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

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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.