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

摘要 由噪音、衰老和化疗引起的听力损失影响着全球7亿人， 目前还没有FDA批准的药物来预防它。这项研究将测试重新利用一个小的 分子BRAF抑制剂，达拉非尼（TAFINLAR），FDA批准的几种癌症的药物，用于新的用途， 预防顺铂引起的听力损失。达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种特异性激酶抑制剂用于顺铂诱导的细胞死亡保护， 内耳细胞系我们发现，达拉非尼完全保护外毛细胞免受顺铂毒性， 小鼠耳蜗外植体，IC 50为30 nM，极好的治疗指数（LD 50/IC 50）>2000。 从机制上讲，我们鉴定了达拉非尼和三种额外的BRAF抑制剂，两种MEK 1/2抑制剂，以及一种 ERK 1/2抑制剂在细胞通路中紧邻BRAF下游，支持BRAF在 顺铂诱导的毛细胞死亡。顺铂治疗内耳细胞系引起磷酸化的上调， BRAF、磷酸化-MEK 1/2和磷酸化-ERK 1/2，其通过与达拉非尼共处理而被抑制。此外，委员会认为， 顺铂处理耳蜗外植体或体内噪声暴露引起磷酸化ERK 1/2上调 支持细胞（内指骨和Deiters细胞）损伤后短时间内， 达拉非尼治疗。此外，在100 nM浓度下，达拉非尼可保护斑马鱼侧线神经瘤， 顺铂诱导的体内死亡，重要的是，口服给药 在小鼠模型中，达拉菲尼连续三天对抗顺铂诱导的听力损失。每日剂量 在批准用于长期人类治疗的范围内。在这 建议，我们将测试dabrafenib提供的保护顺铂诱导的听力损失在多个低 剂量顺铂方案，其紧密模拟临床中癌症患者的顺铂治疗。功能 将评估听觉性能和内耳形态。将测试两个剂量的达拉非尼， 评价药物的体内治疗窗。我们还将确定达拉非尼对 顺铂在肿瘤细胞系和以顺铂为标准的小鼠肿瘤模型中的肿瘤杀伤功效 治疗，神经母细胞瘤和肺癌。我们将证实BRAF激酶是达拉非尼的分子靶点 通过产生支持细胞特异性条件性敲除小鼠来治疗顺铂诱导的听力损失，其中 从出生后第28天开始，BRAF在内耳的支持细胞中特异性缺失，并测试其 顺铂耐药。支持细胞的ERK磷酸化可以作为顺铂的体内生物标志物 损伤和评估BRAF抑制剂治疗，并可用于确定达拉非尼的PK/PD 特性.我们的研究将揭示一个新的细胞途径和分子靶点BRAF激酶的耳保护 并将为达拉菲尼进入人体临床试验提供关键数据， 顺铂引起的听力损失

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.