Abstract Neuroblastoma (NB) is a pediatric cancer that originates from precursor cells of the sympathetic nervous system. Despite advances in treatment and disease management, the overall 5-year survival rates remain poor, depending on patient age, histopathology and genetic subtype. Low expression of miR-497 was identified as being significantly (p<0.015) associated with poor event free and overall NB patient survival, indicating a potential tumor suppressor role (Bray et al., 2009). Furthermore, down-regulation of miR-497 has been demonstrated in a number of multi-drug resistant cancer cell lines. Here we present our findings on the functional effects of exogenous miR-497 expression in NB cell lines, and we identify WEE1 as a direct target of miR-497. Ectopic over-expression of miR-497 resulted in significantly decreased cell viability for MYCN amplified (MNA) Kelly cells (p<0.04) and NB1691 cells (p<0.03) (MTS assay), indicating that this miRNA must be targeting mRNA sequences from gene(s) with significant anti-proliferative effects. We examined the list of putative target genes generated by the TargetScan miRNA Target prediction algorithm and determined that, WEE1, a tyrosine kinase with a key role as an inhibitory regulator of the G2/M checkpoint, is a predicted target of miR-497. Expression analysis from our cohort of NB patients (n=47) identified that higher expression of WEE1 was significantly associated with poor overall survival and event free survival (p<0.03). In silico analysis (web based R2 microarray analysis platform from the Academic Medical Center (www.r2.humangenetics-amc.nl)) from a large independent data set (n=283) supports this association of WEE1 with less favourable outcome in NB (p<0.0001). miR-497 expression was found to be inversely correlated with WEE1 in the tumor cohort, consistent with direct targeting. Ectopic over-expression of miR-497 did not affect WEE1 mRNA levels but resulted in reduced WEE1 protein levels in both Kelly and NB1691 cells. Direct targeting of the 3′UTR of WEE1 by miR-497 was validated using luciferase reporter assays (p<0.002). In order to elucidate the mechanism of miR-497 down-regulation in poor prognosis tumors, we examined methylation DNA immunoprecipitation (MeDIP) data from 15 primary NB tumors and identified a hypermethylated site 166 bp up-stream of the miR-497. There was a significant inverse correlation between methylation at this site and levels of miR-497 expression. The miRNA methylation status was validated using bisulphite sequencing, and we also determined that miR-497 expression could be significantly increased following treatment of NB cell lines with the de-methylating agent 5-aza-cytidine. We conclude that miR-497 is down-regulated in neuroblastoma by hypermethylation and that functional studies indicate a tumor suppressive function for this miRNA, potentially through direct targeting of the G2/M checkpoint tyrosine kinase WEE1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2289. doi:1538-7445.AM2012-2289