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

摘要 致癌性人乳头瘤病毒（HPV）是宫颈癌的病原体， 部分头颈部鳞状细胞癌（HNSCC），但HNSCC几乎完全与 HPV 16型。HPV 16型的致癌特性主要归因于两种主要的HPV致癌基因，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的抑制因子导致不受控制的增殖，减少的凋亡和增加的基因组 易导致恶性转化的不稳定性。E6/E7在HPV相关癌变中的作用 使其成为抗癌治疗的有吸引力的靶点，因为降低其表达或活性的方法 将恢复由HPV驱动的肿瘤中的p53和RB活性。我们的初步结果表明， HPV阳性（HPV+）HNSCC与去甲基化剂，5-氮杂胞苷（5-aza）在临床相关 浓度，导致所有HPV基因表达的显著下调，包括E6和E7。5-氮杂 治疗恢复了HPV+头颈癌细胞中p53的表达和活性，这部分地 负责这些细胞对5-aza的敏感性。除了p53的恢复，5-aza也对 HPV+ HNSCC通过产生DNA双链断裂（DSB）。5-氮杂诱导的DNA HPV+ HNSCC中的DSB依赖于转录和复制以及胞苷的过表达， 脱氨酶，APOBEC 3B（A3 B）。实验性A3 B耗竭抑制5-氮杂毒性并减少DSB 这不仅表明未处理的HPV+ HNSCC的克隆形成依赖于A3 B，而且还表明未处理的HPV+ HNSCC的克隆形成生长依赖于A3 B。的 观察到未经治疗的HPV+ HNSCC依赖于A3 B，但A3 B有助于5-氮杂毒性， DSB，表明在用5-aza处理后A3 B依赖性合成致死性，并且A3 B可以作为一种抑制剂。 反应的生物标志物。用5-aza治疗携带HPV+肿瘤的小鼠显示出显著的肿瘤生长 抑制和阻止检测循环肿瘤细胞。在HNSCC患者中进行的窗口临床试验， 标准给药5天实现了与我们的体外实验中所见相似的去甲基化（LINE-1）， 证实了5-aza治疗：1）显著降低HPV基因表达; 2）重新激活p53; 3） 激活半胱天冬酶，4）并抑制HPV+ HNSCC中的基质金属蛋白酶表达。这项建议是 旨在确定去甲基化诱导的HPV癌基因下调的分子机制， 阐明A3 B在5-氮杂诱导的合成致死性和DNA双链断裂形成中的作用， 在HPV+ HNSCC中去甲基化对免疫细胞浸润的影响，并探索5-aza单独或联合 与化疗剂组合以抑制HPV相关的HNSCC转移并抑制生长 使用患者来源的异种移植物。这些研究将为HPV+的新的合理靶向治疗提供依据 HNSCC，迫切需要治疗复发性或转移性HPV+ HNSCC患者， 降低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.