Mentor Name: Adam Wolpaw

Neuroblastoma accounts ~12% of childhood cancer mortality in addition to significant morbidity among survivors. Recent studies have shown that neuroblastomas are composed of cells in distinct lineage states. Adrenergic state cells compose the bulk tumor, while mesenchymal state cells are a smaller subpopulation that is chemoresistant and enriched at relapse. Thus, mesenchymal state cells are at the root of treatment failure and curing neuroblastoma will require approaches to target these cells. No current therapies preferentially target the mesenchymal state. We found that compared with adrenergic state cells, mesenchymal state cells express higher levels of inflammatory sensing proteins, present more antigens, and are more susceptible to killing by NK and T cells. What underlies these differences and how to therapeutically exploit them is unknown. We found that only mesenchymal state cells express STING (Stimulator of Interferon Genes), a component of the pathway that senses cytosolic DNA. STING activates interferon signaling and can promote anti-tumor immunity. We performed a genome-wide CRISPR-Cas9 screen to uncover regulators of STING signaling in mesenchymal state cells. We identified candidate genes that are either required for STING signaling or limit it. Our hypothesis is that by understanding how STING signaling is regulated, we can understand how mesenchymal cells differ from adrenergic cells and how we can better target them with immunotherapy. In the current proposal, we will study how the Nuclear Factor Kappa-B (NF-?B) pathway regulates STING signaling in mesenchymal neuroblastoma cells. NF-?B is classically described as activated by STING, but this does not happen in neuroblastoma cells. However, our screen identified multiple components of the NF-?B pathway that are required for STING signaling, implying that in neuroblastoma NF-?B regulates STING rather than STING regulating NF-?B. NF-?B can be divided into two separate signaling pathways, a canonical and a noncanonical pathway. Based on our screen data, both pathways are required for STING signaling, but the non-canonical is more important. To understand this better, we will individually knockout components of both canonical and non-canonical NF-?B signaling, then measure how this changes translocation into the nucleus of key transcription factors from both NF-?B pathways, how transcriptional NF-?B activity changes, and how STING expression and signaling changes. This will help us understand how to target NF-?B to promote immune detection of mesenchymal cells.