Although metabolic adaptation is a cornerstone of the hypoxia response, very little is known about hypoxia-induced metabolic changes in neuroblastoma or how the metabolome influences phenotype. Identifying genes that regulate neuroblastoma metabolism and also influence clinical behavior may provide clues for novel therapeutic targets. Mixed linear models were used to identify differentially expressed genes between neuroblastoma cells grown in hypoxia or normoxia in two independent experiments (GEO accessions GSE17714 and GSE55391). Linear models were also used in to identify differentially expressed genes from tumors of two independent cohorts of neuroblastoma patients (GEO accession GSE16254 and EMBL accession E-MTAB-179) who survived compared to those that did not. We utilized a false discovery rate of 0.01 and fold change in the top or bottom 10% of all genes as a significance cutoff. qPCR was used to validate expression differences in multiple neuroblastoma cell lines. 157 genes, significantly exceeding chance (p < 1x10-6) on permutation testing, were present in both neuroblastoma cell line data sets, all but five of which showed consistent directionality of expression change from normoxia to hypoxia, including 44 involved in metabolism. These genes were enriched for hypoxia and metabolic pathways: HIF-1α transcription factor network (p = 1.3x10-11), glycolysis (p = 1.9x10-8) and fructose metabolism (p = 1.0x10-3). 826 genes, significantly exceeding chance (p < 1x10-6) were differentially expressed in both patient cohorts of 478 and 88 patients. These genes were enriched for cell cycle pathways (p = 4.5x10-7). Of these genes, nine of them were also differentially expressed in hypoxia compared to normoxia with consistent directionality, again more than expected by chance (p < 1x10-6). High expression in eight of the nine genes was also significantly associated with poor outcome in a Kaplan-Meier analysis of both of the patient cohorts evaluated. Three of these genes are part of the glycolytic pathway and three more are directly involved in cellular metabolism. In the SK-N-BE2 cell line, all eight of our identified genes are up-regulated in hypoxia (p < 0.05). Analysis of the LAN-5, La1-55n, SK-N-DZ cell lines and show similar results. MTT assays of proliferation show the expected decreased proliferation for all of these cell lines in 48 hours of hypoxia compared to normoxia (p < 0.05). Analysis of cell cycle by flow cytometry in SK-N-BE2 cells shows an increase of cells in G0/G1 in normoxia compared to hypoxia (58.3% vs. 71.5% p = 0.04) and decrease of cells in S phase (24.5% vs. 13.6%, p = 0.001). We have identified eight genes with increased expression in hypoxia and are associated with poor survival in patients. Efforts to use shRNA to alter cellular phenotype are ongoing. Citation Format: Mark A. Aplebaum, Aashish Jha, Alexandre Chlenski, Christopher Mariani, Clara Kao, Mildred Nelson, Kyle Hernandez, Helen Salwen, Marija Dobratic, Kevin White, Barbara Stranger, Susan L. Cohn. Evaluation of hypoxia adaptation in neuroblastoma identifies reproducible transcriptional and phenotypic responses. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A01.

Background: In cancer, hypoxia leads to a clinically aggressive phenotype by inducing angiogenesis, altering metabolism, and promoting cell infiltration, invasion and metastasis. Hypoxia also results in specific adaptive genetic changes in Drosophila melanogaster and many other species. We hypothesized that an evolutionarily conserved transcriptional program exists in Drosophila, neuroblastoma cell lines, and primary neuroblastoma tumors conferring both a survival advantage in Drosophila bred in hypoxia and aggressive tumor behavior. Methods: RNA-seq data from neuroblastoma cells grown in normoxia or hypoxia were analyzed to identify differentially expressed genes (DEG). These data were compared to previously identified human orthologous DEG from Drospohila bred in normoxia or hypoxia. The analysis was validated using public microarray expression data from 11 additional neuroblastoma cell lines cultured in normal or hypoxic conditions (GEO accession GSE17714). In order to evaluate these genes in patients, gene expression microarray data from 479 primary neuroblastoma tumors (EMBL accession E-MTAB-179) were analyzed with respect to patient survival status. Results: Pathway analysis of genes differentially expressed in Drosophila bred in hypoxia revealed enrichment for genes of the citric acid cycle (p = 13.1x10-17), pyruvate metabolism (p = 5.4x10-10), and glycolysis (p = 4.1x10-7). Analysis of neuroblastoma cells grown in hypoxia identified DEG in steroid biosynthesis (p = 1.3x10-13), HIF-1α transcription network (p = 3.3x10-13), and glycolysis (p = 3.2x10-6). 222 common DEG in neuroblastoma cells and Drosophila were enriched for glycolysis (p = 2.1x10-8), gluconeogenesis (p = 1.2x10-5) and the HIF-1α transcription network (p = 7.2x10-5). Up-regulation of glycolysis genes was also detected in the 11 additional neuroblastoma cell lines cultured in hypoxia. These cell lines showed additional differential expression in Ataxia telangiectasia and Rad3 (ATR) DNA damage sensing genes and general cell cycle related genes (p = 2.9x10-9 and p = 1.5x10-5, respectively). Of the 479 primary tumor samples with median follow up time of 3.75 years, there were 119 stage 1, 80 stage 2, 69 stage 3, 148 stage 4, and 62 stage 4S patients of whom 91 are deceased. In the analysis of primary tumors, decreased expression of cell cycle and metabolism genes (p = 4.8x10-13 and p = 1.6x10-12, respectively) was associated with survival. Thirty-nine of the DEG from patients, including multiple metabolism genes such as PGK1, GPI, and ACSS2, were common for both Drosophila and cell lines exposed to hypoxic conditions. Conclusions: Hypoxia alters metabolism in Drosophila, and neuroblastoma cell lines, and similar changes are associated with clinically aggressive phenotype in primary neuroblastoma tumors. Changes in cell cycle gene expression were also common in neuroblastoma cell lines grown in hypoxia and in clinically aggressive primary tumors. Further analysis of these pathways and their regulation will allow us to identify patients at high risk and provide insight to personalize therapy. Citation Format: Mark A. Aplebaum, Aashish R. Jha, Alexandre Chlenski, Kyle Hernandez, Christopher J. Mariani, Barbara E. Stranger, Susan L. Cohn. Identification of evolutionarily conserved hypoxia-induced genomic pathways responsible for aggressive neuroblastoma phenotypes. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr B1-15.