Methionine metabolism and NOP2 methyltransferase are essential for MYC-Driven liver tumorigenesis

Hepatocellular carcinoma (HCC) represents the third leading cause of cancer-related death worldwide and has been increasing in recent years in developed nations.1,2 The MYC oncogene or its paralogs are frequently amplified or overexpressed in particularly aggressive subtypes of cancer associated with stem cell-like features and worse clinical outcomes,3,4 including in liver cancer.5 Unfortunately, selective inhibitors that target MYC or its transcriptional program are not yet clinically available for therapy of HCC. Here, we identified methionine metabolism as a selective vulnerability for MYC but not RAS-driven liver cancers. MYC-driven liver cancer cells are methionine dependent and S-adenosylmethionine (SAM), the predominant methyl donor, partially rescues methionine depletion. A low methionine diet, or the methylation inhibitor 5-azacytidine limited MYC-driven tumor formation, but RAS-driven liver cancer was resistant to a low methionine diet. Metabolic tracing of methionine catabolism in MYC high cells identified increased m5C methylation of genomic DNA or ribosomal RNA. We found NOP2, an rRNA m5C-methyltransferase, was regulated by both MYC and methionine. RNA bisulfite seq demonstrated that methionine depletion reduced methylation levels at 28S rRNA C4099, C3438 and C3683. NOP2 knockdown decreased the methylation at C4099, and slightly affected C3438 methylation, suggesting the involvement of NOP2 in methionine dependence. Depletion of NOP2 selectively inhibited MYC liver cancer cell proliferation, tumorigenesis and in vivo tumor growth. Thus, methionine catabolism is critical for MYC-driven liver tumorigenesis and NOP2 may serve as a new therapeutic target in liver cancer. Overall design: RNA bisulfite seq in EC4 cells. MYC high and MYC low EC4 cells were cultured with either 10 µM or 200 µM methionine medium for 48 h, and RNA was isolated for bisulfite sequencing. Four groups (in triplicate), MYC high methionine high, MYC low methionine high, MYC high methionine low, MYC low methionine low. For NOP2 knockdown assay, EC4 cells were treated with a pool of siRNAs (20nM) for 72h, and total RNA were isolated for bisulfite sequencing. Two groups (in triplicate), siNOP2 and siNon-Target.