Single cell transcriptomics reveals early photoreceptor states, cell-specific transcript isoforms, and cancer-predisposing features.

Human cone photoreceptors differ from rods and serve as the retinoblastoma cell-of-origin. Here, we used deep full-length single-cell RNA-sequencing to distinguish post-mitotic cone and rod developmental states and cone-specific features that contribute to retinoblastomagenesis. The analyses revealed early post-mitotic cone- and rod-directed populations characterized by higher THRB or NRL regulon activities, an immature photoreceptor precursor population with concurrent cone and rod gene and regulon expression, and distinct early and late cone and rod maturation states distinguished by maturation-associated declines in RAX regulon activity. Unexpectedly, both L/M cone and rod precursors co-expressed NRL and THRB RNAs, yet they differentially expressed functionally antagonistic NRL isoforms and prematurely terminated THRB transcripts. Early L/M cone precursors exhibited successive expression of lncRNAs along with MYCN, which composed the seventh most L/M-cone-specific regulon, and SYK, which contributed to the early cone precursors' proliferative response to RB1 loss. These findings reveal previously unrecognized photoreceptor precursor states and a role for early cone-precursor-intrinsic SYK expression in retinoblastoma initiation.

Detection of diagnostic and prognostic methylation-based signatures in liquid biopsy specimens from patients with meningiomas.

Recurrence of meningiomas is unpredictable by current invasive methods based on surgically removed specimens. Identification of patients likely to recur using noninvasive approaches could inform treatment strategy, whether intervention or monitoring. In this study, we analyze the DNA methylation levels in blood (serum and plasma) and tissue samples from 155 meningioma patients, compared to other central nervous system tumor and non-tumor entities. We discover DNA methylation markers unique to meningiomas and use artificial intelligence to create accurate and universal models for identifying and predicting meningioma recurrence, using either blood or tissue samples. Here we show that liquid biopsy is a potential noninvasive and reliable tool for diagnosing and predicting outcomes in meningioma patients. This approach can improve personalized management strategies for these patients.

SETD2 Deficiency Confers Sensitivity to Dual Inhibition of DNA Methylation and PARP in Kidney Cancer.

SETD2 deficiency alters the epigenetic landscape by causing depletion of H3K36me3 and plays an important role in diverse forms of cancer, most notably in aggressive and metastatic clear-cell renal cell carcinomas (ccRCC). Development of an effective treatment scheme targeting SETD2-compromised cancer is urgently needed. Considering that SETD2 is involved in DNA methylation and DNA repair, a combination treatment approach using DNA hypomethylating agents (HMA) and PARP inhibitors (PARPi) could have strong antitumor activity in SETD2-deficient kidney cancer. We tested the effects of the DNA HMA 5-aza-2'-dexoxydytidine (DAC), the PARPi talazoparib (BMN-673), and both in combination in human ccRCC models with or without SETD2 deficiency. The combination treatment of DAC and BMN-673 synergistically increased cytotoxicity in vitro in SETD2-deficient ccRCC cell lines but not in SETD2-proficient cell lines. DAC and BMN-673 led to apoptotic induction, increased DNA damage, insufficient DNA damage repair, and increased genomic instability. Furthermore, the combination treatment elevated immune responses, upregulated STING, and enhanced viral mimicry by activating transposable elements. Finally, the combination effectively suppressed the growth of SETD2-deficient ccRCC in in vivo mouse models. Together, these findings indicate that combining HMA and PARPi is a promising potential therapeutic strategy for treating SETD2-compromised ccRCC. SIGNIFICANCE: SETD2 deficiency creates a vulnerable epigenetic status that is targetable using a DNA hypomethylating agent and PARP inhibitor combination to suppress renal cell carcinoma, identifying a precision medicine-based approach for SETD2-compromised cancers.

Potential role of fructose on human colon DNA methylation in racial disparities observed for colorectal cancer risk.

