Whole-Genome Mapping of Epigenetic Modification of 5-Formylcytosine at Single-Base Resolution by Chemical Labeling Enrichment and Deamination Sequencing.

DNA cytosine methylation (5-methylcytosine, 5mC) is a predominant epigenetic modification that plays a critical role in a variety of biological and pathological processes in mammals. In active DNA demethylation, the 10-11 translocation (TET) dioxygenases can sequentially oxidize 5mC to generate three modified forms of cytosine, 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Beyond being a demethylation intermediate, recent studies have shown that 5fC has regulatory functions in gene expression and chromatin organization. While some methods have been developed to detect 5fC, genome-wide mapping of 5fC at base resolution is still highly desirable. Herein, we propose a chemical labeling enrichment and deamination sequencing (CLED-seq) method for detecting 5fC in genomic DNA at single-base resolution. The CLED-seq method utilizes selective labeling and enrichment of 5fC-containing DNA fragments, followed by deamination mediated by apolipoprotein B mRNA-editing catalytic polypeptide-like 3A (APOBEC3A or A3A) and sequencing. In the CLED-seq process, while all C, 5mC, and 5hmC are interpreted as T during sequencing, 5fC is still read as C, enabling the precise detection of 5fC in DNA. Using the proposed CLED-seq method, we accomplished genome-wide mapping of 5fC in mouse embryonic stem cells. The mapping study revealed that promoter regions enriched with 5fC overlapped with H3K4me1, H3K4me3, and H3K27ac marks. These findings suggest a correlation between 5fC marks and active gene expression in mESCs. In conclusion, CLED-seq is a straightforward, bisulfite-free method that offers a valuable tool for detecting 5fC in genomes at a single-base resolution.

Preliminary Study of Whole-Genome Bisulfite Sequencing and Transcriptome Sequencing in VHL Disease-Associated ccRCC.

BACKGROUND: Von Hippel-Lindau (VHL) disease is an autosomal dominant hereditary tumor syndrome with an incidence of approximately 1/36,000. VHL disease-associated clear cell renal cell carcinoma (ccRCC) is the most common congenital RCC. Although recent advances in treating RCC have improved the long-term prognosis of patients with VHL disease, kidney cancer is still the leading cause of death in these patients. Therefore, finding new targets for diagnosing and treating VHL disease-associated ccRCC is still essential. METHODS: In this study, we collected matched tumor tissues and normal samples from 25 patients with VHL disease-associated ccRCC, diagnosed and surgically treated in the Department of Urology, Peking University First Hospital. After screening, we performed whole genome bisulfite sequencing (WGBS) on 23 pairs of tissues and RNA-seq on 6 pairs of tissues. And we also compared the VHL disease-associated ccRCC transcriptome data with the sporadic ccRCC transcriptome data from the The Cancer Genome Atlas (TCGA) public database RESULTS: We found that the methylation level of VHL disease-associated ccRCC tumor tissues was significantly lower than that of normal tissues. The tumor tissues showed a difference in the copy number of 3p loss and 5q and 7q gain compared with normal tissues. We integrated RNA-seq and WGBS data to reveal methylation candidate genes associated with VHL disease-associated ccRCC; our results showed 124 hypermethylated and downregulated genes, and 245 hypomethylated and upregulated genes. By comparing the VHL disease-associated ccRCC transcriptome data with the sporadic ccRCC transcriptome data from the TCGA public database, we found that the major pathways of differential gene enrichment differed between them. CONCLUSIONS: Our study mapped the multiomics of copy number variation, methylation and mRNA level changes in tumor and normal tissues of clear cell renal cell carcinoma with VHL syndrome, which provides a solid foundation for the mechanistic study, biomarker screening, and therapeutic target discovery of clear cell renal cell carcinoma.

UCseek: ultrasensitive early detection and recurrence monitoring of urothelial carcinoma by shallow-depth genome-wide bisulfite sequencing of urinary sediment DNA.

