Defining the Role of Glutamine Anaplerosis in High Risk MYCN-Driven Neuroblastoma

Background

MYCN amplification is a prognostic marker of tumor aggression and poor outcome in neuroblastoma patients. Neuroblastoma cells with MYCN-amplification are addicted to glutamine and they utilize it to fuel the TCA cycle (glutamine anaplerosis). Our preliminary data demonstrates that the expression of two genes, DLST and IDH2, are highly predictive of overall survival in neuroblastoma patients. These two genes are critical mediators of glutamine intake in the TCA cycle. We have demonstrated that Dlst is required during MYCN-driven neuroblastoma tumorigenesis, as MYCN;dlst+/- fish exhibit a significant delay in tumor onset.

Project Goal

To understand the cellular mechanisms responsible for delay in tumor onset, we will examine the differentiation, proliferation and apoptotic status of MYCN;dlst+/- and MYCN tumors (Goal 1). Utilizing markers of neural crest differentiation (Huc, TH, etc), we will use immunofluorescence to determine the differentiation status of MYCN;dlst+/- and MYCN tumors. Inactivation of Dlst in MYCN-driven tumors may result in the induction of apoptosis or slowing of the cell cycle, therefore we will use immunofluorescence to examine the expression of cleaved caspase 3 and phoso-histone 3 in MYCN;dlst+/- and MYCN tumors. Next, we will determine whether heterozygous-loss of idh2 disrupts MYCN-driven tumor onset or progression (Goal 2). Our MYCN fish will be bred to idh2+/- fish and their progeny will be monitored by fluorescent microscopy for tumor development. Fish will be genotyped by PCR and segregated according to MYCN and idh2 allele status. Statistical analysis will be performed to determine if idh2-loss disrupts either tumor onset or progression.