Discovering targets for neuroblastoma differentiation therapy from the human druggable genome - Discovering targets for neuroblastoma differentiation therapy from the human druggable genome Abstract Neuroblastoma (NBL), an aggressive childhood cancer, arises from differentiation-defective neural crest precursor cells. Differentiation therapy, an approach to halt tumor growth by inducing differentiation of the malignant cells, plays a critical role in treating NBL. There is a high demand for more effective differentiation agents because > 50 % of the high-risk NBLs are resistant to currently available differentiation agents. However, the mechanisms that block NBL cell differentiation have not been fully elucidated, which set an obstacle to developing new differentiation agents. The objective of this study is to discover novel druggable differentiation- blocking oncogenes from the human druggable genome and to preliminarily investigate inhibitors for protein products of the identified key oncogenes as differentiation agents in NBL cells. The hypothesis is that novel druggable oncogenes which function as blockers of NBL cell differentiation can be discovered via a siRNA-based high-content screening (HCS) of the druggable genome and that inhibitors for protein products of the identified genes are potential candidates for differentiation therapy. The hypothesis is strongly supported by a pilot HCS of siRNAs against 352 druggable genes in a NBL cell line BE(2)-C, which identified 6 differentiation-blocking oncogenes. Three of the 6 genes are novel discoveries, whereas the remaining 3 genes are previously identified oncogenes in NBL and FDA-approved inhibitors for their protein products have been shown to have differentiation-inducing activities. Four Specific Aims are proposed: Aim 1, systematically investigate the druggable genome and identify differentiation-blocking oncogenes. This will be done by (1) performing HCS of a library of siRNAs against the human druggable genome and identify genes with their knockdown inducing neurite-outgrowth and cell growth arrest, the differentiation marker of NBL cells, in BE(2)-C, and then (2) validating the identified genes using additional siRNAs and by measuring NBL molecular differentiation markers. Aim 2, discover novel differentiation-blocking oncogenes and novel differentiation-regulating pathways by informatics analysis of the identified genes. Aim 3, investigate the generic differentiation-blocking functions of the key novel genes in a panel of NBL cell lines with different genetic backgrounds. Six novel genes will be investigated in this grant period, which will be selected based on the potency ranking from HCS. Aim 4, investigate the differentiation-inducing activities of protein inhibitors for the identified key novel genes in NBL cell lines. Six novel genes will be investigated in this grant period, which will be selected based on the potency ranking from HCS, genericity shown in multiple cell lines and availability of FDA-approved inhibitors for their protein products. The study is innovative because the proposed HCS approach has not been used to investigate the entire druggable genome for NBL cell differentiation. The study is significant because success of this study will not only advance our understanding of the mechanisms that control NBL cell differentiation but will also directly provide a group of novel druggable targets for developing new differentiation agents.
