Targeting HMGB1 to improve hearing andenhance therapy for Vestibular Schwannomas

摘要 2型神经纤维瘤病(NF2)是一种破坏性疾病，需要有效的治疗。NF2的标志是 双侧前庭神经鞘瘤(VSS)，渐进性增大并导致听力损失和实质性 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 听力损失是一个主要的未得到满足的医疗需求。管理NF2相关性听力障碍的最大障碍是 我们对肿瘤如何导致听力损失的不完全理解。我们的目标是研究VS肿瘤的机制- 导致听力损失，并制定策略，以控制VS肿瘤生长，同时防止听力损失。我们的预赛 研究表明，1)神经炎症水平，包括巨噬细胞浸润和NLRP3 VS患者的炎性小体激活与听力功能呈负相关，II)患者的VSS 分泌炎性细胞因子，造成耳蜗损伤。因此，我们探索了 神经炎性VS。我们发现高迁移率族蛋白1(HMGB1)是一种强有力的炎症 启动子和放大因子，由VS肿瘤细胞大量分泌。此外，我们发现HMGB1封锁：III) 巨噬细胞趋化因子CCL基序趋化因子配体2表达降低与肿瘤相关 巨噬细胞()募集，iv)抑制NLRP3炎性小体激活和IL-1b的产生。 V)阻断HMGB1激活表皮生长因子(EGF)信号，从而可能补偿肿瘤 成长。基于这些发现，我们假设VS衍生的HMGB1：i)招募炎症性 巨噬细胞通过上调CCL2趋化因子，II)通过以下途径驱动巨噬细胞炎症级联反应 激活NLRP3炎症性小体，导致耳蜗损伤和听力损失；以及iii)联合 HMGB1和EGF受体(EGFR)阻断将同时抑制VS生长和防止肿瘤诱导 听力损失。在目标1中，我们将使用HMGB1和Nlrp3的遗传沉默来研究HMGB1的因果作用 在调节CCL2表达以募集巨噬细胞和激活NLRP3炎症小体以触发 巨噬细胞炎症反应。在目标2中，使用忠实复制VS肿瘤的VS小鼠模型- 致聋，我们将表征HMGB1药物抑制对听力功能的影响 和耳蜗骨损伤。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HMGB1和EGFR抑制剂在小鼠体内的药代动力学/药效学特性 肿瘤、小鼠脑和耳蜗骨。然后，使用确定的最有效的HMGB1和EGFR抑制剂，我们将 HMGB1和EGFR联合阻断对VS小鼠肿瘤生长和听力的影响 老鼠模型。影响：本研究将为以下方面提供关键的见解：1)HMGB1在脑内的作用和机制 推动神经炎症，II)HMGB1阻断后的治疗潜力和听力反应， 以及iii)使用与HMGB1的新组合策略设计NF2相关VS的未来临床试验 和EGFR阻断，以同时控制肿瘤生长和防止VS导致的听力损失。

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.