Project 3: Demethylation of HPV-associated head and neck cancer to trigger APOBEC synthetic lethality and enhance immune response

总结 人乳头瘤病毒（HPV）相关的颈部鳞状细胞癌（HNSCC）代表了越来越多的 HNSCC的比例。HPV+ HNSCC的发病率在过去20年中急剧增加， 在2012年超过宫颈癌成为美国最常见的HPV相关恶性肿瘤。 HPV疫苗，估计HPV引起的HNSCC“流行”要到2060年才会减弱。 HPV+ HNSCC发生在年轻个体中，与HPV + HNSCC相比， 经典HNSCC患者;然而，约25%的患者复发，有效的治疗选择很少。 基于从TCGA观察到的HPV+ HNSCC的超甲基化，并理解HPV使用 超甲基化阻碍先天性免疫应答，去甲基化剂5-氮杂胞苷（5- azaC），对HPV+ HNSCC进行了测试。我们发现，培养和异种移植中的HPV+ HNSCC细胞， 对5-azaC敏感，并且5-azaC引起双链断裂（DSB），而在5-azaC后未观察到 HPV阴性HNSCC的治疗，即使使用高得多的剂量。我们发现在5-azaC治疗后， 载脂蛋白B mRNA编辑酶催化多肽3 B（APOBEC 3 B）与 HPV+ HNSCC中的染色质，而不是HPV阴性细胞。A3 B的CRISPR敲低阻止了DSB， 保护细胞免于5-azaC诱导的死亡。尽管需要DSB和由5-氨基丁酸引起的细胞毒性， azaC、A3 B也是未经治疗的HPV+ HNSCC的克隆形成存活所必需的。这些数据表明，A3 B 是HPV+ HNSCC细胞存活所必需的，但在去甲基化之后，A3 B介导毒性和DSB。 此外，5-azaC治疗增加了I型干扰素信号传导，如通过增加的表达所测量的。 干扰素刺激基因。这些令人兴奋的临床前数据导致了5天的5-azaC窗口试验。分析 的肿瘤标本证实了体外数据显示5-azaC导致细胞毒性。免疫荧光 HPV+患者肿瘤在5-azaC治疗前和治疗后的染色显示，肿瘤相关的 淋巴细胞，可能是通过激活I型干扰素结合增加的 新抗原在这个YHN-SPORE项目中，我们假设5-azaC治疗将增强对nivolumab的反应， （Nivo）通过其引起细胞死亡、增加新抗原表达、增加A3 B驱动的突变的能力， 负载，并通过增加I型干扰素信号传导增强T细胞浸润。这些假设将是 使用已建立的和新的体外试验进行测试，以及通过治疗前和治疗后的检查 来自三臂临床试验的肿瘤标本。在目标1中，来自SPORE窗口试验的肿瘤标本将被 分析以确定5-azaC、Nivo或其组合对细胞死亡、细胞增殖、免疫应答和/或细胞凋亡的影响。 浸润和免疫激活。目的2将采用标准和新的检测方法来探索A3 B在以下方面的作用： 5-azaC治疗暴露的细胞毒性。在目标3中，我们将确定5-azaC对以下活化剂的影响： 在存在或不存在Nivo的情况下的免疫识别和应答。

SUMMARY Human papillomavirus (HPV)-associated neck squamous cell carcinoma (HNSCC) represents an increasing proportion of HNSCC. The incidence of HPV+ HNSCC has dramatically increased over the last 2 decades and in 2012 surpassed uterine cervical cancer as the most common HPV-related malignancy in the U.S. Despite the HPV vaccine, it is estimated that the “epidemic” of HNSCC caused by HPV will not diminish until 2060. HPV+ HNSCCs occur in younger individuals and prognosis for patients with these tumors is better compared to patients with classical HNSCC; however, ~25% of patients recur with few effective therapeutic options. Based on observed hypermethylation of HPV+ HNSCC from TCGA, and understanding that HPV uses hypermethylation to impede the innate immune response, effects of the demethylating agent, 5-azacytidine (5- azaC), were tested on HPV+ HNSCC. We found that HPV+ HNSCC cells in culture and xenografts are sensitive to 5-azaC, and that 5-azaC caused double strand breaks (DSB) that were not observed after 5-azaC therapy in HPV-negative HNSCC, even with much higher doses. We found that following 5-azaC therapy, APOlipoprotein B mRNA-Editing enzyme Catalytic polypeptide 3B (APOBEC3B) was associated with chromatin in HPV+ HNSCC, but not HPV-negative cells. CRISPR knockdown of A3B prevented DSB and protected cells from 5-azaC-induced death. Despite being required for DSBs and cellular toxicity caused by 5- azaC, A3B was also required for clonogenic survival of untreated HPV+ HNSCC. These data showing that A3B is required for survival of HPV+ HNSCC cells, but that following demethylation A3B mediates toxicity and DSB. In addition, 5-azaC therapy increased type I interferon signaling as measured by increased expression of interferon-stimulated genes. These exciting pre-clinical data led to a window trial of 5days of 5-azaC. Analysis of tumor specimens confirmed in vitro data showing that 5-azaC resulted in cellular toxicity. Immunofluorescent staining of an HPV+ patient tumors pre- and post-5-azaC showed a marked increase in tumor-associated lymphocytes, possibly driven through activation of type I interferon combined with increased expression of neoantigens. In this YHN-SPORE project, we hypothesize 5-azaC therapy will enhance response to nivolumab (Nivo) through its ability to cause cell death, increase neoantigen expression, increase A3B-driven mutational load, and enhance T cell infiltration through increased type I interferon signaling. These hypotheses will be tested using established and novel in vitro assays, as well as through examination of pre- and post-therapy tumor specimens from a 3-armed clinical trial. In Aim 1, tumor specimens from the SPORE window trial will be analyzed to determine effects of 5-azaC, Nivo, or the combination on cell death, cell proliferation, immune infiltration and immune activation. Aim 2 will employ standard and novel assays to explore the role of A3B in cellular toxicity exposed by 5-azaC therapy. In Aim 3, we will determine effects of 5-azaC on activators of immune recognition and response in the presence or absence of Nivo.