Background and aims: An increasing body of observational studies has linked fructose intake to colorectal cancer (CRC). African Americans (AAs) are significantly more likely than European Americans to consume greater quantities of fructose and to develop right-side colon cancer. Yet, a mechanistic link between these two associations remains poorly defined. We aimed to identify differentially methylated regions (DMRs) associated with dietary fructose consumption measures obtained from food frequency questionnaires in a cohort of normal colon biopsies derived from AA men and women (n=79). Methods: DNA methylation data from this study was obtained using the Illumina Infinium MethylationEPIC kit and is housed under accession GSE151732. DMR analysis was carried out using DMRcate in right and matched left colon, separately. Secondary analysis of CRC tumors was carried out using data derived from TCGA-COAD, GSE101764 and GSE193535. Differential expression analysis was carried out on CRC tumors from TCGA-COAD using DESeq2 . Results: We identified 4,263 right-side fructose-DMRs. In contrast, only 24 DMRs survived multiple testing corrections (FDR<0.05) in matched, left colon. To identify targets by which dietary fructose drives CRC risk, we overlaid these findings with data from three CRC tumor datasets. Remarkably, almost 50% of right-side fructose-DMRs overlapped regions associated with CRC in at least one of three datasets. TNXB and CDX2 ranked among the most significant fructose risk DMRs in right and left colon respectively that also displayed altered gene expression in CRC tumors. Conclusions: Our mechanistic data support the notion that fructose has a greater CRC-related effect in right than left AA colon, alluding to a potential role for fructose in contributing to racial disparities in CRC.

Reversal of dual epigenetic repression of non-canonical Wnt-5a normalises diabetic corneal epithelial wound healing and stem cells.

AIMS/HYPOTHESIS: Diabetes is associated with epigenetic modifications including DNA methylation and miRNA changes. Diabetic complications in the cornea can cause persistent epithelial defects and impaired wound healing due to limbal epithelial stem cell (LESC) dysfunction. In this study, we aimed to uncover epigenetic alterations in diabetic vs non-diabetic human limbal epithelial cells (LEC) enriched in LESC and identify new diabetic markers that can be targeted for therapy to normalise corneal epithelial wound healing and stem cell expression. METHODS: Human LEC were isolated, or organ-cultured corneas were obtained, from autopsy eyes from non-diabetic (59.87±20.89 years) and diabetic (71.93±9.29 years) donors. The groups were not statistically different in age. DNA was extracted from LEC for methylation analysis using Illumina Infinium 850K MethylationEPIC BeadChip and protein was extracted for Wnt phospho array analysis. Wound healing was studied using a scratch assay in LEC or 1-heptanol wounds in organ-cultured corneas. Organ-cultured corneas and LEC were transfected with WNT5A siRNA, miR-203a mimic or miR-203a inhibitor or were treated with recombinant Wnt-5a (200 ng/ml), DNA methylation inhibitor zebularine (1-20 µmol/l) or biodegradable nanobioconjugates (NBCs) based on polymalic acid scaffold containing antisense oligonucleotide (AON) to miR-203a or a control scrambled AON (15-20 µmol/l). RESULTS: There was significant differential DNA methylation between diabetic and non-diabetic LEC. WNT5A promoter was hypermethylated in diabetic LEC accompanied with markedly decreased Wnt-5a protein. Treatment of diabetic LEC and organ-cultured corneas with exogenous Wnt-5a accelerated wound healing by 1.4-fold (p<0.05) and 37% (p<0.05), respectively, and increased LESC and diabetic marker expression. Wnt-5a treatment in diabetic LEC increased the phosphorylation of members of the Ca2+-dependent non-canonical pathway (phospholipase Cγ1 and protein kinase Cß; by 1.15-fold [p<0.05] and 1.36-fold [p<0.05], respectively). In diabetic LEC, zebularine treatment increased the levels of Wnt-5a by 1.37-fold (p<0.01)and stimulated wound healing in a dose-dependent manner with a 1.6-fold (p<0.01) increase by 24 h. Moreover, zebularine also improved wound healing by 30% (p<0.01) in diabetic organ-cultured corneas and increased LESC and diabetic marker expression. Transfection of these cells with WNT5A siRNA abrogated wound healing stimulation by zebularine, suggesting that its effect was primarily due to inhibition of WNT5A hypermethylation. Treatment of diabetic LEC and organ-cultured corneas with NBC enhanced wound healing by 1.4-fold (p<0.01) and 23.3% (p<0.05), respectively, with increased expression of LESC and diabetic markers. CONCLUSIONS/INTERPRETATION: We provide the first account of epigenetic changes in diabetic corneas including dual inhibition of WNT5A by DNA methylation and miRNA action. Overall, Wnt-5a is a new corneal epithelial wound healing stimulator that can be targeted to improve wound healing and stem cells in the diabetic cornea. DATA AVAILABILITY: The DNA methylation dataset is available from the public GEO repository under accession no. GSE229328 ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE229328 ).

