Genomic sequencing of thousands of tumors has revealed many genes associated with specific types of cancer. Similarly, large scale CRISPR functional genomics efforts have mapped genes required for cancer cell proliferation or survival in hundreds of cell lines. Despite this, for specific disease subtypes, such as metastatic prostate cancer, there are likely a number of undiscovered tumor specific driver genes that may represent potential drug targets. To identify such genetic dependencies, we performed genome-scale CRISPRi screens in metastatic prostate cancer models. We then created a pipeline in which we integrated pan-cancer functional genomics data with our metastatic prostate cancer functional and clinical genomics data to identify genes that can drive aggressive prostate cancer phenotypes. Our integrative analysis of these data reveals known prostate cancer specific driver genes, such as AR and HOXB13, as well as a number of top hits that are poorly characterized. In this study we highlight the strength of an integrated clinical and functional genomics pipeline and focus on two top hit genes, KIF4A and WDR62. We demonstrate that both KIF4A and WDR62 drive aggressive prostate cancer phenotypes in vitro and in vivo in multiple models, irrespective of AR-status, and are also associated with poor patient outcome.

Metastatic prostate cancer remains a major clinical challenge and metastatic lesions are highly heterogeneous and difficult to biopsy. Liquid biopsy provides opportunities to gain insights into the underlying biology. Here, using the highly sensitive enrichment-based sequencing technology, we provide analysis of 60 and 175 plasma DNA methylomes from patients with localized and metastatic prostate cancer, respectively. We show that the cell-free DNA methylome can capture variations beyond the tumor. A global hypermethylation in metastatic samples is observed, coupled with hypomethylation in the pericentromeric regions. Hypermethylation at the promoter of a glucocorticoid receptor gene NR3C1 is associated with a decreased immune signature. The cell-free DNA methylome is reflective of clinical outcomes and can distinguish different disease types with 0.989 prediction accuracy. Finally, we show the ability of predicting copy number alterations from the data, providing opportunities for joint genetic and epigenetic analysis on limited biological samples.

Prostate cancer (PCa) risk-associated SNPs are enriched in noncoding cis-regulatory elements (rCREs), yet their modi operandi and clinical impact remain elusive. Here, we perform CRISPRi screens of 260 rCREs in PCa cell lines. We find that rCREs harboring high risk SNPs are more essential for cell proliferation and H3K27ac occupancy is a strong indicator of essentiality. We also show that cell-line-specific essential rCREs are enriched in the 8q24.21 region, with the rs11986220-containing rCRE regulating MYC and PVT1 expression, cell proliferation and tumorigenesis in a cell-line-specific manner, depending on DNA methylation-orchestrated occupancy of a CTCF binding site in between this rCRE and the MYC promoter. We demonstrate that CTCF deposition at this site as measured by DNA methylation level is highly variable in prostate specimens, and observe the MYC eQTL in the 8q24.21 locus in individuals with low CTCF binding. Together our findings highlight a causal mechanism synergistically driven by a risk SNP and DNA methylation-mediated 3D genome architecture, advocating for the integration of genetics and epigenetics in assessing risks conferred by genetic predispositions.

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.