Abstract PR16: Whole-genome analysis of the epigenetic mark 5-hydroxymethylcytosine reveals differential profiles in low- , intermediate- , and high-risk neuroblastomas
Authors: Mark A. Applebaum, Erin Barr, Ji Nie, Sakshi Uppal, Wei Zhang, Alexandre Chlenski, Helen Salwen, Marija Dobratic, Barbara Stranger, Lucy Godley, Chuan He, Susan L. Cohn
Journal: Cancer Research (2018)

Abstract Background: Neuroblastoma is characterized by a paucity of somatic mutations, suggesting epigenetic modifications likely play a key role in regulating gene expression driving tumor phenotype. 5-Methylcytosine is an epigenetic DNA modification that decreases transcription and silences several neuroblastoma tumor suppressors. New technology allows evaluation of genome-wide 5-hydroxymethylcytosine (5hmC), a marker of activated transcription. We hypothesize that linking whole-genome 5hmC profiling with tumor transcriptomes will identify epigenetically regulated oncogenic drivers of neuroblastoma phenotype, and may ultimately lead to the discovery of new therapeutic targets. Methods: 5hmC was quantified by nano-hmC-Seal-Seq using 100ng of DNA from locally banked diagnostic neuroblastoma tumors. 50bp sequencing reads underwent quality control with Trimmomatic and were aligned to hg19 with Bowtie2. Homer software quantified peaks by genomic feature. Raw fragment counts were called using featureCounts and normalized with DeSeq2, which also identified genes with differential 5hmC. Gene Ontology (GO) pathway analysis of differential 5hmC genes was performed with the goseq package in R. 5hmC patterns were compared to gene expression in the E-MTAB-1781 cohort from ArrayExpress. Results: 53 patients (27 low- , 12 intermediate- , and 14 high-risk) were evaluated. Low-risk tumors had increased 5hmC peaks per sample compared to high-risk tumors (140,062 vs. 79,727, p=0.014). Peaks were predominantly intronic in all risk groups. Global 5hmC profiles distinguished four groups of patients, low- , intermediate- , MYCN-amplified high-risk, and MYCN-nonamplified high-risk. After controlling for sex, batch, and MYCN-amplification status, 578 genes had differential 5hmC peaks between low- and high-risk tumors (padjusted&amp;lt;0.05). Genes with increased 5hmC in low-risk tumors were enriched for GO pathways of neuronal differentiation, including cell morphogenesis involved in differentiation (p=9.72e-6), neuron projection morphogenesis (p=1.15e-5), and cell projection morphogenesis (p=1.89e-5). High-risk tumor genes were enriched genes in embryonal GO pathways, including anatomic structure formation involved in morphogenesis (p=9.72e-6), chordate embryonic development (p=1.15e-5), and embryo development (p=1.89e-5). Of the 578 genes with differential 5hmC, 149 were differentially expressed genes between 367 low-risk and 254 high-risk patients (poverlap&amp;lt;1e-5) and 158 were significantly differentially expressed between survivors and deceased patients (poverlap&amp;lt;1e-5). These 149 and 158 genes were enriched for neuronal pathways. MYCN-amplified high-risk tumors have divergent 5hmC patterns with MYCN-nonamplified tumors having decreased 5hmC on chromosomes 1p, 4q, 12q, and 17p. Conclusions: We demonstrate that 5hmC profiles are able to different neuroblastoma risk groups and highlight cellular mechanisms important for tumor growth. These results suggest that neuroblastoma epigenetics play a contributory role to gene expression and tumor phenotype. Further analysis is ongoing to identify biologic drivers of these differences, which may be used to identify new therapeutic approaches. Citation Format: Mark A. Applebaum, Erin Barr, Ji Nie, Sakshi Uppal, Wei Zhang, Alexandre Chlenski, Helen Salwen, Marija Dobratic, Barbara Stranger, Lucy Godley, Chuan He, Susan L. Cohn. Whole-genome analysis of the epigenetic mark 5-hydroxymethylcytosine reveals differential profiles in low- , intermediate- , and high-risk neuroblastomas [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr PR16.