A Multi-Omics Overview of Colorectal Cancer to Address Mechanisms of Disease, Metastasis, Patient Disparities and Outcomes.

Human colorectal cancer (CRC) is one of the most common malignancies in men and women across the globe, albeit CRC incidence and mortality shows a substantial racial and ethnic disparity, with the highest burden in African American patients. Even with effective screening tools such as colonoscopy and diagnostic detection assays, CRC remains a substantial health burden. In addition, primary tumors located in the proximal (right) or distal (left) sides of the colorectum have been shown to be unique tumor types that require unique treatment schema. Distal metastases in the liver and other organ systems are the major causes of mortality in CRC patients. Characterizing genomic, epigenomic, transcriptomic and proteomic (multi-omics) alterations has led to a better understanding of primary tumor biology, resulting in targeted therapeutic advancements. In this regard, molecular-based CRC subgroups have been developed that show correlations with patient outcomes. Molecular characterization of CRC metastases has highlighted similarities and differences between metastases and primary tumors; however, our understanding as to how to improve patient outcomes based on metastasis biology is lagging and remains a major obstacle to improving CRC patient outcomes. In this review, we will summarize the multi-omics features of primary CRC tumors and their metastases across racial and ethnic groups, the differences in proximal and distal tumor biology, molecular-based CRC subgroups, treatment strategies and challenges for improving patient outcomes.

Multi-omic analysis in normal colon organoids highlights MSH4 as a novel marker of defective mismatch repair in Lynch syndrome and microsatellite instability.

INTRODUCTION: Lynch syndrome (LS) is a hereditary condition that increases the risk of colorectal (CRC) and extracolonic cancers that exhibit microsatellite instability (MSI-H). MSI-H is driven by defective mismatch repair (dMMR), and approximately 15% of nonhereditary CRCs also exhibit MSI-H. Here, we aimed to better define mechanisms underlying tumor initiation in LS and MSI-H cancers through multi-omic analyses of LS normal colon organoids and MSI-H tumors. METHODS: Right (n = 35) and left (n = 23) colon organoids generated from normal colon biopsies at routine colonoscopy of LS and healthy individuals were subjected to Illumina EPIC array. Differentially methylated region (DMR) analysis was performed by DMRcate. RNA-sequencing (n = 16) and bisulfite-sequencing (n = 15) were performed on a subset of right colon organoids. CRISPR-cas9-mediated editing of MMR genes in colon organoids of healthy individuals was followed by quantitative PCR of MSH4. The relationship between MSH4 expression and tumor mutational burden was further explored in three independent tumor data sets. RESULTS: We identified a hypermethylated region of MSH4 in both the right and left colon organoids of LS versus healthy controls, which we validated using bisulfite-sequencing. DMR analysis in three gastrointestinal and one endometrial data set revealed that this region was also hypermethylated in MSI-H versus microsatellite stable (MSS) tumors. MSH4 expression was increased in colon organoids of LS versus healthy subjects and in publicly available MSI-H versus MSS tumors across four RNA-seq and four microarray data sets. CRISPR-cas9 editing of MLH1 and MSH2, but not MSH6, in normal colon organoids significantly increased MSH4 expression. MSH4 expression was significantly associated with tumor mutational burden in three publicly available data sets. CONCLUSIONS: Our findings implicate DNA methylation and gene expression differences of MSH4 as a marker of dMMR and as a potential novel biomarker of LS. Our study of LS colon organoids supports the hypothesis that dMMR exists in the colons of LS subjects prior to CRC.

