Targeting HMGB1 to improve hearing andenhance therapy for Vestibular Schwannomas

摘要 2型神经纤维瘤病（NF 2）是一种需要有效治疗的毁灭性疾病。NF 2的特点是 双侧前庭神经鞘瘤（VS），进行性扩大并导致听力损失和大量 对生活质量的负面影响。确定耐受性良好的药物，以阻止VS生长并改善VS相关性 听力损失是一个主要的未满足的医疗需求。管理NF 2相关听觉障碍的最大障碍是 我们对肿瘤如何导致听力损失的不完全理解。我们的目的是研究VS肿瘤的机制- 诱发听力损失，并制定策略，以控制VS肿瘤生长，同时防止听力损失。我们的初步 研究表明，i）神经炎症的水平，包括巨噬细胞浸润和NLRP 3 炎性小体激活，与VS患者的听力功能呈负相关，以及ii）患者的VS 分泌炎性细胞因子并导致耳蜗损伤。因此，我们探讨了 我们发现，高迁移率族蛋白盒1（HMGB 1），一种有效的炎症， 启动子和放大器，由VS肿瘤细胞大量分泌。此外，我们发现HMGB 1阻断：iii） 巨噬细胞趋化因子CCL基序趋化因子配体2（CCL 2）表达减少， 巨噬细胞（TAM）募集，iv）消除NLRP 3炎性体活化和IL-1b产生，然而， v）HMGB 1阻断激活表皮生长因子（EGF）信号传导，这可以补偿肿瘤生长。 增长基于这些发现，我们假设VS衍生的HMGB 1：i）募集炎性细胞， ii）通过上调CCL 2趋化因子驱动巨噬细胞炎症级联反应， 激活NLRP 3炎性体，导致耳蜗损伤和听力损失;和iii）组合 HMGB 1和表皮生长因子受体（EGFR）的阻断将同时抑制VS的生长，并防止肿瘤诱导的细胞凋亡。 听力损失在目的1中，我们将使用HMGB 1和Nlrp 3的遗传沉默来研究HMGB 1的因果作用。 在调节CCL 2表达以募集巨噬细胞和激活NLRP 3炎性体以触发巨噬细胞凋亡中， 巨噬细胞炎症反应。在目标2中，使用忠实地再现VS肿瘤的VS小鼠模型， 我们将描述HMGB 1药理学抑制对听力功能的影响 和耳蜗损伤在目标3中，我们将确定最有效的EGFR抑制剂来控制肿瘤生长 并表征HMGB 1和EGFR抑制剂在受试者中的药代动力学/药效学特性。 肿瘤、小鼠脑和耳蜗。然后，使用最有效的HMGB 1和EGFR抑制剂确定，我们将 评估HMGB 1和EGFR联合阻断对VS中肿瘤生长和听力的治疗效果 小鼠模型。影响：这项研究将提供关键的洞察i）HMGB 1的作用和机制， 驱动神经炎症，ii）HMGB 1阻断后的治疗潜力和听力反应， 以及iii）设计使用与HMGB 1的新组合策略的NF 2相关VS的未来临床试验 和EGFR阻断，以同时控制肿瘤生长和预防VS诱导的听力损失。

Abstract Neurofibromatosis type 2 (NF2) is a devastating disease that needs effective treatments. The hallmark of NF2 is bilateral vestibular schwannomas (VSs), which progressively enlarge and lead to hearing loss and substantial negative impacts to quality of life. Identifying well-tolerated drugs to halt VS growth and ameliorate VS-associated hearing loss is a major unmet medical need. The greatest barrier to managing NF2-related auditory impairment is our incomplete understanding of how tumors cause hearing loss. We aim to investigate the mechanisms of VS tumor- induced hearing loss and develop strategies to control VS tumor growth while preventing hearing loss. Our preliminary studies showed that i) the level of neuroinflammation, including macrophage infiltration and NLRP3 inflammasome activation, negatively correlates with hearing function in patients with VS, and ii) patients’ VSs secrete inflammatory cytokines and cause cochlear damage. Therefore, we explored the drivers of neuroinflammation in VS. We identified that Highly Mobility Group Box1 (HMGB1), a potent inflammation initiator and amplifier, is abundantly secreted by VS tumor cells. Further, we found that HMGB1 blockade: iii) reduced macrophage chemokine CCL motif chemokine ligand 2 (CCL2) expression and tumor-associated macrophage (TAM) recruitment, iv) abolished NLRP3 inflammasome activation and IL-1b production, however, v) HMGB1 blockade activates epidermal growth factor (EGF) signaling, which may compensate for tumor growth. Based on these findings, we hypothesize that VS-derived HMGB1: i) recruits inflammatory macrophages by upregulating the CCL2 chemokine, ii) drives the macrophage inflammatory cascade by activating the NLRP3 inflammasome, contributing to cochlear damage and hearing loss; and iii) combined HMGB1 and EGF receptor (EGFR) blockade will concurrently suppress VS growth and prevent tumor-induced hearing loss. In Aim 1, we will use genetic silencing of HMGB1 and Nlrp3 to investigate the causal role of HMGB1 in regulating CCL2 expression to recruit macrophages and in activating NLRP3 inflammasome to trigger the macrophage inflammatory response. In Aim 2, using VS mouse models that faithfully reproduce VS tumor- induced hearing loss, we will characterize the effects of HMGB1 pharmacologic inhibition on hearing function and cochlear damage. In Aim 3, we will determine the most effective EGFR inhibitor to control tumor growth and characterize the pharmacokinetic/pharmacodynamic properties of HMGB1 and EGFR inhibitors in the tumor, mouse brain, and cochlea. Then, using the most effective HMGB1 and EGFR inhibitor identified, we will evaluate the treatment efficacy of combined HMGB1 and EGFR blockade on tumor growth and hearing in VS mouse models. Impact: This study will provide pivotal insight into i) the role and mechanisms of HMGB1 in driving neuroinflammation, ii) the treatment potential and the hearing response following HMGB1 blockade, and iii) the design of a future clinical trial for NF2-related VS using a novel combination strategy with HMGB1 and EGFR blockade to concurrently control tumor growth and prevent VS-induced hearing loss.