BACKGROUND: Current methods for the detection and surveillance of urothelial carcinomas (UCs) are often invasive, costly, and not effective for low-grade, early-stage, and minimal residual disease (MRD) tumors. We aimed to develop and validate a model from urine sediments to predict different grade and stage UCs with low cost and high accuracy. METHODS: We collected 167 samples, including 90 tumors and 77 individuals without tumors, as a discovery cohort. We assessed copy number variations and methylation values for them and constructed a diagnostic classifier to detect UC, UCseek, by using an individual read-based method and support vector machine. The performance of UCseek was validated in an independent cohort derived from three hospitals (n = 206) and a relapse cohort (n = 42) for monitoring recurrence. FINDINGS: We constructed UCseek, which could predict UCs with high sensitivity (92.7%), high specificity (90.7%), and high accuracy (91.7%) in the independent validation set. The accuracy of UCseek in low-grade and early-stage patients reached 91.8% and 94.3%, respectively. Notably, UCseek retained great performance at ultralow sequencing depths (0.3X-0.5X). It also demonstrated a powerful ability to monitor recurrence in a surveillance cohort compared with cystoscopy (90.91% vs. 59.09%). INTERPRETATION: We optimized an improved approach named UCseek for the noninvasive diagnosis and monitoring of UCs in both low- and high-grade tumors and in early- and advanced-stage tumors, even at ultralow sequencing depths, which may reduce the burden of cystoscopy and blind second surgery. FUNDING: A full list of funding bodies that contributed to this study can be found in the Acknowledgments section.

Whole-Genome Sequencing of 5-Hydroxymethylcytosine at Base Resolution by Bisulfite-Free Single-Step Deamination with Engineered Cytosine Deaminase.

The epigenetic modification 5-hydroxymethylcytosine (5hmC) plays a crucial role in the regulation of gene expression. Although some methods have been developed to detect 5hmC, direct genome-wide mapping of 5hmC at base resolution is still highly desirable. Herein, we proposed a single-step deamination sequencing (SSD-seq) method, designed to precisely map 5hmC across the genome at single-base resolution. SSD-seq takes advantage of a screened engineered human apolipoprotein B mRNA-editing catalytic polypeptide-like 3A (A3A) protein, known as eA3A-v10, to selectively deaminate cytosine (C) and 5-methylcytosine (5mC) but not 5hmC. During sequencing, the deaminated C and 5mC are converted to uracil (U) and thymine (T), read as T in the sequencing data. However, 5hmC remains unaffected by eA3A-v10 and is read as C during sequencing. Consequently, the presence of C in the sequence reads indicates the original 5hmC. We applied SSD-seq to generate a base-resolution map of 5hmC in human lung tissue. Our findings revealed that 5hmC was predominantly localized to CpG dinucleotides. Furthermore, the base-resolution map of 5hmC generated by SSD-seq demonstrated a strong correlation with prior ACE-seq results. The advantages of SSD-seq are its single-step process, absence of bisulfite treatment or DNA glycosylation, cost effectiveness, and ability to detect and quantify 5hmC directly at single-base resolution.

Single-cell atlases link macrophages and CD8+ T-cell subpopulations to disease progression and immunotherapy response in urothelial carcinoma.

Rationale: Immune checkpoint inhibitors (ICIs) have revolutionized the management of locally advanced or metastatic urothelial carcinoma. Strikingly, compared to urothelial carcinoma of the bladder (UCB), upper tract urothelial carcinoma (UTUC) has a higher response rate to ICIs. The stratification of patients most likely to benefit from ICI therapy remains a major clinical challenge. Methods: In this study, we performed the first single-cell RNA sequencing (scRNA-seq) study of 13 surgical tissue specimens from 12 patients with UTUC. The key results were validated by the analysis of two independent cohorts with bulk RNA-seq data for UCB (n = 404) and UTUC (n = 158) and one cohort of patients with metastatic urothelial carcinoma (mUC) who were treated with atezolizumab (n = 348). Results: Using scRNA-seq, we observed a higher proportion of tumor-infiltrating immune cells in locally advanced UTUC. Similar prognostically relevant intrinsic basal and luminal-like epithelial subtypes were found in both UTUC and UCB, although UTUC is predominantly of the luminal subtype. We also discovered that immunosuppressive macrophages and exhausted T-cell subpopulations were enriched in the basal subtype and showed enhanced interactions. Furthermore, we developed a gene expression signature (Macro-C3 score) capturing the immunosuppressive macrophages that better predicts outcomes than the currently established subtypes. We also developed a computational method to model immune evasion, and the Macro-C3 score predicted therapeutic response of mUC treated with first-line anti-PD-L1 inhibitors in patients with lower basal scores. Conclusions: Overall, the distinct microenvironment and Macro-C3 score provide an explanation for ICI efficacy in urothelial carcinoma and reveal new candidate regulators of immune evasion, suggesting potential therapeutic targets for improving antitumor immunity in the basal subtype.