Analysis of Pregnancy Complications and Epigenetic Gestational Age of Newborns.

Importance: Preeclampsia, gestational hypertension, and gestational diabetes, the most common pregnancy complications, are associated with substantial morbidity and mortality in mothers and children. Little is known about the biological processes that link the occurrence of these pregnancy complications with adverse child outcomes; altered biological aging of the growing fetus up to birth is one molecular pathway of increasing interest. Objective: To evaluate whether exposure to each of these 3 pregnancy complications (gestational diabetes, gestational hypertension, and preeclampsia) is associated with accelerated or decelerated gestational biological age in children at birth. Design, Setting, and Participants: Children included in these analyses were born between 1998 and 2018 and spanned multiple geographic areas of the US. Pregnancy complication information was obtained from maternal self-report and/or medical record data. DNA methylation measures were obtained from blood biospecimens collected from offspring at birth. The study used data from the national Environmental Influences on Child Health Outcomes (ECHO) multisite cohort study collected and recorded as of the August 31, 2021, data lock date. Data analysis was performed from September 2021 to December 2022. Exposures: Three pregnancy conditions were examined: gestational hypertension, preeclampsia, and gestational diabetes. Main Outcomes and Measures: Accelerated or decelerated biological gestational age at birth, estimated using existing epigenetic gestational age clock algorithms. Results: A total of 1801 child participants (880 male [48.9%]; median [range] chronological gestational age at birth, 39 [30-43] weeks) from 12 ECHO cohorts met the analytic inclusion criteria. Reported races included Asian (49 participants [2.7%]), Black (390 participants [21.7%]), White (1026 participants [57.0%]), and other races (92 participants [5.1%]) (ie, American Indian or Alaska Native, Native Hawaiian or other Pacific Islander, multiple races, and other race not specified). In total, 524 participants (29.0%) reported Hispanic ethnicity. Maternal ages ranged from 16 to 45 years of age with a median of 29 in the analytic sample. A range of maternal education levels, from less than high school (260 participants [14.4%]) to Bachelor's degree and above (629 participants [34.9%]), were reported. In adjusted regression models, prenatal exposure to maternal gestational diabetes (ß, -0.423; 95% CI, -0.709 to -0.138) and preeclampsia (ß, -0.513; 95% CI, -0.857 to -0.170), but not gestational hypertension (ß, 0.003; 95% CI, -0.338 to 0.344), were associated with decelerated epigenetic aging among exposed neonates vs those who were unexposed. Modification of these associations, by sex, was observed with exposure to preeclampsia (ß, -0.700; 95% CI, -1.189 to -0.210) and gestational diabetes (ß, -0.636; 95% CI, -1.070 to -0.200), with associations observed among female but not male participants. Conclusions and Relevance: This US cohort study of neonate biological changes related to exposure to maternal pregnancy conditions found evidence that preeclampsia and gestational diabetes delay biological maturity, especially in female offspring.

RNA mis-splicing drives viral mimicry response after DNMTi therapy in SETD2-mutant kidney cancer.

Tumors with mutations in chromatin regulators present attractive targets for DNA hypomethylating agent 5-aza-2'-deoxycytidine (DAC) therapy, which further disrupts cancer cells' epigenomic fidelity and reactivates transposable element (TE) expression to drive viral mimicry responses. SETD2 encodes a histone methyltransferase (H3K36me3) and is prevalently mutated in advanced kidney cancers. Here, we show that SETD2-mutant kidney cancer cells are especially sensitive in vitro and in vivo to DAC treatment. We find that the viral mimicry response are direct consequences of mis-splicing events, such as exon inclusions or extensions, triggered by DAC treatment in an SETD2-loss context. Comprehensive epigenomic analysis reveals H3K9me3 deposition, rather than DNA methylation dynamics, across intronic TEs might contribute to elevated mis-splicing rates. Through epigenomic and transcriptomic analyses, we show that SETD2-deficient kidney cancers are prone to mis-splicing, which can be therapeutically exacerbated with DAC treatment to increase viral mimicry activation and provide synergy with combinatorial immunotherapy approaches.

