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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.SignificanceIn 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.

We are now in an era of molecular medicine, where specific DNA alterations can be used to identify patients who will respond to specific drugs. However, there are only a handful of clinically used predictive biomarkers in oncology. Herein, we describe an approach utilizing in vitro DNA and RNA sequencing and drug response data to create TreAtment Response Generalized Elastic-neT Signatures (TARGETS). We trained TARGETS drug response models using Elastic-Net regression in the publicly available Genomics of Drug Sensitivity in Cancer (GDSC) database. Models were then validated on additional in-vitro data from the Cancer Cell Line Encyclopedia (CCLE), and on clinical samples from The Cancer Genome Atlas (TCGA) and Stand Up to Cancer/Prostate Cancer Foundation West Coast Prostate Cancer Dream Team (WCDT). First, we demonstrated that all TARGETS models successfully predicted treatment response in the separate in-vitro CCLE treatment response dataset. Next, we evaluated all FDA-approved biomarker-based cancer drug indications in TCGA and demonstrated that TARGETS predictions were concordant with established clinical indications. Finally, we performed independent clinical validation in the WCDT and found that the TARGETS AR signaling inhibitors (ARSI) signature successfully predicted clinical treatment response in metastatic castration-resistant prostate cancer with a statistically significant interaction between the TARGETS score and PSA response (p = 0.0252). TARGETS represents a pan-cancer, platform-independent approach to predict response to oncologic therapies and could be used as a tool to better select patients for existing therapies as well as identify new indications for testing in prospective clinical trials.

Although infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has pleiotropic and systemic effects in some individuals1-3, many others experience milder symptoms. Here, to gain a more comprehensive understanding of the distinction between severe and mild phenotypes in the pathology of coronavirus disease 2019 (COVID-19) and its origins, we performed a whole-blood-preserving single-cell analysis protocol to integrate contributions from all major immune cell types of the blood-including neutrophils, monocytes, platelets, lymphocytes and the contents of the serum. Patients with mild COVID-19 exhibit a coordinated pattern of expression of interferon-stimulated genes (ISGs)3 across every cell population, whereas these ISG-expressing cells are systemically absent in patients with severe disease. Paradoxically, individuals with severe COVID-19 produce very high titres of anti-SARS-CoV-2 antibodies and have a lower viral load compared to individuals with mild disease. Examination of the serum from patients with severe COVID-19 shows that these patients uniquely produce antibodies that functionally block the production of the ISG-expressing cells associated with mild disease, by activating conserved signalling circuits that dampen cellular responses to interferons. Overzealous antibody responses pit the immune system against itself in many patients with COVID-19, and perhaps also in individuals with other viral infections. Our findings reveal potential targets for immunotherapies in patients with severe COVID-19 to re-engage viral defence.

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.

PurposeAutoantibody responses in cancer are of great interest, as they may be concordant with T-cell responses to cancer antigens or predictive of response to cancer immunotherapies. Thus, we sought to characterize the antibody landscape of metastatic castration-resistant prostate cancer (mCRPC).Experimental designSerum antibody epitope repertoire analysis (SERA) was performed on patient serum to identify tumor-specific neoepitopes. Somatic mutation-specific neoepitopes were investigated by associating serum epitope enrichment scores with whole-genome sequencing results from paired solid tumor metastasis biopsies and germline blood samples. A protein-based immunome-wide association study (PIWAS) was performed to identify significantly enriched epitopes, and candidate serum antibodies enriched in select patients were validated by ELISA profiling. A distinct cohort of patients with melanoma was evaluated to validate the top cancer-specific epitopes.ResultsSERA was performed on 1,229 serum samples obtained from 72 men with mCRPC and 1,157 healthy control patients. Twenty-nine of 6,636 somatic mutations (0.44%) were associated with an antibody response specific to the mutated peptide. PIWAS analyses identified motifs in 11 proteins, including NY-ESO-1 and HERVK-113, as immunogenic in mCRPC, and ELISA confirmed serum antibody enrichment in candidate patients. Confirmatory PIWAS, Identifying Motifs Using Next-generation sequencing Experiments (IMUNE), and ELISA analyses performed on serum samples from 106 patients with melanoma similarly revealed enriched cancer-specific antibody responses to NY-ESO-1.ConclusionsWe present the first large-scale profiling of autoantibodies in advanced prostate cancer, utilizing a new antibody profiling approach to reveal novel cancer-specific antigens and epitopes. Our study recovers antigens of known importance and identifies novel tumor-specific epitopes of translational interest.

