的HNSCC病例来自其他亚位点。高危HPV病毒株， 最常见的HPV 16和HPV 18引起HPV阳性HNSCC的优势。目前的数据显示， 在HNSCC中，HPV 16通过阻止HPV 16主要抗原的表面呈递来限制免疫检测。 组织相容性复合物I类（MHC-I）。在HPV阳性的HNSCC中，MHC-I表面呈递水平是 与细胞毒性T细胞介导的抗肿瘤免疫应答相关， 预防HPV介导的MHC-I抑制的策略可能对患者具有显著的治疗益处 接受免疫治疗有资料表明E7负调控MHC-I的转录 然而，E7介导的MHC-I抑制的详细机制的特点很差。 在这里，我已经建立了两个E7过表达HPV阴性的HNSCC细胞系，并证实E7蛋白 下调MHC-I的转录。我们已经制定了系统和逻辑的方法，包括 我们建议利用CRISPR/CAS9分析来表征E7- 在这些模型和HPV+ HNSCC模型中抑制MHC-I。此外，我们还开发了一种基于3D类器官的 共培养试验，其中这些模型与患者匹配的外周血单核细胞共培养 （PBMC）以测试MHC-I表达的去阻遏的功能效应。我的核心假设是 HPV16_E7减少MHC-I表达的分子机制的鉴定将 导致治疗策略的进步，通过活化的T- 细胞本文将从以下几个方面对这一假说进行阐述：1）阐明MHC的分子机制 HNSCC中HPV16_E7的基因座阻遏，和2）鉴定HPV16_E7依赖性MHC I类调节基因 HNSCC中的通路。我的长期目标是开发新的治疗方法， HPV+患者的生存率，在这样做的过程中，我希望描述HPV16_E7 可抑制HPV阳性HNSCC中MHC-I表面表达。

Abstract Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer type in the world, and is responsible for over 8,000 deaths in the United States each year. The number of HNSCC cases caused by human papilloma virus (HPV) is on the rise, especially in patients 40-65 years old. HPV is currently associated with 80% of oropharyngeal cancers and 5-10% of HNSCC cases from other sub-sites. High-risk HPV strains, most commonly HPV16 and HPV18, cause the preponderance of HPV-positive HNSCC. Current data suggests that in HNSCC, HPV16 limits immune detection by preventing surface presentation of the major histocompatibility complex class I (MHC-I). In HPV-positive HNSCC, MHC-I surface presentation level is associated with the cytotoxic T cell-mediated anti-tumor immune response and thus the identification of strategies to prevent HPV-mediated MHC-I repression may have a significant therapeutic benefit for patients receiving immunotherapies. There some data that suggest E7 negatively regulates transcription of MHC-I constituent genes; however, the detailed mechanism of E7-mediated MHC-I repression is poorly characterized. Here, I have generated two E7-overexpressing HPV-negative HNSCC cell lines and confirmed that E7 protein downregulates transcription of MHC-I. We have developed systematic and logical approaches including CRISPR/CAS9 profiling that we propose to leverage to characterize the molecular mechanism(s) by which E7- represses MHC-I in these models, and HPV+ HNSCC models. Further, we have developed a 3D organoid-based co-culture assay in which these models are co-cultured with patient-matched peripheral blood mononuclear cells (PBMCs) to test the functional effects of de-repression of MHC-I expression. My central hypothesis is that identification of the molecular mechanisms by which HPV16_E7 diminish the expression of MHC-I will lead to the advancement of therapeutic strategies that enhance tumor cell recognition by activated T- cells. I will address this hypothesis through the following aims: 1) Detail the molecular mechanism(s) of MHC locus repression by HPV16_E7 in HNSCC, and 2) Qualify HPV16_E7-dependent MHC class I regulatory pathways in HNSCC. My long-term goal is to develop new therapeutic approaches that improve the overall survival of HPV+ patients, and in doing so, I hope to characterize the specific mechanisms by which HPV16_E7 can prevent MHC-I surface expression in HPV-positive HNSCC.