Significance: Neuroblastoma is the most common extra cranial solid tumor in childhood. DOT1L is the only known histone methyltransferase that induces histone H3K79 methylation. We aimed to investigate whether DOT1L plays an important role in neuroblastoma.
Methods: siRNAs transfections were used to knockdown DOT1L and N-Myc expression in two neuroblastoma cell lines, BE2C and Kelly. Gene and protein expression was analysed by real-time RT-PCR and immunoblot. Chromatin immunoprecipitation assays were employed to examine N-Myc protein binding to the DOT1L gene promoter region. Affymetrix microarray was utilised to identify DOT1L target genes. Alamar blue assays were used to investigate the role of DOT1L on cell proliferation. Human neuroblastoma tissue microarray datasets were examined to evaluate the prognostic value of DOT1L gene expression.
Results: Knocking-down N-Myc expression reduced DOT1L mRNA and protein expression in MYCN oncogene-amplified neuroblastoma cells, BE2C and Kelly. Forced over-expression of N-Myc up-regulated DOT1L expression in MYCN non-amplified neuroblastoma cells, and chromatin immunoprecipitation assays showed that N-Myc bound to the DOT1L gene promoter. Affymetrix microarray revealed that E2F2 and ODC1 expression was significantly reduced by DOT1L siRNAs. Alamar blue assays showed that knocking-down DOT1L or E2F2 expression reduced neuroblastoma cell proliferation. A high level of DOT1L gene expression in human neuroblastoma tissues predicted poor patient survival independent of patient age, sex, disease stage and MYCN oncogene amplification status in 476 neuroblastoma patients.
Conclusions: Our data suggest that N-Myc up-regulates DOT1L gene expression by binding to its gene promoter, and that DOT1L induces neuroblastoma cell proliferation at least partly through regulating the expression of E2F2 and ODC1. Moreover, high levels of DOT1L expression in human neuroblastoma tissues independently predict poor prognosis, showing that DOT1L is a potential therapeutic target in neuroblastoma patients.