Regional gain and global loss of 5-hydroxymethylcytosine coexist in genitourinary cancers and regulate different oncogenic pathways.

BACKGROUND: DNA 5-hydroxymethylcytosine (5hmC) is produced by dynamic 5mC oxidation process contributing to tissue specification, and loss of 5hmC has been reported in multiple cancers including genitourinary cancers. However, 5hmC is also cell-type specific, and its variability may exist between differentiated tumor cells and cancer stem cells. Thus, cancer-associated changes in 5hmC may be contributed by distinct sets of tumor cells within the tumor tissues. RESULTS: Here, we applied a sensitive immunoprecipitation-based method (hMeDIP-seq) to analyze 5hmC changes during genitourinary carcinogenesis (including prostate, urothelial and kidney). We confirmed the tissue-specific distribution of 5hmC in genitourinary tissues and identified regional gain and global loss of 5hmC coexisting in genitourinary cancers. The genes with gain of 5hmC during tumorigenesis were functionally enriched in regulating stemness and hypoxia, whereas were associated with poor clinical prognosis irrespective of their differences in tumor type. We identified that gain of 5hmC occurred in soft fibrin gel-induced 3D tumor spheres with a tumor-repopulating phenotype in two prostate cancer cell lines, 22RV1 and PC3, compared with conventional two-dimensional (2D) rigid dishes. Then, we defined a malignant signature derived from the differentially hydroxymethylated regions affected genes of cancer stem-like cells, which could predict a worse clinical outcome and identified phenotypically malignant populations of cells from prostate cancer tumors. Notably, an oxidation-resistant vitamin C derivative, ascorbyl phosphate magnesium, restored 5hmC and killed the cancer stem cell-like cells leading to apoptosis in prostate cancer cell lines. CONCLUSIONS: Collectively, our study dissects the regional gain of 5hmC in maintaining cancer stem-like cells and related to poor prognosis, which provides proof of concept for an epigenetic differentiation therapy with vitamin C by 5hmC reprogramming.

DNA methylation subtypes guiding prognostic assessment and linking to responses the DNA methyltransferase inhibitor SGI-110 in urothelial carcinoma.

BACKGROUND: At present, the extent and clinical relevance of epigenetic differences between upper tract urothelial carcinoma (UTUC) and urothelial carcinoma of the bladder (UCB) remain largely unknown. Here, we conducted a study to describe the global DNA methylation landscape of UTUC and UCB and to address the prognostic value of DNA methylation subtype and responses to the DNA methyltransferase inhibitor SGI-110 in urothelial carcinoma (UC). METHODS: Using whole-genome bisulfite sequencing (n = 49 samples), we analyzed epigenomic features and profiles of UTUC (n = 36) and UCB (n = 9). Next, we characterized potential links between DNA methylation, gene expression (n = 9 samples), and clinical outcomes. Then, we integrated an independent UTUC cohort (Fujii et al., n = 86) and UCB cohort (TCGA, n = 411) to validate the prognostic significance. Furthermore, we performed an integrative analysis of genome-wide DNA methylation and gene expression in two UC cell lines following transient DNA methyltransferase inhibitor SGI-110 treatment to identify potential epigenetic driver events that contribute to drug efficacy. RESULTS: We showed that UTUC and UCB have very similar DNA methylation profiles. Unsupervised DNA methylation classification identified two epi-clusters, Methy-High and Methy-Low, associated with distinct muscle-invasive statuses and patient outcomes. Methy-High samples were hypermethylated, immune-infiltrated, and enriched for exhausted T cells, with poor clinical outcome. SGI-110 inhibited the migration and invasion of Methy-High UC cell lines (UMUC-3 and T24) by upregulating multiple antitumor immune pathways. CONCLUSIONS: DNA methylation subtypes pave the way for predicting patient prognosis in UC. Our results provide mechanistic rationale for evaluating SGI-110 in treating UC patients in the clinic.

Pyroptosis-Related Signature Predicts Prognosis and Immunotherapy Efficacy in Muscle-Invasive Bladder Cancer.

