Neuroblastoma (NB) is the most common non-CNS paediatric cancer. Fifty-percent of children with high-risk disease succumb to relapse. Alteration in MYCN and ALK genes correlates with aggressive tumour biology, increased tumour vascularisation and relapse. Concurrent ALK point mutations and MYCN gene amplification occur in patients with therapy-resistant tumours and poor clinical survival (15% at 5 years), implying a functional interaction between these two genes that induces an aggressive tumour phenotype. We characterised the first MYCN-driven transgenic model of NB and used it to show that PI3-kinase pathway inhibitors destabilise Mycn protein by altering n-terminal phosphorylation. We have now developed the first ALK-driven NB transgenic model in which tumours arise exclusively in mice doubly-transgenic for MYCN and ALK. Compared to MYCN-driven tumours, MYCN/ALK-driven tumours have increased Mycn protein, reduced neovascularisation and an aggressive phenotype mirroring that of human MYCN/ALK tumours. Since ALK is known to stimulate PI3K-patwhay activity, we hypothesise that ALK potentiates MYCN-driven oncogenesis, in part, through PI3K-driven Mycn protein stabilisation. The three aims of this project are to examine: 1) whether ALK potentiates MYCN-driven NB, by stabilising Mycn protein, 2) whether ALK inhibits MYCN-driven tumour neovascularisation, and 3) whether ALK- and/or Mycn-targeted therapeutics are effective against these tumours. Established human and murine NB cell lines with varying expression of MYCN and phospho-mutated (stabilised) Mycn proteins will be transfected with ALK mutants to assess whether stabilisation of Mycn protein occurs through PI3K-pathway induction and Mycn phosphorylation. I will examine in parallel whether MYCN-driven angiogenesis is inhibited using in vitro angiogenesis assays with which I have extensive prior experience. In vivo tissue from MYCN and MYCN/ALK transgenic tumours will be examined for stabilisation of Mycn through assessment of PI3K pathway expression and ratio of Mycn to pMycn protein. Vessels will be analysed by pathology and expression of major angiogenic signalling proteins will be characterised. MRI will be used to quantitate tumour blood flow and size, response in preclinical studies of Mycn and ALK-targeted therapeutics (BEZ235 and PF2341066). Specifically the ability of PF-2341066 alone or in combination with BEZ235 to regress tumours and destabilise Mycn will be assessed. This project presents a unique combination of reagents and validated approaches that will allow mechanistic dissection of the interaction between MYCN and ALK in NB, and will guide the development of targeted-therapeutics for ultra high-risk NB patients.