Project Summary Neuroblastoma (NB) is an often fatal form of childhood cancer caused by aberrant development of the sympathetic nervous system (1). The majority of patients have widely disseminated disease at diagnosis, and despite intense multi-modal therapy, the survival rate for high-risk NB remains less than 50%, and relapsed NB is almost universally incurable. There is an urgent and unmet need to improve the outcomes of children with NB, which constitutes our long-term goal. Recently, antibody-based immunotherapeutic targeting of GD2 in NB showed significant survival advantages in a phase III trial, and is now being administered to help prevent disease recurrence (2). However, identification of ideal anticancer agents that specifically target tumor cells and minimize adverse effects resulting from injury to healthy cells remains a significant challenge in solid tumors, including NB where GD2 also expressed on pain fibers. Our objective here is to identify and deliver new immunotherapeutic targets for high-risk and/or relapsed NB. Our central hypothesis is that an integrative proteogenomic strategy utilizing highly annotated patient-derived NB samples coupled with rigorous experimental validation will define clinically actionable immunotherapeutic targets with a large therapeutic index. Our initial studies have identified DLL3, CNTN1, GFRA2, UNC5C and ADAM22 as proteins preferentially expressed on the cell surface of NB. We will build on these data, and test our central hypothesis in specific aims: (1) Define the cell surface protein landscape of high-risk and relapsed NB using DNA/RNA-sequencing and mass spectrometry (MS). Specifically, we will profile 10 NB patient-derived cell lines and 20 NB tumors (10 patient derived xenografts, 5 primary and 5 relapse patient tumors) by MS. Computational integration of proteomic and transcriptomic with UniProt annotations have allowed us to filter lowly expressed proteins and those lacking an extracellular domain. In addition, RNA-sequencing data (2,242 NB primary and relapsed tumors and 7,859 normal tissues) allows us to ensure no, or minimal, expression in normal tissues and prioritize candidate targets for further study. (2) Validate and assess biological relevance of candidate immunotherapeutic targets. Here, we will verify cell surface expression of candidate targets (see preliminary data) using a combination of western blot, immunofluorescence (IF), flow cytometry and immunohistochemistry (IHC). We will validate cell surface expression in a panel of normal tissues and NB samples by staining tissue microarrays (TMA) in collaboration with the CHOP Pathology Core. To determine the biological relevance, we will perform in vitro studies following genetic manipulation of candidate genes including CNTN1. This work...