Pyroptosis has profound impacts on tumor cell proliferation, invasion, and metastasis and is of great clinical significance for different cancers. However, the role of pyroptosis in the progression and prognosis of muscle invasive bladder cancer (MIBC) remains poorly characterized. Here, we collected multicenter MIBC data and performed integrated analysis to dissect the role of pyroptosis in MIBC and provide an optimized treatment for this disease. Based on transcriptomic data, we developed a novel prognostic model named the pyroptosis-related gene score (PRGScore), which summarizes immunological features, genomic alterations, and clinical characteristics associated with the pyroptosis phenotype. Samples with high PRGScore showed enhancement in CD8+ T cell effector function, antigen processing machinery and immune checkpoint and better response to immunotherapy by programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PD-L1) inhibitors, which indicates that PRGScore is a valuable signature in the identification of populations sensitive to immune checkpoint inhibitors. Collectively, our study provides insights into further research targeting pyroptosis and its tumor immune microenvironment (TME) and offers an opportunity to optimize the treatment of MIBC.

Metabolism-Related Signature Analysis Uncovers the Prognostic and Immunotherapeutic Characteristics of Renal Cell Carcinoma.

Renal cell carcinoma (RCC) is one of the most common urological cancers. RCC has a poor prognosis and is considered a metabolic disease. It has been reported that many metabolic pathways are associated with the development of RCC. However, the prognostic value of metabolism-related genes in RCC is unclear. We herein aimed to establish a scoring system based on the gene expression profile of metabolic genes to evaluate the response to immunotherapy and predict the prognosis of RCC. In this study, we collected multicentre RCC data and performed integrated analysis to characterize the role of tumour metabolism in RCC and explore the relationship between metabolism and prognosis and immune therapy. Based on transcriptomic data, metabolism-related genes were used for nonnegative matrix factorization clustering. We obtained three subclasses of RCC (M1, M2, and M3), and they are associated with different prognoses and immune infiltrate levels. Then, based on the pathway activity of 113 metabolism-related gene signatures, we classified patients into three distinct metabolism-related signatures. Finally, we provide a metabolism-related pathway score (MRPScore) that is significantly associated with RCC prognosis and the response to immunotherapy. Taken together, in this study, we established an RCC classification system based on metabolic gene expression profiles that could further the understanding of the diversity of RCC. We also present the MRPScore, which may be used as an indicator to predict the response to clinical immune therapy.

Decoding the multicellular ecosystem of vena caval tumor thrombus in clear cell renal cell carcinoma by single-cell RNA sequencing.

BACKGROUND: Vascular invasion with tumor thrombus frequently occurs in advanced renal cell carcinoma (RCC). Thrombectomy is one of the most challenging surgeries with high rate of perioperative morbidity and mortality. However, the mechanisms driving tumor thrombus formation are poorly understood which is required for designing effective therapy for eliminating tumor thrombus. RESULTS: We perform single-cell RNA sequencing analysis of 19 surgical tissue specimens from 8 clear cell renal cell carcinoma (ccRCC) patients with tumor thrombus. We observe tumor thrombus has increased tissue resident CD8+ T cells with a progenitor exhausted phenotype compared with the matched primary tumors. Remarkably, macrophages, malignant cells, endothelial cells and myofibroblasts from TTs exhibit enhanced remodeling of the extracellular matrix. The macrophages and malignant cells from primary tumors represent proinflammatory states, but also increase the expression of immunosuppressive markers compared to tumor thrombus. Finally, differential gene expression and interaction analyses reveal that tumor-stroma interplay reshapes the extracellular matrix in tumor thrombus associated with poor survival. CONCLUSIONS: Our comprehensive picture of the ecosystem of ccRCC with tumor thrombus provides deeper insights into the mechanisms of tumor thrombus formation, which may aid in the design of effective neoadjuvant therapy to promote downstaging of tumor thrombus and decrease the perioperative morbidity and mortality of thrombectomy.

Single-Cell RNA-seq Reveals a Developmental Hierarchy Super-Imposed Over Subclonal Evolution in the Cellular Ecosystem of Prostate Cancer.

