MYCN and HIF-1 directly regulate TET1 expression to control 5-hmC gains and enhance neuroblastoma cell migration in hypoxia

Ten-Eleven-Translocation 5-methylcytosine dioxygenases 1-3 (TET1-3) convert 5-methylcytosine to 5-hydroxymethylcytosine (5-hmC), using oxygen as a co-substrate. Contrary to expectations, hypoxia induces 5-hmC gains in MYCN-amplified neuroblastoma (NB) cells via upregulation of TET1. Here, we show that MYCN directly controls TET1 expression in normoxia, and in hypoxia, HIF-1 augments TET1 expression and TET1 protein stability. Through gene-editing, we identify two MYCN and HIF-1 binding sites within TET1 that regulate gene expression. Bioinformatic analyses of 5-hmC distribution and RNA-sequencing data from hypoxic cells implicate hypoxia-regulated genes important for cell migration, including CXCR4. We show that hypoxic cells lacking the two MYCN/HIF-1 binding sites within TET1 migrate slower than controls. Treatment of MYCN-amplified NB cells with a CXCR4 antagonist results in slower migration under hypoxic conditions, suggesting that inclusion of a CXCR4 antagonist into NB treatment regimens could be beneficial for children with MYCN-amplified NBs. In MYCN-amplified neuroblastoma cell lines, MYCN directly controls TET1 expression in normoxia. In MYCN-amplified neuroblastoma cell lines exposed to hypoxia, HIF-1 augments TET1 expression and TET1 protein stability. Hypoxic MYCN-amplified neuroblastoma cell lines have increased cell migration, mediated by genes including CXCR4 that gain 5-hydroxymethylcytosine density. Treatment of MYCN-amplified NB cells with a CXCR4 antagonist slows hypoxia-associated migration, suggesting a CXCR4 antagonist could be beneficial in treatment regimens for children with MYCN-amplified neuroblastomas.