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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. It is hypothesized that there are a number of tumor specific driver genes for metastatic prostate cancer. Here, the authors perform genome-wide CRISPRi screens and integrate these data with metastatic prostate cancer functional and clinical genomics data to show that KIF4A and WDR62 drive aggressive prostate cancer phenotypes.

The impact of variations in the three-dimensional structure of the genome has been recognized, but solid cancer tissue studies are limited. Here, we performed integrated deep Hi-C sequencing with matched whole-genome sequencing, whole-genome bisulfite sequencing, 5-hydroxymethylcytosine (5hmC) sequencing and RNA sequencing across a cohort of 80 biopsy samples from patients with metastatic castration-resistant prostate cancer. Dramatic differences were present in gene expression, 5-methylcytosine/5hmC methylation and in structural variation versus mutation rate between A and B (open and closed) chromatin compartments. A subset of tumors exhibited depleted regional chromatin contacts at the AR locus, linked to extrachromosomal circular DNA (ecDNA) and worse response to AR signaling inhibitors. We also identified topological subtypes associated with stark differences in methylation structure, gene expression and prognosis. Our data suggested that DNA interactions may predispose to structural variant formation, exemplified by the recurrent TMPRSS2 – ERG fusion. This comprehensive integrated sequencing effort represents a unique clinical tumor resource. A multiomic approach profiles the three-dimensional, epigenetic and mutational landscapes of 80 metastatic prostate cancer biopsies. Hi-C experiments identify an extrachromosomal circular DNA at the AR locus associated with therapy resistance.

Xenobiotic-induced interstrand DNA–DNA cross-links (ICL) interfere with transcription and replication and can be converted to toxic DNA double strand breaks. In this work, we investigated cellular responses to 1,4-bis-(guan-7-yl)-2,3-butanediol (bis-N7G-BD) cross-links induced by 1,2,3,4-diepoxybutane (DEB). High pressure liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ESI+-MS/MS) assays were used to quantify the formation and repair of bis-N7G-BD cross-links in wild-type Chinese hamster lung fibroblasts (V79) and the corresponding isogenic clones V-H1 and V-H4, deficient in the XPD and FANCA genes, respectively. Both V-H1 and V-H4 cells exhibited enhanced sensitivity to DEB-induced cell death and elevated bis-N7G-BD cross-links. However, relatively modest increases of bis-N7G-BD adduct levels in V-H4 clones did not correlate with their hypersensitivity to DEB. Further, bis-N7G-BD levels were not elevated in DEB-treated human clones with defects in the XPA or FANCD2 genes. Comet assays and γ-H2AX focus analyses conducted with hamster cells revealed that ICL removal was associated with chromosomal double strand break formation, and that these breaks persisted in V-H4 cells as compared to control cells. Our findings suggest that ICL repair in cells with defects in the Fanconi anemia repair pathway is associated with aberrant re-joining of repair-induced double strand breaks, potentially resulting in lethal chromosome rearrangements.

Despite extensive investigation, the factors promoting aggressive prostate cancer are poorly understood. By performing a comprehensive analysis of whole-genome transcriptome data to identify differential expression across 1,567 patients with prostate cancer, we now report the identification of a novel lncRNA, Prostate Locus of Uncharacterized Transcript Outlier 201 (PLUTO-201), which is strongly associated with metastasis and poor overall survival in men with prostate cancer. We find that overexpression/knockdown of PLUTO-201 in pre-clinical models of prostate cancer modulates proliferation rates and markers of an aggressive phenotype through regulation of steroid biosynthesis and expression of the MHC class I complex, driving increased growth in androgen-depleted conditions and decreased susceptibility to T cell-mediated cytotoxicity. We further find that the heterogeneous nuclear ribonucleoprotein hnRNPK directly binds PLUTO-201 and is indispensable for its activity. Overall, our findings indicate that PLUTO-201 is a driver of aggressive prostate cancer phenotypes and poor clinical outcomes. Identification and characterization of PLUTO-201, a novel lncRNA driving aggressive biology in prostate cancer, sheds new light on the mechanisms driving aggressive prostate cancer and will motivate therapeutic and biomarker development. The factors promoting prostate cancer progression and metastasis are poorly understood, resulting in a lack of therapeutic targets and prognostic biomarkers for this disease. Here, we have identified the novel long non-coding RNA (lncRNA) PLUTO-201 as strongly associated with prostate cancer progression and metastasis in patients with localized prostate cancer undergoing prostatectomy. We show that PLUTO-201 promotes proliferation, invasion, and metastasis in multiple prostate cancer models both and . Mechanistically, we find that PLUTO-201 downregulates MHC class 1 and upregulates steroid biosynthesis by interacting with the heterogeneous nuclear ribonucleoprotein K (hnRNPK), leading to decreased T cell-mediated cytotoxicity and increased resistance to androgen receptor inhibition. Altogether, this study provides strong evidence for a critical role of PLUTO-201 in prostate cancer progression and metastasis, and a rationale for further exploration of PLUTO-201 as a therapeutic target and prognostic biomarker for patients with prostate cancer.

The androgen receptor (AR) is a critical driver of prostate cancer (PCa). To study regulators of AR protein levels and oncogenic activity, we created the first live cell quantitative endogenous AR fluorescent reporters. Leveraging this novel AR reporter, we performed genome-scale CRISPRi flow cytometry sorting screens to systematically identify genes that modulate AR protein levels. We identified and validated known AR protein regulators including HOXB13 and GATA2 and also unexpected top hits including PTGES3, a poorly characterized gene in PCa. PTGES3 repression resulted in loss of AR protein, cell cycle arrest, and cell death in AR-driven PCa models. PTGES3 is not a commonly essential gene, and our data nominate it as a prime PCa therapeutic target. Clinically, analysis of PCa data demonstrate that PTGES3 expression is associated with AR-directed therapy resistance. Mechanistically, we show PTGES3 binds directly to AR, forms a protein complex with AR in the nucleus, regulates AR protein stability and and modulates AR function in the nucleus at AR target genes. PTGES3 represents a novel therapeutic target for overcoming known mechanisms of resistance to existing AR-directed therapies in PCa.

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. Whole-genome bisulfite sequencing along with whole-genome and transcriptome sequencing of 100 prostate cancer metastases identifies genomic regions that are differentially methylated during disease progression and a novel epigenomic subtype.

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 only detectable with integrated whole-genome approaches. Notably, we observed that 22% of tumors exhibited a novel epigenomic subtype associated with hyper-methylation 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 and provides a comprehensive overview of the important regulatory role of methylation in metastatic castration-resistant prostate cancer.