Prostate cancer (PCa) is a complex disease. An ongoing accumulation of mutations results in increased genetic diversity, with the tumor acquiring distinct subclones. However, non-genetic intra-tumoral heterogeneity, the cellular differentiation state and the interplay between subclonal evolution and transcriptional heterogeneity are poorly understood. Here, the authors perform single-cell RNA sequencing from 14 untreated PCa patients. They create an extensive cell atlas of the PCa patients and mapped developmental states onto tumor subclonal evolution. They identify distinct subclones across PCa patients and then stratify tumor cells into four transcriptional subtypes, EMT-like (subtype 0), luminal A-like (subtype 1), luminal B/C-like (subtype 2), and basal-like (subtype 3). These subtypes are hierarchically organized into stem cell-like and differentiated status. Strikingly, multiple subclones within a single primary tumor present with distinct combinations of preferential subtypes. In addition, subclones show different communication strengths with other cell types within the tumor ecosystem, which may modulate the distinct transcriptional subtypes of the subclones. Notably, by integrating TCGA data, they discover that both tumor cell transcriptional heterogeneity and cellular ecosystem diversity correlate with features of a poor prognosis. Collectively, their study provides the analysis of subclonal and transcriptional heterogeneity and its implication for patient prognosis.

Ascorbic acid induced TET2 enzyme activation enhances cancer immunotherapy efficacy in renal cell carcinoma.

Exploring the regulatory mechanism of PD-L1 in renal cancer is one of the key strategies to improve the response of renal cancer patients to checkpoint blockade therapy. In this study, the synergistic effect of ascorbic acid (vitamin C) supplementation and the impact of TET2 depletion on anti-PD-L1 therapy were determined in xenograft model experiments. Lymphocyte infiltration and chemokine expression were determined using flow cytometry and qRT-PCR. To determine the downstream targets of TET2, we performed hMeDip-seq and RNA-seq analyses. The molecular mechanism was further confirmed by hMeDip-qPCR, MeDip-qPCR, bisulfite sequencing, Western blotting, qRT-PCR and xenograft model experiments in vitro and in vivo. The present study demonstrated that ascorbic acid enhanced the efficacy of immunotherapy and that the loss of TET2 function enabled renal cancer cells to evade antitumor immunity. Ascorbic acid treatment significantly increased the intratumoral infiltration of T cells and the expression of cytokines and chemokines, while the loss of TET2 impaired the infiltration of T cells and the expression of cytokines and chemokines. TET2 was recruited to IRF1 by IFN-γ-STAT1 signaling, thereby maintaining IRF1 demethylation and ultimately inducing PD-L1 expression. These results suggest a new strategy of stimulating TET activity to improve immunotherapy for renal cell carcinoma.

The RNA N6-Methyladenosine Methyltransferase METTL3 Promotes the Progression of Kidney Cancer via N6-Methyladenosine-Dependent Translational Enhancement of ABCD1.

The role of N6-methyladenosine (m6A)-modifying proteins in cancer progression depends on the cell type and mRNA affected. However, the biological role and underlying mechanism of m6A in kidney cancer is limited. Here, we discovered the variability in m6A methyltransferase METTL3 expression was significantly increased in clear cell renal cell carcinoma (ccRCC) the most common subtype of renal cell carcinoma (RCC), and high METTL3 expression predicts poor prognosis in ccRCC patients using a dataset from The Cancer Genome Atlas (TCGA). Importantly, knockdown of METTL3 in ccRCC cell line impaired both cell migration capacity and tumor spheroid formation in soft fibrin gel, a mechanical method for selecting stem-cell-like tumorigenic cells. Consistently, overexpression of METTL3 but not methyltransferase activity mutant METTL3 can promote cell migration, spheroid formation in cell line and tumor growth in xenograft model. Transcriptional profiling of m6A in ccRCC tissues identified the aberrant m6A transcripts were enriched in cancer-related pathways. Further m6A-sequencing of METTL3 knockdown cells and functional studies confirmed that translation of ABCD1, an ATP-binding cassette (ABC) transporter of fatty acids, was inhibited by METTL3 in m6A-dependent manner. Moreover, knockdown of ABCD1 in ccRCC cells decreased cancer cell migration and spheroid formation, and upregulation of ABCD1 acts as an adverse prognosis factor of kidney cancer patients. In summary, our study identifies that METTL3 promotes ccRCC progression through m6A modification-mediated translation of ABCD1, providing an epitranscriptional insight into the molecular mechanism in kidney cancer.

Copy Number Signatures and Clinical Outcomes in Upper Tract Urothelial Carcinoma.

