PROJECT SUMMARY/ABSTRACT More effective therapy is needed for children with high-risk neuroblastoma as 5-year survival rates remain less than 50%. The current risk classification is unable to predict which high-risk patients are likely to respond to upfront treatment or relapse. Thus, an improved understanding of the molecular mechanisms important to the growth and survival of neuroblastoma is critical to refining risk stratification and the development of novel therapeutics. Regions of severe hypoxia are known to be present in solid tumors, affecting numerous cellular pathways and increasing resistance to common therapeutics. I have previously identified transcriptional pathways induced by hypoxia that promote malignant behavior in neuroblastoma and are possible therapeutic targets. Hypoxia also induces epigenetic changes including down-regulation of TET1, an enzyme that converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). In contrast, recent studies have shown 5hmC, a marker of activated RNA transcription, is increased by hypoxia in neuroblastoma. My preliminary data show that this unique hypoxia response is present in tumorigenic neuroblastoma cell lines but not in non-tumorigenic cell lines. I hypothesize that up-regulated TET1 and increased 5hmC levels in hypoxia induce transcriptional changes driving clinically aggressive neuroblastoma tumor growth can be used to risk stratify patients and may ultimately lead to the discovery of new therapeutic targets. Specific Aim 1 of my proposal will generate and validate a neuroblasotma risk classifier based on 5hmC-regulated transcriptional profiles and identify potential therapeutic targets regulated by the these hypoxia-induced epigenetic modifications. In Specific Aim 2, I will perform an in depth epigenetic and transcriptional analysis of several phenotypically distinct neuroblastoma cell lines that exhibit heterogeneous TET1 responses to hypoxia. I will then be able to test the functional role of hypoxia-induced, epigenetically regulated genes and pathways to identify novel therapeutic strategies. These experiments will establish preclinical data for further in vivo and clinical studies critical for future R01-level stud- ies to identify drugs that inhibit the epigenetically driven hypoxia-response in neuroblastoma. I propose a four- year career development plan to establish myself as an independent investigator in neuroblastoma cancer genomics. My development plan is comprised of didactics, institutional resources, and supervised research in algorithmic approaches to data clustering, statistical methods, and the epigenetic regulation of cancer. I have assembled a first-class team internationally recognized experts in bioinformatics (Robert Grossman, PhD), epigenetics (Lucy Godley, MD, PhD), neuroblastoma (Susan Cohn, MD), and statistical genetics (Barbara Stranger, PhD) as mentors to oversee this training. With a career development plan which takes advantage of the rich academic environment at the University of Chicago, I will develop into an independent physician- scientist who can bridge the gulf between therapeutics, bioinformatics, and neuroblastoma.

PROJECT NARRATIVE Fewer than half of children with high-risk neuroblastoma are cured with current treatments and neuroblastoma tumors become clinically aggressive and therapy resistant when they outgrow their blood supply and adapt to hypoxia. In neuroblastoma cell lines, hypoxia induces activation of TET1, an enzyme that leads to epigenetic markers of activated RNA transcription. Understanding which critical genes are induced by this unique biology would enable the identification of new markers of aggressive disease in children with neuroblastoma.