Neuroblastoma is a common and lethal tumor of childhood. Amplification of MYCN occurs commonly in high-risk disease. We generated a model for primary neuroblastoma by directing expression of a MYCN transgene to the murine peripheral neural crest. Over the past 4y, we validated and established mice transgenic for TH-MYCN as a platform for developmental therapeutics, demonstrating that MYCN protein plays a central role in neuroblastoma, that signaling through PI3K critically stabilizes MYCN, that clinica inhibitors of PI3K destabilize MYCN and improve survival in mice transgenic for TH-MYCN, and that a clinical PI3K inhibitor drives degradation of Mycn cells in-vivo, with secondary paracrine vascular blockade. Our observations, that PI3K signaling stabilizes Mycn protein and that Mycn in tumor cells contributes prominently to the tumor microenvironment, raise new questions. In a tumor with few prominent kinases, how is PI3K activated? How do activation of ALK and loss of NF1 (which contribute to progression in neuroblastoma) influence response to PI3K inhibition? Can we identify translatable combination therapies that promote durable responses? How do these therapies affect the tumor microenvironment? In this application, we will evaluate candidate MYCN-driven miRNAs as activators of PI3K in neuroblastoma. We will generate TH-MYCN mice mutant at Nf1 or Alk, and determine how these genetic abnormalities influence response to PI3K/mTOR inhibitors. We will also identify targeted therapies that cooperate with inhibition of PI3K to induce cytotoxicity, a translatable approach to promote durable responses. The specific aims of this application are: Aim1. To evaluate miR-8208;17-8208;92 as a Mycn target that blocks PTEN, activates PI3K, promoting an autocrine loop which subsequently stabilizes MYCN in neuroblastoma. Aim 2. To evaluate how loss of NF1 and activation of ALK influence response to PI3K/mTOR inhibitors in MYCN-driven neuroblastoma, Aim 3. To identify combination therapies which cooperate with inhibitors of PI3K to drive apoptosis resulting in durable remissions in neuroblastoma. Successful completion of this proposal establishes a mechanistic basis for PI3K activation in neuroblastoma, and identifies new miR targets for therapy, extends the utility of mice transgenic for TH-MYCN, adds new GEM mouse models as platforms for personalized medicine in neuroblastoma, and provides a preclinical rationale to test effective combination therapies in clinical trials, in children with neuroblastoma.