Abstract 3808: MYCN coordinately regulates the entire polyamine gene pathway to drive polyamine biosynthesis in neuroblastoma, with expression of every synthetic and catabolic polyamine pathway gene being highly prognostic of clinical outcome

Background: Polyamines are highly regulated essential cations that are elevated in rapidly proliferating tissues. We have previously shown that the MYC/MYCN target ornithine decarboxylase (ODC1), rate-limiting for polyamine biosynthesis, is a therapeutic target for neuroblastoma (Hogarty et al, Cancer Res 2008; 68: 9735). We have also shown that targeting multiple steps in the polyamine pathway enhances therapeutic efficacy in complementary pre-clinical models (Haber et al, Adv Neurobl Res, 2012, OR 88). An international Phase I trial for refractory neuroblastoma, coordinated by the New Approaches to Neuroblastoma Treatment (NANT) consortium USA, will open shortly using ODC1 inhibition by high-dose difluoromethylornithine and SAT1 induction by celecoxib. We have now examined the prognostic impact of all polyamine pathway genes in a large cohort of neuroblastoma tumors. Methods: Gene-expression profiles of 650 primary untreated neuroblastomas were analyzed for all polyamine pathway genes, and related to clinical outcome. Gene expression was evaluated by Real-Time PCR (RT-PCR) and correlated with MYCN levels in neuroblastoma cell lines. Activation and repression of polyamine genes by MYCN were analyzed by chromatin immunoprecipitation (ChIP). Polyamine gene promoters were cloned into luciferase reporter vectors and tested as a function of MYCN expression. Results: Expression of all polyamine pathway genes was highly prognostic of neuroblastoma outcome. High levels of each synthetic gene and low levels of each catabolic gene predicted poor outcome. Multivariate analysis showed 6/11 genes retained independent prognostic significance following adjustment for MYCN, age and stage. In neuroblastoma cell lines, in the presence or absence of MYCN expression, RT-PCR analysis demonstrated a correlation of high MYCN with high biosynthetic polyamine gene expression, but an inverse correlation of high MYCN with low catabolic polyamine gene expression. ChIP assays confirmed that MYCN associates with biosynthetic gene promoters by binding to E-box sites in order to activate transcription. In contrast, MYCN binds the promoters of the catabolic genes in proximity to the transcription start sites by interacting with the Sp1 protein in order to repress transcription of these genes. Direct activation by MYCN of biosynthetic genes and down-regulation by MYCN of catabolic genes was confirmed by luciferase activity assays. Conclusions: These results provide a remarkable demonstration of an oncogene coordinately regulating the expression of every gene in a metabolic pathway in order to drive cell proliferation. The findings highlight the importance of polyamines in neuroblastoma and identify target enzymes in addition to ODC1 for potential therapeutic intervention. Citation Format: Murray D. Norris, Laura D. Gamble, Jayne Murray, Stefania Purgato, Giorgio Milazzo, Lesley J. Ashton, Andrei Oberthuer, Wendy B. London, Mike D. Hogarty, Mattias Fischer, David Ziegler, Glenn M. Marshall, Giovanni Perini, Michelle Haber. MYCN coordinately regulates the entire polyamine gene pathway to drive polyamine biosynthesis in neuroblastoma, with expression of every synthetic and catabolic polyamine pathway gene being highly prognostic of clinical outcome. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3808. doi:10.1158/1538-7445.AM2013-3808