PurposeThe purpose of this study was to measure genomic changes that emerge with enzalutamide treatment using analyses of whole-genome sequencing and RNA sequencing.Experimental designOne hundred and one tumors from men with metastatic castration-resistant prostate cancer (mCRPC) who had not been treated with enzalutamide (n = 64) or who had enzalutamide-resistant mCRPC (n = 37) underwent whole genome sequencing. Ninety-nine of these tumors also underwent RNA sequencing. We analyzed the genomes and transcriptomes of these mCRPC tumors.ResultsCopy number loss was more common than gain in enzalutamide-resistant tumors. Specially, we identified 124 protein-coding genes that were more commonly lost in enzalutamide-resistant samples. These 124 genes included eight putative tumor suppressors located at nine distinct genomic regions. We demonstrated that focal deletion of the 17q22 locus that includes RNF43 and SRSF1 was not present in any patient with enzalutamide-naïve mCRPC but was present in 16% (6/37) of patients with enzalutamide-resistant mCRPC. 17q22 loss was associated with lower RNF43 and SRSF1 expression and poor overall survival from time of biopsy [median overall survival of 19.3 months in 17q22 intact vs. 8.9 months in 17q22 loss, HR, 3.44 95% confidence interval (CI), 1.338-8.867, log-rank P = 0.006]. Finally, 17q22 loss was linked with activation of several targetable factors, including CDK1/2, Akt, and PLK1, demonstrating the potential therapeutic relevance of 17q22 loss in mCRPC.ConclusionsCopy number loss is common in enzalutamide-resistant tumors. Focal deletion of chromosome 17q22 defines a previously unappreciated molecular subset of enzalutamide-resistant mCRPC associated with poor clinical outcome.

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.

IntroductionGermline variants in androgen metabolism genes may influence clinical response to androgen deprivation therapy (ADT) in advanced prostate cancer. We sought to investigate the prognostic significance of germline variants in androgen metabolism genes with respect to overall survival (OS) after ADT, and to associate germline variants with tumor genomic features.MethodsGermline and somatic whole-genome sequencing (WGS) data were evaluated in a cohort of 101 men with metastatic castration-resistant prostate cancer (mCRPC). Survival analyses were performed to identify polymorphisms associated with impaired OS after primary ADT. Germline variants found to be prognostic of OS were associated with tumor somatic DNA-sequence alterations based on WGS performed on paired metastasis biopsies from the same 101 patients. Gene set enrichment analysis was performed based on tumor RNA-sequencing data to identify genomic pathways differentially expressed in patients with germline variants.ResultsA comprehensive literature review identified 17 candidate polymorphisms in nine androgen metabolism genes that have been previously shown to have an association with response to ADT in prostate cancer. Of these, the variant rs1856888 allele located 13 kb upstream of HSD3B1 was found to be significantly associated with impaired OS (P = 0.029). Variant rs1856888 was commonly co-inherited with the well-characterized HSD3B1(1245A>C) polymorphism, and there was a trend toward shorter median OS in patients with HSD3B1(1245A>C) compared with homozygous wild-type patients (P = 0.052). While HSD3B1 germline variants were not associated with common somatic tumor DNA alterations, they were associated with increased tumor expression of cell proliferation and cell cycle genes.ConclusionsThis study presents a comprehensive assessment of germline variants in androgen metabolism genes and highlights HSD3B1 polymorphisms as prognostic of OS after ADT and associated with an aggressive gene expression tumor profile in mCRPC.

BackgroundMetastatic castration-resistant prostate cancer (mCRPC) is the lethal form of the disease. Several recent studies have identified genomic alterations in mCRPC, but the clinical implications of these genomic alterations have not been fully elucidated.ObjectiveTo use whole-genome sequencing (WGS) to assess the association between key driver gene alterations and overall survival (OS), and to use whole-transcriptome RNA sequencing to identify genomic drivers of enzalutamide resistance.Design, setting, and participantsWe performed survival analyses and gene set enrichment analysis (GSEA) on WGS and RNA sequencing results for a cohort of 101 mCRPC patients.Outcome measurements and statistical analysisOS was the clinical endpoint for all univariate and multivariable survival analyses. Candidate drivers of enzalutamide resistance were identified in an unbiased manner, and mutations of the top candidate were further assessed for enrichment among enzalutamide-resistant patients using Fisher's exact test.Results and limitationsHarboring two DNA alterations in RB1 was independently predictive of poor OS (median 14.1 vs 42.0mo; p=0.007) for men with mCRPC. GSEA identified the Wnt/β-catenin pathway as the top differentially modulated pathway among enzalutamide-resistant patients. Furthermore, β-catenin mutations were exclusive to enzalutamide-resistant patients (p=0.01) and independently predictive of poor OS (median 13.6 vs 41.7mo; p=0.025).ConclusionsThe presence of two RB1 DNA alterations identified in our WGS analysis was independently associated with poor OS among men with mCRPC. The Wnt/β-catenin pathway plays an important role in enzalutamide resistance, with differential pathway expression and enrichment of β-catenin mutations in enzalutamide-resistant patients. Moreover, β-catenin mutations were predictive of poor OS in our cohort.Patient summaryWe observed a correlation between genomic findings for biopsy samples from metastases from men with metastatic castration-resistant prostate cancer (mCRPC) and clinical outcomes. This work sheds new light on clinically relevant genomic alterations in mCRPC and provides a roadmap for the development of new personalized treatment regimens in mCRPC.

