Neuroblastoma (NBL) is a pediatric solid tumor that arises from deregulated development of the sympathetic nervous system. The five-year survival of patients with high-risk NBL is only 40% and relapse-free survival is extremely rare. This underscores the need to identify biologically-relevant and clinically-actionable targets for children with high-risk disease. One genomic aberration found in high-risk NBL is amplification of a lineage- specific oncogene, MYCN. Once amplified, the MYCN transcription factor can invade enhancers of other lineage oncogenes and establish a core regulatory circuit (CRC), whereby these transcription factors autoregulate themselves and each other to maintain expression of the circuit. Due to the developmental nature of NBL, these tumors display unique cell-surface molecules and may be susceptible to immunotherapy. An antibody-based therapy (dinutuximab) targeting the glycosphingolipid molecule, GD2, extends the 5-year survival rates of patients with aggressive, high-risk NBL. However, GD2 is also found on cells of the peripheral nervous system and treatment with dinutuximab causes agonizing pain. To avoid these on-target / off-tumor side effects, we aim to identify NBL tumor-specific surface molecules that are regulated by the CRC and associated with the undifferentiated, malignant state of NBL. Our preliminary analysis of CRC-bound genes revealed both novel and known immunotherapeutic targets, including ALK and L1CAM. Another CRC-regulated cell-surface protein identified in our analysis is Delta-like canonical notch ligand 3 (DLL3). DLL3 has NBL-specific cell-surface expression, and is known as an inhibitor of Notch signaling in other cell types. Clinical trials with DLL3-targeted immunotherapies in adult cancers are currently ongoing, however, the oncogenic mechanism of DLL3 in these tumor-types, and in NBL, is uncharacterized and poorly understood. In this proposal, we also aim to uncover the relevant biology of DLL3 in NBL in order to credential DLL3 as a viable immunotherapeutic target for this pediatric malignancy. Our preliminary data shows that shRNA-depletion of DLL3 reduces the cellular viability in NBL cell line models with high expression of DLL3. Furthermore, CRISPR-Cas9 genomic depletion of DLL3 results in increased expression of a canonical Notch target gene, HES1. Therefore, we postulate that DLL3 inhibits Notch signaling to promote persistent NBL cell survival, and that the CRC transcriptionally regulates DLL3 to cause overexpression. This project will combine integrative approaches in bioinformatics and molecular biology to 1) describe differentially expressed, cell-surface targets driven by the CRC and 2) determine the role of DLL3 in the malignant state of NBL. We also anticipate that we can apply our CRC computational workflow to other cancers with established transcriptional circuits. This NRSA F31 will provide insight into NBL biology and the regulation of candidate immunotherapeutic targets in NBL, both of which will inform preclinical and clinical studies with targeted immunotherapies.
The research proposed here is relevant to public health because it seeks to identify candidate immunotherapeutic targets that are regulated by the oncogenic core regulatory circuit (CRC) and determine if DLL3, a CRC target, functions as an oncoprotein in the pediatric cancer neuroblastoma. The research is highly relevant to the NIH mission and the urgent unmet need of developing rational evidence-based therapeutic strategies to reduce the burden of cancer as well as in developing young scientists in a rigorous translational academic environment.
