Analysis of DNA methylation is a valuable tool to understand disease progression and is increasingly being used to create diagnostic and prognostic clinical biomarkers. While conversion of cytosine to 5-methylcytosine (5mC) commonly results in transcriptional repression, further conversion to 5-hydroxymethylcytosine (5hmC) is associated with transcriptional activation. Here we perform the first study integrating whole-genome 5hmC with DNA, 5mC, and transcriptome sequencing in clinical samples of benign, localized, and advanced prostate cancer. 5hmC is shown to mark activation of cancer drivers and downstream targets. Furthermore, 5hmC sequencing revealed profoundly altered cell states throughout the disease course, characterized by increased proliferation, oncogenic signaling, dedifferentiation, and lineage plasticity to neuroendocrine and gastrointestinal lineages. Finally, 5hmC sequencing of cell-free DNA from patients with metastatic disease proved useful as a prognostic biomarker able to identify an aggressive subtype of prostate cancer using the genes TOP2A and EZH2, previously only detectable by transcriptomic analysis of solid tumor biopsies. Overall, these findings reveal that 5hmC marks epigenomic activation in prostate cancer and identify hallmarks of prostate cancer progression with potential as biomarkers of aggressive disease.

Significance

In prostate cancer, 5-hydroxymethylcytosine delineates oncogene activation and stage-specific cell states and can be analyzed in liquid biopsies to detect cancer phenotypes. See related article by Wu and Attard, p. 3880.

Although DNA methylation is a key regulator of gene expression, the comprehensive methylation landscape of metastatic cancer has never been defined. Through whole-genome bisulfite sequencing paired with deep whole-genome and transcriptome sequencing of 100 castration-resistant prostate metastases, we discovered alterations affecting driver genes that were detectable only with integrated whole-genome approaches. Notably, we observed that 22% of tumors exhibited a novel epigenomic subtype associated with hypermethylation and somatic mutations in TET2, DNMT3B, IDH1 and BRAF. We also identified intergenic regions where methylation is associated with RNA expression of the oncogenic driver genes AR, MYC and ERG. Finally, we showed that differential methylation during progression preferentially occurs at somatic mutational hotspots and putative regulatory regions. This study is a large integrated study of whole-genome, whole-methylome and whole-transcriptome sequencing in metastatic cancer that provides a comprehensive overview of the important regulatory role of methylation in metastatic castration-resistant prostate cancer.