PurposeRb-pathway disruption is of great clinical interest, as it has been shown to predict outcomes in multiple cancers. We sought to develop a transcriptomic signature for detecting biallelic RB1 loss (RBS) that could be used to assess the clinical implications of RB1 loss on a pan-cancer scale.Experimental designWe utilized data from the Cancer Cell Line Encyclopedia (N = 995) to develop the first pan-cancer transcriptomic signature for predicting biallelic RB1 loss (RBS). Model accuracy was validated using The Cancer Genome Atlas (TCGA) Pan-Cancer dataset (N = 11,007). RBS was then used to assess the clinical relevance of biallelic RB1 loss in TCGA Pan-Cancer and in an additional metastatic castration-resistant prostate cancer (mCRPC) cohort.ResultsRBS outperformed the leading existing signature for detecting RB1 biallelic loss across all cancer types in TCGA Pan-Cancer (AUC, 0.89 vs. 0.66). High RBS (RB1 biallelic loss) was associated with promoter hypermethylation (P = 0.008) and gene body hypomethylation (P = 0.002), suggesting RBS could detect epigenetic gene silencing. TCGA Pan-Cancer clinical analyses revealed that high RBS was associated with short progression-free (P < 0.00001), overall (P = 0.0004), and disease-specific (P < 0.00001) survival. On multivariable analyses, high RBS was predictive of shorter progression-free survival in TCGA Pan-Cancer (P = 0.03) and of shorter overall survival in mCRPC (P = 0.004) independently of the number of DNA alterations in RB1.ConclusionsOur study provides the first validated tool to assess RB1 biallelic loss across cancer types based on gene expression. RBS can be useful for analyzing datasets with or without DNA-sequencing results to investigate the emerging prognostic and treatment implications of Rb-pathway disruption.See related commentary by Choudhury and Beltran, p. 4199.

Off-label drug use, defined as use of a drug in a manner that deviates from its approved use defined by the drug's FDA label, is problematic because such uses have not been evaluated for safety and efficacy. Studies estimate that 21% of prescriptions are off-label, and only 27% of those have evidence of safety and efficacy. We describe a data-mining approach for systematically identifying off-label usages using features derived from free text clinical notes and features extracted from two databases on known usage (Medi-Span and DrugBank). We trained a highly accurate predictive model that detects novel off-label uses among 1,602 unique drugs and 1,472 unique indications. We validated 403 predicted uses across independent data sources. Finally, we prioritize well-supported novel usages for further investigation on the basis of drug safety and cost.

Author(s): Alexis J. Combes [sup.1] [sup.2] [sup.3] , Tristan Courau [sup.1] [sup.2] [sup.3] , Nicholas F. Kuhn [sup.1] [sup.2] , Kenneth H. Hu [sup.1] [sup.2] , Arja Ray [sup.1] [sup.2] [...]

Akt kinases control essential cellular functions, including proliferation, apoptosis, metabolism and transcription, and have been proposed as promising targets for treatment of angiogenesis-dependent pathologies, such as cancer and ischemic injury. But their precise roles in neovascularization remain elusive. Here we show that Akt1 is the predominant isoform in vascular cells and describe the unexpected consequences of Akt1 knockout on vascular integrity and pathological angiogenesis. Angiogenic responses in three distinct in vivo models were enhanced in Akt1[sup.-/-] mice; these enhanced responses were associated with impairment of blood vessel maturation and increased vascular permeability. Although impaired vascular maturation in Akt1[sup.-/-] mice may be attributed to reduced activation of endothelial nitric oxide synthase (eNOS), the major phenotypic changes in vascular permeability and angiogenesis were linked to reduced expression of two endogenous vascular regulators, thrombospondins 1 (TSP-1) and 2 (TSP-2). Re-expression of TSP-1 and TSP-2 in mice transplanted with wild-type bone marrow corrected the angiogenic abnormalities in Akt1[sup.-/-] mice. These findings establish a crucial role of an Akt-thrombospondin axis in angiogenesis., Author(s): Juhua Chen [1, 4]; Payaningal R Somanath [1, 4]; Olga Razorenova [1]; William S Chen [2]; Nissim Hay [2]; Paul Bornstein [3]; Tatiana V Byzova (corresponding author) [1] The [...]

Growth retardation and greater apoptosis in mice having homozygous disruption of the akt1 gene are discussed in this research communication. The most ubiquitously expressed member of the akt family of genes in mice has been disrupted for the study.