Abstract A025: Assessing the role of bivalency in MYCN-amplified neuroblastoma

Abstract Introduction: Neuroblastoma is an embryonic pediatric cancer with a broad spectrum of clinical behavior. In aggressive neuroblastoma tumors, we previously demonstrated enrichment of 5-hydroxymethylcytosine (5-hmC), a marker of transcriptional activation, on genes targeted by the repressive Polycomb Repressive Complex 2 (PRC2). PRC2 has been shown to be a dependency of MYCN-amplified neuroblastoma. In embryonic stem cells, 5-hmC is disproportionately enriched at the transcription start sites (TSS) of bivalent genes that are characterized by the presence of both repressive and activating histone modifications (H3K27me3 and H3K4me3). Bivalent genes are postulated to be poised for rapid activation during embryogenesis. We hypothesized that bivalent genes enriched for 5-hmC at the TSS play a key role in regulating the undifferentiated phenotype of MYCN-amplified neuroblastoma. Methods: Tumor derived 5-hmC profiles were obtained previously. Nano-hmC-seal was used to generate genome-wide 5-hmC profiles in the MYCN-amplified or overexpressed SK-N-BE2, LA1-55n, LA1-5s, NBLW-N, NBLW-S, NBL-S, and MYCN-non-amplified SH-SY5Y, and SHEP cell lines. H3K27me3, H3K4me3, and H3K27ac profiles were generated in these cell lines using ChIP-Seq. KAS-Seq, a chemical pulldown of ssDNA that marks active enhancers and transcribed genes, and RNA-Seq profiling were performed. MACS2 and deepTools were used to analyze ChIP-Seq, 5-hmC, and KAS-Seq data. Results: A comparison of 5-hmC profiles derived from diagnostic tumor biopsy samples with and without MYCN-amplification demonstrated increased 5-hmC deposition on SUZ12 and EZH2 (PRC2 subunits) target genes. MYCN-amplified and MYC overexpressing cell lines but not MYCN-non-amplified lines bore a TSS specific association between 5-hmC deposition and H3K27me3 enrichment. Using the SK-N-BE2 and NBLW-N cell lines, we identified 313 genes with both H3K4me3 and H3K27me3 deposition, which we defined as bivalent, and 806 genes with only H3K27me3 deposition. Both H3K27me3-only and bivalent genes had TSS specific 5-hmC deposition, were enriched in development and differentiation pathways, and had significantly lower expression in cell lines with high MYCN expression. However, unlike H3K27me3-only genes, bivalent genes were also enriched for transcription factor biding sites of AP-2 and E2F which co-regulate MYCN. Using a publicly available dataset of 498 tumors, we found that bivalent genes were transcriptionally repressed in MYCN-amplified tumor samples. Within the cohort of 175 high-risk patients, those with lower expression of bivalent genes (N=55) had worse overall survival (OS) from diagnosis than those (N=120) with high expression (5-year OS: 27.2% vs. 52.0%; 95% CI: 17.1%-43.3% vs 42.9%-63.1%, p=0.001 by log-rank test). Conclusion: The low-level expression of bivalent genes enriched for 5-hmC at the TSS in MYCN-amplified neuroblastoma cells may represent a novel mechanism by which dedifferentiated states and aggressive phenotype is maintained in MYCN-amplified neuroblastoma. Citation Format: Mohansrinivas Chennakesavalu, Gepoliano Chaves, Kelley Moore, Tong Wu, Ruitu Lyu, Rachel TerHaar, Alexandre Chlenski, Chuan He, Susan Cohn, Andrea Piunti, Mark Applebaum. Assessing the role of bivalency in MYCN-amplified neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr A025.