Integrated DNA Methylation/RNA Profiling in Middle Temporal Gyrus of Alzheimer's Disease.

Alzheimer's disease is a neurodegenerative disorder clinically defined by gradual cognitive impairment and alteration in executive function. We conducted an epigenome-wide association study (EWAS) of a clinically and neuropathologically characterized cohort of 296 brains, including Alzheimer's disease (AD) and non-demented controls (ND), exploring the relationship with the RNA expression from matched donors. We detected 5246 CpGs and 832 regions differentially methylated, finding overlap with previous EWAS but also new associations. CpGs previously identified in ANK1, MYOC, and RHBDF2 were differentially methylated, and one of our top hits (GPR56) was not previously detected. ANK1 was differentially methylated at the region level, along with APOE and RHBDF2. Only a small number of genes showed a correlation between DNA methylation and RNA expression statistically significant. Multiblock partial least-squares discriminant analysis showed several CpG sites and RNAs discriminating AD and ND (AUC = 0.908) and strongly correlated with each other. Furthermore, the CpG site cg25038311 was negatively correlated with the expression of 22 genes. Finally, with the functional epigenetic module analysis, we identified a protein-protein network characterized by inverse RNA/DNA methylation correlation and enriched for "Regulation of insulin-like growth factor transport", with IGF1 as the hub gene. Our results confirm and extend the previous EWAS, providing new information about a brain region not previously explored in AD DNA methylation studies. The relationship between DNA methylation and gene expression is not significant for most of the genes in our sample, consistently with the complexities in the gene expression regulation.

The Role of DNA Methylation and DNA Methyltransferases in Cancer.

The malignant transformation of normal cells is driven by both genetic and epigenetic changes. With the advent of next-generation sequencing and large-scale international consortia, it is now possible to profile the genomes and epigenomes of thousands of primary tumors from nearly every cancer type. These studies clearly demonstrate that the dynamic regulation of DNA methylation is a critical epigenetic mechanism of cancer initiation, maintenance, and progression. Proper control of DNA methylation is not only crucial for regulating gene transcription and tissue-specific cellular functions, but its broader consequences include maintaining the integrity of the genome and modulating the immune response. Here, we describe the aberrant DNA methylation changes in human cancers and how they contribute to the disease phenotypes. Aside from CpG island promoter DNA hypermethylation-based gene silencing, human cancers also display gene body DNA hypomethylation that is also associated with downregulated gene expression. In addition, the implementation of whole genome bisulfite sequencing (WGBS) has unveiled DNA hypomethylation of large blocks of the genome, known as partially methylated domains (PMDs), as well as cancer-specific DNA methylation aberrancies at enhancers and super-enhancers. Integrating WGBS and DNA methylation array data with mutation, copy number, and gene expression data has allowed for the identification of novel tumor suppressor genes and candidate driver genes of the disease state. Finally, we highlight potential clinical implications of these changes in the context of prognostic and diagnostic biomarkers, as well as therapeutic targets. Mounting evidence shows that DNA methylation data are effective and highly-sensitive disease classifiers, not only from analyses of the primary tumor but also from tumor-derived, cell free DNA (cfDNA) in blood of cancer patients. These findings highlight the power of DNA methylation aberrancies in providing efficacious biomarkers for clinical utility in improving patient diagnostics and their reversal using DNA methylation inhibitors in cancer treatment may be key in surveillance, treatment, and quality of life for cancer patients.

DNMT and EZH2 inhibitors synergize to activate therapeutic targets in hepatocellular carcinoma.