Tumor staging of upper tract urothelial carcinomas (UTUCs) is relatively difficult to assert accurately before surgery. Here, we used copy number (CN) signatures as a tool to explore their clinical significance of molecular stratification in UTUC. CN signatures were extracted by non-negative matrix factorization from the whole-genome sequencing (WGS) data of 90 Chinese UTUC primary tumor samples. A validation UTUC cohort (n = 56) and a cohort from urinary cell-free DNA (cfDNA) of urothelial cancer patients (n = 94) and matched primary tumors were also examined. Survival analyses were measured using the Kaplan-Meier, and Cox regression was used for multivariate analysis. Here, we identified six CN signatures (Sig1-6). Patients with a high contribution of Sig6 (Sig6high) were associated with higher microsatellite instability level and papillary architecture and had a favorable outcome. Patients with a low weighted genome integrity index were associated with positive lymph node and showed the worst outcome. Sig6high was identified to be an independently prognostic factor. The predictive significance of CN signature was identified by a validation UTUC cohort. CN signatures retained great concordance between primary tumor and urinary cfDNA. In conclusion, our results reveal that CN signature assessment for risk stratification is feasible and provides a basis for clinical studies that evaluate therapeutic interventions and prognosis.

Enhanced Detection of Genitourinary Cancers Using Fragmentation and Copy Number Profiles Obtained from Urinary Cell-Free DNA.

BACKGROUND: Recent studies have reported that examining the fragmentation profiles (FP) of plasma cell-free DNA (cfDNA) further improves the clinical sensitivity of tumor detection. We hypothesized that considering the differences of the FP of urinary cfDNA would increase the clinical sensitivity of genitourinary (GU) cancer detection. METHODS: 177 patients with GU cancer and 94 individuals without tumors were enrolled in the discovery cohort. An independent validation dataset comprising 30 patients without tumors and 66 patients with GU cancer was also collected. We constructed an ensemble classifier, GUIDER, to detect and localize GU cancers using fragmentation and copy number profiles obtained from shallow whole-genome sequencing of urinary cfDNA. RESULTS: Urinary cfDNA of patients with GU cancer had a higher proportion of long fragments (209-280 bp) and a lower proportion of short fragments (140-208 bp) compared to controls. The overall mean classification accuracy of the FP was 74.62%-85.39% for different algorithms, and integration of the FP and copy number alteration (CNA) features further enhanced the classification of samples from patients with GU cancer. The mean diagnostic accuracy was further improved by the ensemble classifier GUIDER, which integrated the FP and CNA profiles and resulted in a higher mean accuracy (87.52%) compared to the analysis performed without FP features (74.62%). GUIDER performed well in an independent validation dataset. CONCLUSIONS: The lengthening and shortening of urinary cfDNA within specific size ranges were identified in patients with GU cancer. Integration of the FP should further enhance the ability to use urinary cfDNA as a molecular diagnostic tool.

Construction of diagnostic and subtyping models for renal cell carcinoma by genome-wide DNA methylation profiles.

BACKGROUND: Renal cell carcinoma (RCC) is one of the most common urological cancers and has a poor prognosis. RCC is classified into several subtypes, among which kidney renal clear cell carcinoma (KIRC) and kidney renal papillary cell carcinoma (KIRP) are the two most common subtypes. Due to the lack of adequate screening and comparative analysis of RCC subtypes, effective diagnosis and treatment strategies have not yet been achieved. METHODS: In this study, 450K methylation array data were collected from The Cancer Genome Atlas (TCGA). The 'limma moderated t-test' and LASSO were used to construct diagnostic and subtyping models, and survival analysis was conducted online by GEPIA. RESULTS: We built a model with 15 methylation sites, which showed high diagnostic and subtyping performance in specificity and sensitivity. At the same time, for potential clinical usability, we calculated the diagnostic and subtyping scores to classify RCC from normal tissue and distinguish the different RCC subtypes. Additionally, the CpG sites were mapped to their corresponding genes, which could also be used to predict the prognosis of RCC. CONCLUSIONS: Different methylation sites can be used as diagnostic and subtyping markers that are specific to RCC and RCC subtypes (KIRC and KIRP) with high sensitivity and accuracy.

Increased chromosomal instability characterizes metastatic renal cell carcinoma.

