Exploring mechanisms of therapeutic demethylation effects in HPV-associated head and neck cancer

摘要 致癌性人乳头状瘤病毒(HPV)是宫颈病变的病原体，其发病率呈上升趋势。 部分头颈部鳞状细胞癌(HNSCC)，但HNSCC几乎只与 HPV 16型的致癌特性很大程度上归因于两个主要的HPV癌基因E6 和E7，分别降解P53和视网膜母细胞瘤(RB)家族成员。这些肿瘤的降解 E6和E7的抑制子导致不受控制的增殖，减少细胞凋亡，增加基因组 易于恶变的不稳定。E6/E7在人乳头瘤病毒致癌中的重要作用 使它们成为抗癌治疗的有吸引力的靶点，因为减少它们表达或活性的方法 将恢复HPV驱动的肿瘤中的P53和Rb活性。我们的初步结果表明，治疗 去甲基化药物5-氮杂胞苷(5-aza)治疗HPV阳性HNSCC的临床研究 浓度，导致所有HPV基因表达显著下调，包括E6和E7。5-杂氮 治疗恢复了HPV头颈部癌细胞中P53的表达和活性，这是部分恢复的 负责这些细胞对5-氮杂氮的敏感性。除了对P53的修复外，5-aza还对 HPV HNSCC通过产生DNA双链断裂(DSB)。从机制上讲，5-氮杂诱导的DNA HPV HNSCC中的双链断裂依赖于转录和复制以及胞苷的过度表达 脱氨酶，APOBEC3B(A3B)。A3B的实验性耗竭抑制5-氮杂毒性和降低DSB 但也表明未经处理的HPV HNSCC依赖于A3B进行克隆生长。这个 观察到未经治疗的HPV HNSCC依赖于A3B，但A3B有助于5-氮杂毒性和 DSB，提示A3B依赖于5-aza治疗后的合成致死性，A3B可能是一种 反应的生物标志物。5-氮杂氮对HPV荷瘤小鼠的治疗作用 抑制和预防检测循环中的肿瘤细胞。一项针对HNSCC患者的窗口临床试验 5天的标准剂量实现了去甲基化(LINE-1)，与我们的体外实验和 证实5-氮杂处理：1)显著降低HPV基因的表达；2)重新激活P53；3) 激活caspase；4)抑制HPV HNSCCs中基质金属蛋白酶的表达。这项建议是 旨在确定去甲基化导致HPV癌基因下调的分子机制， 阐明A3B在5-氮杂诱导的合成杀伤力和DNA双链断裂形成中的作用，确定A3B的作用 去甲基化对HPV HNSCC免疫细胞浸润的影响，并探讨5-aza单独或联合应用的可能性。 联合化疗药物抑制HPV相关性HNSCC转移和生长 使用病人来源的异种移植物。这些研究将为新的合理靶向治疗HPV提供基础。 HNSCC，这是治疗复发或转移性HPV HNSCC患者急需的 减少与当前治疗相关的毒性。

Abstract Oncogenic human papillomaviruses (HPV) are the causative agents of uterine cervical and an increasing portion of head and neck squamous cell carcinomas (HNSCC), but HNSCC is almost exclusively associated with HPV type 16. The oncogenic properties of HPV type 16 are largely attributed to two major HPV oncogenes, E6 and E7, that degrade p53 and retinoblastoma (RB) family members, respectively. Degradation of these tumor suppressors by E6 and E7 results in uncontrolled proliferation, diminished apoptosis and increased genomic instability that predisposes to malignant transformation. The crucial roles of E6/E7 in HPV-related carcinogenesis make them an attractive target for anti-cancer therapy, since methods for decreasing their expression or activities would restore p53 and RB activity in tumors driven by HPV. Our preliminary results indicate that treatment of HPV-positive (HPV+) HNSCC with the demethylating agent, 5-azacytidine (5-aza) at clinically relevant concentrations, resulted in remarkable downregulation of all HPV gene expression, including E6 and E7. 5-aza treatment restored p53 expression and activity in HPV+ head and neck cancer cells, which was partially responsible for the sensitivity of these cells to 5-aza. In addition to restoration of p53, 5-aza also was toxic to HPV+ HNSCC through creation of DNA double strand breaks (DSBs). Mechanistically, 5-aza-induced DNA DSBs in HPV+ HNSCC were dependent on transcription and replication and on overexpression of the cytidine deaminase, APOBEC3B (A3B). Experimental depletion of A3B inhibited 5-aza toxicity and diminished DSB formation, but also indicated that untreated HPV+ HNSCC depend on A3B for clonogenic growth. The observations that untreated HPV+ HNSCC dependent on A3B, but that A3B contributes to 5-aza toxicity and DSBs, suggests an A3B-dependent synthetic lethality upon treatment with 5-aza, and that A3B may serve as a biomarker of response. Treatment of mice bearing HPV+ tumors with 5-aza revealed significant tumor growth inhibition and prevented detection of circulating tumor cells. A window clinical trial in patients with HNSCC using standard dosing for 5 days achieved demethylation (LINE-1) similar to that seen in our in vitro experiments and confirmed that 5-aza treatment: 1) significantly decreased expression of HPV genes; 2) reactivated p53; 3) activated caspases, 4) and inhibited matrix metalloproteinase expression in HPV+ HNSCCs. This proposal is designed to determine molecular mechanisms of demethylation-induced downregulation of HPV oncogenes, elucidate the role of A3B in 5-aza-induced synthetic lethality and DNA DSBs formation, determine effect of demethylation on immune cell infiltration in HPV+ HNSCC, and explore the potential of 5-aza alone or in combination with chemotherapeutic agents to suppress HPV-associated HNSCC metastasis and inhibit growth using patient-derived xenografts. These studies will provide a basis for a new rational targeted therapy for HPV+ HNSCC, which is desperately needed to treat patients with recurrent or metastatic HPV+ HNSCC and to decrease the toxicity associated with current therapy.