Project Summary Neuroblastoma is a cancer derived from the developing sympathetic nervous system and is the most commonly diagnosed extracranial solid tumor of childhood. Despite an intensive treatment regimen of chemotherapy, surgery, radiation, and immunotherapy, the five-year survival rate of high-risk neuroblastoma patients remains at only 50%. The low tumor mutational burden of neuroblastoma has challenged the development of targeted- and immuno-therapies, however moderate success has been achieved by targeting GD2 with monoclonal antibody therapy, credentialing immunotherapeutic treatment strategies for neuroblastoma as a promising approach. The success of GD2-targeted therapy to date has been hindered due to debilitating side effects from on-target/off-tumor toxicity since GD2 is also expressed on pain fibers, and antigen loss as a mechanism of therapy resistance. Therefore, there is an unmet need for the discovery of new therapeutic targets in neuroblastoma. B7-H3, encoded by the CD276 gene, is a type 1 transmembrane protein in the B7 family of immunoregulatory proteins and is highly expressed in many adult and pediatric cancers, including neuroblastoma. In addition to being implicated in immunoinhibition, B7-H3 may also mediate tumor migration and metastasis. Preclinical success of several immunotherapeutic strategies directed toward B7-H3, including CAR-T cells and antibody drug conjugates, suggest that B7-H3 is a targetable tumor-associated antigen with several pediatric clinical trials ongoing or planned. Therefore, it is critical to understand the oncogenic functions of B7-H3 and how its expression is regulated to anticipate mechanisms of therapy resistance. Our central hypothesis is that B7-H3 promotes neuroblastoma metastasis and immune evasion, and its expression is regulated by tumor microenvironment-derived cytokines and neuroblastoma-specific transcription factors. Our preliminary data shows that B7-H3 knockdown using CRISPRi in neuroblastoma cell lines inhibits cellular proliferation. Additionally, ChIP-sequencing data identifies regions of MYC and MYCN binding at the B7-H3 promoter indicating a potential role of the MYC transcription factors in regulating B7-H3 expression. B7-H3 expression may also be regulated by inflammatory cytokines, as neuroblastoma cell lines upregulate B7-H3 expression following TNF-α or TGF-β exposure. Finally, recombinant human B7-H3 inhibits T cell activation, TNF-α, and IFN-y. Uncovering how B7-H3 promotes immune evasion in neuroblastoma is crucial given the T and NK cell-based immunotherapies undergoing clinical testing. We propose that B7-H3 is a multifunctional protein that serves as a promising therapeutic target in neuroblastoma. This NRSA F31 will define the mechanisms of neuroblastoma dependance on B7-H3 for metastasis and immune evasion, while also defining mechanisms of overexpression, to inform future B7-H3-targeting therapies and ultimately improve outcomes for patients with high-risk ne...