The evolutionary trajectories of treatment-naïve metastatic tumour are largely unknown. Such knowledge is crucial for cancer prevention and therapeutic interventions. Herein, we performed whole genome or exome sequencing of 19 tumour specimens and 8 matched normal kidney tissues from 8 clear cell renal cell carcinoma (ccRCC) patients. The clonal origin and parallel evolution of the metastatic lesions and primary tumour is identified in all 8 patients. But the evolutionary branches of primary and metastatic clones diverge early in the development of the tumour. More importantly, larger scale genomic aberrations including somatic copy number alteration (SCNA) or loss of heterozygosity (LOH) differentiate the metastasis lesions from primary tumour. Based on it, we identify that LOH at 14q, loss of 14q32.31 and gain of 6p22.2 are highly selected events during metastatic evolution. Further functional validations of multiple genes within the SCNA regions indicated that these selected events interact to drive metastatic risk with potential therapeutic relevance. Collectively, we described increased genome instability in metastatic ccRCC and validated it via molecular biology, providing an evolution pattern which may facilitate the translation of basic finding.

METTL3 counteracts premature aging via m6A-dependent stabilization of MIS12 mRNA.

N6-Methyladenosine (m6A) messenger RNA methylation is a well-known epitranscriptional regulatory mechanism affecting central biological processes, but its function in human cellular senescence remains uninvestigated. Here, we found that levels of both m6A RNA methylation and the methyltransferase METTL3 were reduced in prematurely senescent human mesenchymal stem cell (hMSC) models of progeroid syndromes. Transcriptional profiling of m6A modifications further identified MIS12, for which m6A modifications were reduced in both prematurely senescent hMSCs and METTL3-deficient hMSCs. Knockout of METTL3 accelerated hMSC senescence whereas overexpression of METTL3 rescued the senescent phenotypes. Mechanistically, loss of m6A modifications accelerated the turnover and decreased the expression of MIS12 mRNA while knockout of MIS12 accelerated cellular senescence. Furthermore, m6A reader IGF2BP2 was identified as a key player in recognizing and stabilizing m6A-modified MIS12 mRNA. Taken together, we discovered that METTL3 alleviates hMSC senescence through m6A modification-dependent stabilization of the MIS12 transcript, representing a novel epitranscriptional mechanism in premature stem cell senescence.

Aristolochic acid mutational signature defines the low-risk subtype in upper tract urothelial carcinoma.

Rationale: Dietary exposure to aristolochic acids and similar compounds (collectively, AA) is a significant risk factor for nephropathy and subsequent upper tract urothelial carcinoma (UTUC). East Asian populations, who have a high prevalence of UTUC, have an unusual genome-wide AA-induced mutational pattern (COSMIC signature 22). Integrating mutational signature analysis with clinicopathological information may demonstrate great potential for risk ranking this UTUC subtype. Methods: We performed whole-genome sequencing (WGS) on 90 UTUC Chinese patients to extract mutational signatures. Genome sequencing data for urinary cell-free DNA from 26 UTUC patients were utilized to noninvasively identify the mutational signatures. Genome sequencing for primary tumors on 8 out of 26 patients was also performed. Metastasis-free survival (MFS) and cancer-specific survival (CSS) were measured using Kaplan-Meier methods. Results: Data analysis showed that a substantial proportion of patients harbored the AA mutational signature and were associated with AA-containing herbal drug intake, female gender, poor renal function, and multifocality. Field cancerization was found to partially contribute to multifocality. Nevertheless, AA Sig subtype UTUC patients exhibited favorable outcomes of CSS and MFS compared to the No-AA Sig subtype. Additionally, AA Sig subtype patients showed a higher tumor mutation burden, higher numbers of predicted neoantigens, and infiltrating lymphocytes, suggesting the potential for immunotherapy. We also confirmed the AA signature in AA-treated human renal tubular HK-2 cells. Notably, the AA subtype could be ascertained using a clinically applicable sequencing strategy (low coverage) in both primary tumors and urinary cell-free DNA as a basis for therapy selection. Conclusion: The AA mutational signature as a screening tool defines low-risk UTUC with therapeutic relevance. The AA mutational signature, as a molecular prognostic marker using either ureteroscopy and/or urinary cell-free DNA, is especially useful for diagnostic uncertainty when kidney-sparing treatment and/or immune checkpoint inhibitor therapy were considered.