High Levels Of 5hmC Research Articles

Decoding the Epigenetic Landscape: Insights into 5mC and 5hmC Patterns in Mouse Cortical Cell Types.

The DNA modifications, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), represent powerful epigenetic regulators of temporal and spatial gene expression. Yet, how the cooperation of these genome-wide, epigenetic marks determine unique transcriptional signatures across different brain cell populations is unclear. Here we applied Nanopore sequencing of native DNA to obtain a complete, genome-wide, single-base resolution atlas of 5mC and 5hmC modifications in neurons, astrocytes and microglia in the mouse cortex (99% genome coverage, 40 million CpG sites). In tandem with RNA sequencing, analysis of 5mC and 5hmC patterns across cell types reveals astrocytes drive uniquely high brain 5hmC levels and support two decades of research regarding methylation patterns, gene expression and alternative splicing, benchmarking this resource. As such, we provide the most comprehensive DNA methylation data in mouse brain as an interactive, online tool (NAM-Me, https://olsenlab.shinyapps.io/NAMME/) to serve as a resource dataset for those interested in the methylome landscape.

Renal ischemia alters the transcriptomic and epigenetic profile of inflammatory genes in swine scattered tubular-like cells.

Scattered tubular-like cells (STCs) are differentiated renal tubular cells that during recovery from ischemic injury dedifferentiate to repair other injured renal cells. Renal artery stenosis (RAS), often associated with chronic inflammatory injury, compromises the integrity and function of STCs, but the underlying mechanisms remain unknown. We hypothesized that RAS alters the transcriptomic and epigenetic profile of inflammatory genes in swine STCs. STCs were harvested from pig kidneys after 10 weeks of RAS or sham (n=6 each). STC mRNA profiles of inflammatory genes were analyzed using high-throughput mRNA-sequencing (seq) and their DNA methylation (5mC) and hydroxymethylation (5hmC) profiles by DNA immunoprecipitation and next-generation sequencing (MeDIP-seq) (n=3 each), followed by an integrated (mRNA-seq/MeDIP-seq) analysis. STC protein expression of candidate differentially expressed (DE) genes and common proinflammatory proteins were subsequently assessed in vitro before and after epigenetic (Bobcat339) modulation. mRNA-seq identified 57 inflammatory genes up-regulated in RAS-STCs versus Normal-STCs (>1.4 or <0.7-fold, P<0.05), of which 14% exhibited lower 5mC and 5% higher 5hmC levels in RAS-STCs versus Normal-STCs, respectively. Inflammatory gene and protein expression was higher in RAS-STCs compared with Normal-STCs but normalized after epigenetic modulation. These observations highlight a novel modulatory mechanism of this renal endogenous repair system and support development of epigenetic or anti-inflammatory therapies to preserve the reparative capacity of STCs in individuals with RAS.

P12.06.A Relationship between ascorbate and DNA methylation markers in clinical glioma tumours

Abstract Background Members of the 2-oxoglutarate-dependent dioxygenase (OGDD) enzyme family play an important role in gliomas as they regulate epigenetic modifications and response to hypoxia. The OGDDs require 2-OG and O2 as substrates, and ferrous iron and ascorbate as cofactors. Both hypoxia and aberrant DNA methylation are prognostic indicators for gliomas. The ten-eleven translocase (TET) DNA demethylases are OGDDs that convert 5-methyl cytosine (5mC) to 5-hydroxymethylcytosine (5hmC), with 5hmC levels related to better prognosis. Despite this, there is limited data on the OGDD enzymes and their substrates/cofactors in glioma tissues. Our previous study showed an association between ascorbate content and markers of the hypoxic response in glioblastoma tissue. Here we determine whether there is an association between ascorbate and DNA methylation in glioma. In addition, we assess whether methylation of the methylguanine-DNA methyltransferase (DNA repair enzyme MGMT) promoter is associated with ascorbate content. Materials and methods Frozen clinical glioma samples from 37 patients (n=11 WHO grade I-III, n=26 glioblastoma) were obtained from the Cancer Society Tissue Bank (Ethics approval H19/163). Isocitrate dehydrogenase 1 (IDH1) mutation status was determined by sequencing. Samples were processed on dry ice in liquid nitrogen and analysed for ascorbate (high-performance liquid chromatography), global DNA methylation (mass spectrometry) and MGMT promoter analyses (methylation specific PCR). Results Many grade I-III tumours were IDH1 R132H mutant (6/11), and most glioblastomas were not (2/26). Glioblastoma had significantly lower ascorbate content than grade I-III tumours (p=0.026). Glioblastoma also had lower global 5hmC levels (p=0.0013). IDH1 R132H tumours tended to have a lower ascorbate content (p=0.09). Ascorbate and 5hmC levels were directly correlated (Spearman r= 0.466, p=0.004). However, cytosine and 5mC showed no association with grade or ascorbate. MGMT promoter methylation status was not associated with global methylation or ascorbate content (p=0.97, p=0.96, respectively). Conclusion Our data suggests that ascorbate supports TET activity in clinical glioma. It also appears that site-specific (promoter) methylation was not affected by ascorbate availability. These findings may have clinical implications, as higher 5hmC levels are associated with improved outcome, whilst continued MGMT suppression suggests chemotherapy responsiveness. However, evidence that raising tumour ascorbate leads to increased 5hmC levels, or an associated improvement in survival, requires intervention trials.

