PROJECT 1 SUMMARY/ABSTRACT Neuroblastoma (NB) remains a leading cause of childhood cancer deaths, and the patients who do survive are left with long-term side effects, many of which can be life threatening. While the paradigm of molecularly targeted therapies holds great promise, genomic studies have revealed that NBs are characterized by extensive intratumor genetic heterogeneity, with subclonal oncogenic drivers often selected for during standard chemoradiotherapy. Our broad hypothesis is that NB therapy resistance is a dynamic and evolving phenomenon that can be serially monitored and rationally treated with targeted small molecules and immunotherapies. Our group discovered gain-of-function mutations in the ALK receptor tyrosine kinase as the etiology for familial NB, and at the same time co-discovered with several other groups identical mutations as the most frequent somatic single nucleotide variants leading to a potent oncogenic driver in up to 25% of newly diagnosed high-risk cases. We have shown that activating mutations in the ALK-RAS-MAPK pathway are highly enriched in the relapse NB genome, providing the impetus for deep and comprehensive characterization of the spatial and temporal heterogeneity underpinning therapy resistance across the continuum of therapy. More recently, we have uncovered epigenetic mechanisms of initial chemotherapy resistance with single cell technologies. Finally, our recent discovery of methods to target non-mutated peptides derived from intracellular oncoproteins with cellular therapies offers a new class of therapeutics to target key molecular drivers of NB cellular persistence. The work proposed in this renewal application will address the challenges of intra-tumoral heterogeneity and persistent NB cells that survive therapy and constitute a major cause of treatment failure. We will test our central hypothesis with three Specific Aims: 1) Identify clonal and subclonal mutations in ctDNA and their evolution during therapy; 2) Develop therapies for patients with ATRX mutant/ALT-driven indolent NB; and 3) Discover therapeutically targetable mechanisms of NB cellular persistence. We will leverage circulating tumor DNA and single cell RNA and ATAC sequencing technologies, as well as paradigm-shifting recent advancements in developing chimeric antigen receptors that precisely target peptides presented on the major histocompatibility complex that have the potential to vastly expand the pool of immunotherapeutic targets and broaden the population of patients who would benefit from such therapy. In parallel, we will develop EZH2 inhibition therapy in combination with immunotherapy for patients with indolent NB, and develop new therapies targeting the NF-κB and other pathways mediating NB cellular persistence. We consider this project significant because it will result in new mechanism- based biomarker-defined therapeutic strategies that ultimately should significantly improve high-risk NB patient outcomes. This will ...