The development of more effective targeted therapies for hepatocellular carcinoma (HCC) patients due to its aggressiveness is urgently needed. DNA methyltransferase inhibitors (DNMTis) represented the first clinical breakthrough to target aberrant cancer epigenomes. However, their clinical efficacies are still limited, in part due to an "epigenetic switch" in which a large group of genes that are demethylated by DNMTi treatment remain silenced by polycomb repressive complex 2 (PRC2) occupancy. EZH2 is the member of PRC2 that catalyzes the placement of H3K27me3 marks. EZH2 overexpression is correlated with poor HCC patient survival. We tested the combination of a DNMTi (5-aza-2'-deoxycytidine, DAC) and the EZH2 inhibitor (EZH2i) GSK126 in human HCC cell lines on drug sensitivity, DNA methylation, nucleosome accessibility, and gene expression profiles. Compared with single agent treatments, all HCC cell lines studied showed increased sensitivity after receiving both drugs concomitant with prolonged anti-proliferative changes and sustained reactivation of nascently-silenced genes. The increased number of up-regulated genes after combination treatment correlated with prolonged anti-proliferation effects and increased nucleosome accessibility. Combination treatments also activate demethylated promoters that are repressed by PRC2 occupancy. Furthermore, 13-31% of genes down-regulated by DNA methylation in primary HCC tumors were reactivated through this combination treatment scheme in vitro. Finally, the combination treatment also exacerbates anti-tumor immune responses, while most of these genes were downregulated in over 50% of primary HCC tumors. We have linked the anti-tumor effects of DAC and GSK126 combination treatments to detailed epigenetic alterations in HCC cells, identified potential therapeutic targets and provided a rationale for treatment efficacy for HCC patients.

The Multi-Omic Landscape of Primary Breast Tumors and Their Metastases: Expanding the Efficacy of Actionable Therapeutic Targets.

Breast cancer (BC) mortality is almost exclusively due to metastasis, which is the least understood aspect of cancer biology and represents a significant clinical challenge. Although we have witnessed tremendous advancements in the treatment for metastatic breast cancer (mBC), treatment resistance inevitably occurs in most patients. Recently, efforts in characterizing mBC revealed distinctive genomic, epigenomic and transcriptomic (multi-omic) landscapes to that of the primary tumor. Understanding of the molecular underpinnings of mBC is key to understanding resistance to therapy and the development of novel treatment options. This review summarizes the differential molecular landscapes of BC and mBC, provides insights into the genomic heterogeneity of mBC and highlights the therapeutically relevant, multi-omic features that may serve as novel therapeutic targets for mBC patients.

Characterizing DNA methylation signatures of retinoblastoma using aqueous humor liquid biopsy.

Retinoblastoma (RB) is a cancer that forms in the developing retina of babies and toddlers. The goal of therapy is to cure the tumor, save the eye and maximize vision. However, it is difficult to predict which eyes are likely to respond to therapy. Predictive molecular biomarkers are needed to guide prognosis and optimize treatment decisions. Direct tumor biopsy is not an option for this cancer; however, the aqueous humor (AH) is an alternate source of tumor-derived cell-free DNA (cfDNA). Here we show that DNA methylation profiling of the AH is a valid method to identify the methylation status of RB tumors. We identify 294 genes directly regulated by methylation that are implicated in p53 tumor suppressor (RB1, p53, p21, and p16) and oncogenic (E2F) pathways. Finally, we use AH to characterize molecular subtypes that can potentially be used to predict the likelihood of treatment success for retinoblastoma patients.

DNA methylation analysis of normal colon organoids from familial adenomatous polyposis patients reveals novel insight into colon cancer development.

