High-throughput identification of molecular targets responsible for drug-induced peripheral neuropathies.

摘要 精确药物疗法已经成为一种有效且越来越常见的癌症治疗形式。 这些疗法经常使用多激酶抑制药(MKI)，每种药物都针对多个受体 酪氨酸激酶，有时与“常规”细胞毒性化疗药物联合使用。一个共同之处 许多癌症药物治疗的副作用是损害患者的周围神经系统，称为药物- 诱发性周围神经病(DIPN)。这些DIPN中的大多数是由远端的“死亡”引起的 感觉轴突支配皮肤，导致感觉疼痛和功能障碍。几种常用的MKI 诱导周围神经病变，然而，负责这些疼痛DIPN的特定靶点尚不清楚。至 为了解决这一知识鸿沟，我们建立了一种高内容筛选方法，允许快速识别 斑马鱼中的神经毒性化合物。使用这种方法，我们展示了三个已知可产生的MKI 患者的DIPN导致斑马鱼远端皮肤体感轴突密度降低。活着 影像显示轴突回缩是真皮轴突密度降低的细胞基础，这与 有着“垂死”的病理生理学。此外，这些结果在小鼠背根神经节中也得到了复制 神经元。对这种神经毒性作用的MKI靶点的初步筛选发现，受体的丢失 酪氨酸激酶c-Kit，而不是其他共享的激酶靶点，导致皮肤轴突密度降低。C-套件是 在胚胎和成体的脊椎动物感觉神经元中表达，其配体SCF在 皮肤，但这种配体受体在轴突维持或DIPN中的具体作用尚未确定。 重要的是，在c-kit突变体中应用其中一种mki并没有加剧轴突密度的丧失，这表明 该试剂盒是周围神经系统MKI的主要靶点。基于我们在报告中提出的初步数据 目标1至：1)在斑马鱼中实施高含量筛选方法，以确定诱导远端 体内感觉轴突毒性，然后确定其分子靶点；以及2)验证这些结果 哺乳动物背根神经节培养。目标2将描述潜在的神经毒性的下游机制 C-Kit受体功能丧失在我们的初步数据中描述。我们的工作将确定和描述 痛性MKI所致周围神经毒性的分子靶点和细胞基础。这反过来又将提供 在一个重大但未解决的临床背景下研究潜在干预的新途径和新要点 癌症药物治疗中的问题。此外，我们还将建立工作流程和试剂，以供将来研究 DIPN的候选目标。

Summary Precision drug therapies have emerged as an effective and increasingly common form of cancer treatment. These therapies frequently employ multi-kinase inhibitor drugs (MKIs) that each target multiple receptor tyrosine kinases, sometime in combination with “conventional” cytotoxic chemotherapy drugs. One common side effect of many cancer drug treatments is damage to the patient's peripheral nervous system, termed drug- induced peripheral neuropathies (DIPNs). Most of these DIPNs are caused by the “dying back” of distal sensory axons that innervate the skin, leading to sensory pain and dysfunction. Several commonly used MKIs induce peripheral neuropathies, however, specific targets responsible for these painful DIPNs are unknown. To address this knowledge gap, we established a high-content screening approach that allows rapid identification of neurotoxic compounds in zebrafish. Using this approach, we showed that three MKIs known to produce DIPNs in patients led to the reduced density of distal cutaneous somatosensory axons in zebrafish. Live imaging demonstrated that axon retraction is the cellular basis for this reduced dermal axon density, consistent with a “dying back” pathophysiology. Furthermore, these results were replicated in mouse dorsal root ganglia neurons. Initial screening for MKI targets underlying this neurotoxic effect found that loss of the receptor tyrosine kinase c-Kit, but not other shared kinase targets, led to reduced cutaneous axon density. c-Kit is expressed in a subset of vertebrate sensory neurons in embryos and adults and its ligand SCF is expressed in the skin, but the specific role of this ligand-receptor in axon maintenance or DIPNs has not been defined. Importantly, application of one of these MKIs in c-kit mutants did not exacerbate axon density loss, indicating that Kit is a major target for an MKI in the peripheral nervous system. Based on preliminary data we propose in Aim 1 to: 1) implement a high-content screening approach in zebrafish to identify MKIs that induce distal sensory axon toxicity in vivo and then identify their molecular targets; and 2) validate these results in mammalian DRG culture. Aim 2 will characterize the downstream mechanisms underlying the neurotoxicity of c-Kit receptor loss-of-function described in our preliminary data. Our work will identify and characterize the molecular targets and cellular bases of painful MKI-induced peripheral neurotoxicity. This, in turn, will provide new pathways and points of potential intervention to study in the context of a major, but unaddressed, clinical problem in cancer drug therapies. In addition, we will also establish a workflow and reagents for future study of candidate targets of DIPNs.