Hypoxia Promotes an Adrenergic to Mesenchymal Transcriptional Program Transition in Neuroblastoma

Adrenergic (ADRN) neuroblastoma cells have a paradoxical increase in TET1 expression and 5-hydroxymethylcytosine (5-hmC) deposition in hypoxia. However, the effect of hypoxia on the mesenchymal (MES) lineage remains less well understood. To identify lineage specific hypoxia responses, we evaluated 5-hmC and RNA expression in a panel of ADRN and MES cells cultured in normoxic or hypoxic conditions. In hypoxia, MES cells had only a modestly elevated expression of hypoxia signaling and no phenotypic changes. In contrast, ADRN cells showed increased 5-hmC deposition and decreased mitochondrial function in hypoxia. In hypoxia, only ADRN cells had increased expression of MES signature genes (p < 0.05) and related pathways including TNF-α signaling. Publicly available RNA-Seq data of diagnostic tumors was accessed (GSE62564 and GSE73517). Each tumor was assigned a hypoxia score using a published hypoxia signature. A MES/hypoxia signature comprising 94 MES signature genes that had increased expression in ADRN cells exposed to hypoxia was also generated. This MES/hypoxia signature was significantly correlated with a T cell infiltration signature (r = 0.66, p = 2.3 × 10^-12). Kaplan-Meier analysis and the log rank test assessed overall survival (OS) differences between groups. High-risk patients with hypoxic tumors that had high MES/hypoxia scores had significantly improved 3-year OS compared to those with low MES/hypoxia scores (71% vs. 40%; p = 0.0054). Superior survival was also observed in high-risk patients with hypoxic tumors that had high MES/hypoxia scores in the validation cohort (GSE73517; n = 28; 3-year OS 78% vs. 26%, p = 0.0012). Our results suggest that hypoxia changes expression of genes in ADRN cells towards MES cells.