BACKGROUND: Familial adenomatous polyposis (FAP) is an inherited colorectal cancer (CRC) syndrome resulting from germ line mutations in the adenomatous polyposis coli (APC) gene. While FAP accounts for less than 1% of all CRC cases, loss of APC expression is seen in > 80% of non-hereditary CRCs. To better understand molecular mechanisms underlying APC-driven CRC, we performed an epigenome-wide analysis of colon organoids derived from normal-appearing colons of FAP patients versus healthy subjects to identify differentially methylated regions (DMRs) that may precede the onset of CRC. RESULTS: We identified 358 DMRs when comparing colon organoids of FAP patients to those of healthy subjects (FDR < 0.05, |mean beta difference| = 5%). Of these, nearly 50% of DMRs were also differentially methylated in at least one of three CRC tumor and normal adjacent tissue (NAT) cohorts (TCGA-COAD, GSE193535 and ColoCare). Moreover, 27 of the DMRs mapped to CRC genome-wide association study (GWAS) loci. We provide evidence suggesting that some of these DMRs led to significant differences in gene expression of adjacent genes using quantitative PCR. For example, we identified significantly greater expression of five genes: Kazal-type serine peptidase inhibitor domain 1 (KAZALD1, P = 0.032), F-Box and leucine-rich repeat protein 8 (FBXL8, P = 0.036), TRIM31 antisense RNA 1 (TRIM31-AS1, P = 0.036), Fas apoptotic inhibitory molecule 2 (FAIM2, P = 0.049) and (Collagen beta (1-0)galactosyltransferase 2 (COLGALT2, P = 0.049). Importantly, both FBXL8 and TRIM31-AS1 were also significantly differentially expressed in TCGA-COAD tumor versus matched NAT, supporting a role for these genes in CRC tumor development. CONCLUSIONS: We performed the first DNA methylome-wide analysis of normal colon organoids derived from FAP patients compared to those of healthy subjects. Our results reveal that normal colon organoids from FAP patients exhibit extensive epigenetic differences compared to those of healthy subjects that appear similar to those exhibited in CRC tumor. Our analyses therefore identify DMRs and candidate target genes that are potentially important in CRC tumor development in FAP, with potential implications for non-hereditary CRC.

Epigenetic dysregulation in meningiomas.

Background: Meningiomas are the most common primary brain tumor. Though typically benign with a low mutational burden, tumors with benign histology may behave aggressively and there are no proven chemotherapies. Although DNA methylation patterns distinguish subgroups of meningiomas and have higher predictive value for tumor behavior than histologic classification, little is known about differences in DNA methylation between meningiomas and surrounding normal dura tissue. Methods: Whole-exome sequencing and methylation array profiling were performed on 12 dura/meningioma pairs (11 WHO grade I and 1 WHO grade II). Single-nucleotide polymorphism (SNP) genotyping and methylation array profiling were performed on an additional 19 meningiomas (9 WHO grade I, 5 WHO grade II, 4 WHO grade III). Results: Using multimodal studies of meningioma/dura pairs, we identified 4 distinct DNA methylation patterns. Diffuse DNA hypomethylation of malignant meningiomas readily facilitated their identification from lower-grade tumors by unsupervised clustering. All clusters and 12/12 meningioma-dura pairs exhibited hypomethylation of the gene promoters of a module associated with the craniofacial patterning transcription factor FOXC1 and its upstream lncRNA FOXCUT. Furthermore, we identified an epigenetic continuum of increasing hypermethylation of polycomb repressive complex target promoters with increasing histopathologic grade. Conclusion: These findings support future investigations of the role of epigenetic dysregulation of FOXC1 and cranial patterning genes in meningioma formation as well as studies of the utility of polycomb inhibitors for the treatment of malignant meningiomas.

Fluctuating methylation clocks for cell lineage tracing at high temporal resolution in human tissues.

Molecular clocks that record cell ancestry mutate too slowly to measure the short-timescale dynamics of cell renewal in adult tissues. Here, we show that fluctuating DNA methylation marks can be used as clocks in cells where ongoing methylation and demethylation cause repeated 'flip-flops' between methylated and unmethylated states. We identify endogenous fluctuating CpG (fCpG) sites using standard methylation arrays and develop a mathematical model to quantitatively measure human adult stem cell dynamics from these data. Small intestinal crypts were inferred to contain slightly more stem cells than the colon, with slower stem cell replacement in the small intestine. Germline APC mutation increased the number of replacements per crypt. In blood, we measured rapid expansion of acute leukemia and slower growth of chronic disease. Thus, the patterns of human somatic cell birth and death are measurable with fluctuating methylation clocks (FMCs).

Genes regulated by DNA methylation are involved in distinct phenotypes during melanoma progression and are prognostic factors for patients.

