Repurposing an FDA approved Drug, B-Raf Kinase Inhibitor Dabrafenib for Protection from Cisplatin- and Noise- induced Hearing Loss

摘要 噪音、衰老和化疗导致的听力损失影响着全球7亿人，但 目前还没有FDA批准的药物来预防这种疾病。这项研究将测试改变小规模 分子BRAF抑制剂达普拉非尼(Tafinlar)，FDA批准的治疗几种癌症的药物，用于治疗 预防顺铂所致听力损失。达普拉非尼在我们无偏见的高通量筛选中是最受欢迎的 4,385种生物活性化合物和187种特异的激酶抑制剂对顺铂诱导的细胞死亡的保护作用 内耳细胞系。我们发现，达普拉非尼完全保护外毛细胞免受顺铂的毒性。 小鼠耳蜗外植体的IC50为30 NM，治疗指数(LD50/IC50)为&gt；2000。 从机制上讲，我们确定了达普拉非尼和另外三种BRAF抑制剂，两种MEK1/2抑制剂和一种 ERK1/2抑制剂直接位于BRAF下游的细胞通路中，支持BRAF在 顺铂诱导毛细胞死亡。顺铂诱导的内耳细胞株磷酸化水平上调 BRAF、磷酸化的MEK1/2和磷酸化的ERK1/2可被达普拉非尼共同抑制。此外， 顺铂处理耳蜗外植体或体内噪声暴露引起磷酸化ERK1/2上调 支持细胞(指骨内侧细胞和Deiters细胞)损伤后的短时间，可通过以下方法减轻 达普拉非尼治疗。此外，在100海里处，达普拉非尼保护斑马鱼侧线神经肥大免受 顺铂诱导的体内死亡，重要的是，口服给药获得了显著的保护 达普拉非尼连续三天在小鼠模型中对抗顺铂所致听力损失。每日剂量 给予小鼠的达普拉非尼的剂量在批准的长期人类治疗范围内。在这 建议，我们将测试达普拉非尼对顺铂所致听力损失的保护作用。 剂量顺铂方案，在临床上与顺铂治疗癌症患者非常相似。功能性 将对听觉表现和内耳形态进行评估。两剂达普拉非尼将被测试为 评价药物在体内的治疗窗。我们还将确定达普拉非尼布对 顺铂对肿瘤细胞系和以顺铂为标准的小鼠肿瘤模型的杀伤作用 治疗、神经母细胞瘤和肺癌。我们将确认BRAF激酶是达普拉非尼的分子靶点 通过产生支持细胞特异性条件性基因敲除小鼠，在顺铂诱导的听力损失中 从出生后第28天开始，BRAF在内耳的支持细胞中被特异性地删除，并检测其 对顺铂耐药。支持细胞ERK磷酸化可作为顺铂的体内生物标志物 BRAF抑制剂的损伤和评价治疗，并可用于测定达普拉非尼的PK/PD 属性。我们的研究将揭示一种新的细胞途径和分子靶点BRAF激酶用于耳保护 并将提供将达普拉非尼推向临床试验所需的关键数据，以预防 顺铂所致听力损失。

Abstract Hearing loss caused by noise, aging and chemotherapy affects seven hundred million people worldwide, but there are no FDA-approved drugs to prevent it. This research will test the potential to repurpose a small molecule BRAF inhibitor, dabrafenib (TAFINLAR), an FDA-approved drug for several cancers, for new use in preventing cisplatin-induced hearing loss. Dabrafenib was a top hit in our unbiased high-throughput screens of 4,385 bioactive compounds and 187 specific kinase inhibitors for cisplatin-induced cell-death protection in an inner ear cell line. We found that dabrafenib fully protected the outer hair cells against cisplatin toxicity in mouse cochlear explants with IC50 of 30 nM and an excellent therapeutic index (LD50/IC50) of >2000. Mechanistically, we identified dabrafenib and three additional BRAF inhibitors, two MEK1/2 inhibitors, and an ERK1/2 inhibitor immediately downstream of BRAF in the cellular pathway, supporting the role of BRAF in cisplatin-induced hair cell death. Cisplatin treatment of the inner ear cell line caused upregulation of phospho- BRAF, phospho-MEK1/2 and phospho-ERK1/2 that was inhibited by co-treatment with dabrafenib. Moreover, cisplatin treatment of cochlear explants or noise exposure in vivo caused up-regulation of phospho-ERK1/2 short time after damage in supporting cells (inner phalangeal and Deiters’ cells) that was mitigated by dabrafenib treatment. Furthermore, at 100 nM dabrafenib protected zebrafish lateral line neuromasts from cisplatin-induced death in vivo and, importantly, significant protection was achieved with oral delivery of dabrafenib for three consecutive days in mouse models against cisplatin-induced hearing loss. The daily dose of dabrafenib administered to the mice was in the range approved for long-term human treatment. In this proposal, we will test the protection provided by dabrafenib for cisplatin-induced hearing loss in a multiple low dose cisplatin regimen that mimics closely the cisplatin treatment of cancer patients in the clinic. Functional auditory performance and inner ear morphology will be assessed. Two doses of dabrafenib will be tested to evaluate the therapeutic window of the drug in vivo. We will also determine dabrafenib’s interference with cisplatin tumor killing efficacy in tumor cell lines and mouse tumor models in which cisplatin is the standard treatment, neuroblastoma and lung cancer. We will confirm BRAF kinase is the molecular target of dabrafenib in cisplatin-induced hearing loss by generating a supporting cell specific conditional knockout mouse in which BRAF is specifically deleted in the supporting cells of the inner ear starting at postnatal day 28 and testing its resistance to cisplatin. Supporting cells’ ERK phosphorylation can serve as an in vivo biomarker for cisplatin damage and evaluating treatment with BRAF inhibitors and can be utilized to determine dabrafenib’s PK/PD properties. Our study will reveal a new cellular pathway and molecular target BRAF kinase for otoprotection and will provide the crucial data needed for advancing dabrafenib to clinical trials in humans for prevention of cisplatin-induced hearing loss.