Abstract 4433: Genome-wide CpG island methylation profiling analysis identifies an epigenetically dysregulated necroptosis mediator in osteosarcoma

BACKGROUND Aberrant CpG island (CGI) methylation with dysregulation of gene expression is a hallmark of cancer. To identify biologically-relevant targets of aberrant epigenetic silencing in osteosarcomas, we analyzed CGI genes exhibiting disproportionate high-frequency gain of methylation. METHODS Infinium HumanMethylation 450 Beadchip profiling quantitated methylation at 485,578 CpG sites in bisulfite-modified DNA of 16 primary human osteosarcomas (OS) samples. Diff scores (D), reflecting difference in prevalence of site-specific methylation for OS samples compared to pooled non-neoplastic controls, were analyzed for gene-associated CGI sites. Genes exhibiting disproportionate high frequency of gain of methylation (+D), were identified utilizing a program to rank order gene CGI loci along an observed/expected (Obs/Exp) hit distribution where “hits” were defined as DAverage > DThreshold. RIPK3 CGI hypermethylation was quantitated by Combined Bisulfite Restriction Analysis (COBRA) and by bisulfite sequencing in 5 OS cell lines (HOS, MNNGHOS, MG-63, G-292, U2OS) and in human mesenchymal cells (hMSC). RIPK3 expression was analyzed by q-RT PCR. To evaluate the effect of RIPK3 expression on cisplatin-associated cytotoxicity, G-292 cells were stably transfected with RIPK3 or the corresponding empty vector (EV). Cisplatin cytotoxicity was assayed in RIPK3 transfectants versus vector-only controls. RESULTS At DTh = 50, >2500/15,000 CGI genes in the OS samples exhibited gain of CGI methylation in excess of what would be expected based on a uniform distribution of methylation (i.e., Obs/Exp > 1). 169 genes exhibited hypermethylation “hits” at all CGI sites assayed (Obs/Exp = 11.95) including the receptor interacting serine-threonine kinase 3 gene (RIPK3). RIPK3 mediates necroptotic cell death and has been associated with cisplatin sensitivity. COBRA analysis of RIPK3 CGI showed extensive methylation in MNNGHOS, MG63 and G292 cells and partial methylation in HOS and U2OS. hMSCs were minimally methylated. COBRA data were congruent with the prevalence of methylation at 18 CpG sites within the CGI determined by bisulfite sequencing. Quantitation of RIPK3 mRNA expression by q-RT PCR confirmed transcriptional silencing in MNNGHOS, MG63, U2OS and G292 cells. G292 stable transfectants expressing RIPK3 were more sensitive (lower LD50) when cultured in cisplatin compared to vector-only controls. CONCLUSIONS Analysis of genome-wide methylation profiling data to identify CGI genes exhibiting a disproportionate gain of methylation in OS identified RIPK3 hypermethylation. Extensive RIPK3 CGI hypermethylation is associated with transcriptional silencing in OS cells. We further show that expression of RIPK3 is associated with enhanced cisplatin cytotoxicity in G-292 cells. These results suggest that methylation of RIPK3 may contribute to cisplatin resistance in OS. Citation Format: Aditya Sharma, Elaine Notis, Christine Mella, Steven J. Kuerbitz. Genome-wide CpG island methylation profiling analysis identifies an epigenetically dysregulated necroptosis mediator in osteosarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4433.