In addition to mutations, epigenetic alterations are important contributors to malignant transformation and tumor progression. The aim of this work was to identify epigenetic events in which promoter or gene body DNA methylation induces gene expression changes that drive melanocyte malignant transformation and metastasis. We previously developed a linear mouse model of melanoma progression consisting of spontaneously immortalized melanocytes, premalignant melanocytes, a nonmetastatic tumorigenic, and a metastatic cell line. Here, through the integrative analysis of methylome and transcriptome data, we identified the relationship between promoter and/or gene body DNA methylation alterations and gene expression in early, intermediate, and late stages of melanoma progression. We identified adenylate cyclase type 3 (Adcy3) and inositol polyphosphate 4-phosphatase type II (Inpp4b), which affect tumor growth and metastatic potential, respectively. Importantly, the gene expression and DNA methylation profiles found in this murine model of melanoma progression were correlated with available clinical data from large population-based primary melanoma cohorts, revealing potential prognostic markers.

Albumin levels predict prognosis in advanced renal cell carcinoma treated with tyrosine kinase inhibitors: a systematic review and meta-analysis.

PURPOSE: With the development of therapy and prognostic criteria for metastatic Renal Cell Carcinoma (mRCC), the prognostic value of serum albumin level has remained in dispute. The aim of this meta-analysis was to evaluate the role of pre-treatment albumin in predicting the prognosis of mRCC patients in the era of tyrosine kinase inhibitor (TKI) treatments. METHODS: The qualitative and quantitative synthesis was conducted of studies retrieved from PubMed, Embase, and Cochrane library from inception of these databases to July 19, 2020. The hazard ratio (HR) and its 95% confidence interval (CI) of overall survival (OS) and progression-free survival (PFS) were extracted from studies comparing different levels of pre-treatment serum albumin (as a dichotomous or continuous variable) in mRCC patients treated with TKI agents. RESULTS: Within 5,638 primitive records, 16 were eligible and 14 had adequate data for quantitative analysis (N = 2,863 participants). Random-effects meta-analysis showed that lower albumin was related to poorer OS (dichotomous: HR = 2.01, 95% CI: 1.64-2.46, P < 0.001, I2 = 28.8%; continuous: HR =0.93, 95% CI: 0.86-1.00, P = 0.040, I2 = 67.5%) and PFS (dichotomous: HR = 1.45, 95% CI: 1.04-2.01, P = 0.029, I2 = 57.4%; continuous: HR = 0.89, 95% CI: 0.80-0.98, P = 0.023, I2 = 93.3%). CONCLUSION: Lower pre-treatment serum albumin level is an independent adverse predictor of prognosis of mRCC patients receiving TKI therapy. REGISTRATION: PROSPERO ID: CRD42020196802 Sep. 2nd, 2020.

A Race-Specific, DNA Methylation Analysis of Aging in Normal Rectum: Implications for the Biology of Aging and Its Relationship to Rectal Cancer.

Approximately 90% of colorectal cancer (CRC) develop over the age of 50, highlighting the important role of aging in CRC risk. African Americans (AAs) shoulder a greater CRC burden than European Americans (EA) and are more likely to develop CRC at a younger age. The effects of aging in AA and EA normal rectal tissue have yet to be defined. Here, we performed epigenome-wide DNA methylation analysis in the first, large-scale biracial cohort of normal rectum (n = 140 samples). We identified increased epigenetic age acceleration in EA than AA rectum (p = 3.91 × 10-4) using linear regression. We also identified differentially methylated regions (DMRs) associated with chronological aging in AA and EA, separately using DMRcate. Next, a consensus set of regions associated with cancer was identified through DMR analysis of two rectal cancer cohorts. The vast majority of AA DMRs were present in our analysis of aging in rectum of EA subjects, though rates of epigenetic drift were significantly greater in AA (p = 1.94 × 10-45). However, 3.66-fold more DMRs were associated with aging in rectum of EA subjects, many of which were also associated with rectal cancer. Our findings reveal a novel relationship between race, age, DNA methylation and rectal cancer risk that warrants further investigation.