Ascorbate content of clinical glioma tissues is related to tumour grade and to global levels of 5-hydroxymethyl cytosine

Gliomas are incurable brain cancers with poor prognosis, with epigenetic dysregulation being a distinctive feature. 5-hydroxymethylcytosine (5-hmC), an intermediate generated in the demethylation of 5-methylcytosine, is present at reduced levels in glioma tissue compared with normal brain, and that higher levels of 5-hmC are associated with improved patient survival. DNA demethylation is enzymatically driven by the ten–eleven translocation (TET) dioxygenases that require ascorbate as an essential cofactor. There is limited data on ascorbate in gliomas and the relationship between ascorbate and 5-hmC in gliomas has never been reported. Clinical glioma samples (11 low-grade, 26 high-grade) were analysed for ascorbate, global DNA methylation and hydroxymethylation, and methylation status of the O-6-methylguanine-DNA methyltransferase (MGMT) promoter. Low-grade gliomas contained significantly higher levels of ascorbate than high-grade gliomas (p = 0.026). Levels of 5-hmC were significantly higher in low-grade than high-grade glioma (p = 0.0013). There was a strong association between higher ascorbate and higher 5-hmC (p = 0.004). Gliomas with unmethylated and methylated MGMT promoters had similar ascorbate levels (p = 0.96). One mechanism by which epigenetic modifications could occur is through ascorbate-mediated optimisation of TET activity in gliomas. These findings open the door to clinical intervention trials in patients with glioma to provide both mechanistic information and potential avenues for adjuvant ascorbate therapy.

Quantification of Global DNA Hydroxymethylation Level Using UHRF2 SRA-Luciferase Based on Bioluminescence Resonance Energy Transfer.

5-Methylcytosine (5mC) plays an important role in the regulation of gene expression. Ten-eleven translocation (TET) continuously oxidizes 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). High levels of 5hmC are found in the brain and embryonic stem cells, while global hydroxymethylation levels are reduced in several cancer cells. Moreover, alterations in hydroxymethylation levels occur in neurological diseases, such as Alzheimer's disease and Parkinson's disease. In this study, a convenient sensing method for the determination of global hydroxymethylation levels was developed. A bioluminescence resonance energy transfer (BRET) assay for global methylation level determination has been previously reported. In the assay, BOBO-3 DNA intercalating dye is excited by the bioluminescence of methyl-CpG-binding domain-fused firefly luciferase (MBD-Fluc); that is, the BRET signal depends on the content of methylated CpG on genomic DNA. To develop a hydroxymethylation level sensing method, SET- and RING-associated (SRA) domain of ubiquitin-like with PHD and RING finger domains 2 (UHRF2)-fused Fluc (UHRF2 SRA-Fluc) was prepared. UHRF2 SRA is known to bind to both hydroxymethylated and methylated CpG sites; thus, MBD was utilized to mask the methylated CpG on genomic DNA. We demonstrated that the BRET signal between UHRF2 SRA-Fluc and BOBO-3 depends on the global hydroxymethylation level in the presence of MBD (R2 = 0.99, and relative standard deviation < 2.3%). The limit of detection for hydroxymethylated genomic DNA was 0.75 ng μL-1. In this assay, the global hydroxymethylation level was quantified within 40 min in a single tube, indicating that the assay would be utilized not only for clinical diagnostics but also for the elucidation of 5hmC functions.

Ten-eleven translocation-2 inactivation restrains IL-10-producing regulatory B cells to enable antitumor immunity in hepatocellular carcinoma.

IL-10-producing regulatory B cells (IL-10 + B cells), a dominant regulatory B cell (Breg) subset, foster tumor progression. However, the mechanisms underlying their generation in HCC are poorly understood. Ten-eleven translocation-2 (TET2), a predominant epigenetic regulatory enzyme in B cells, regulates gene expression by catalyzing demethylation of 5-methylcytosine into 5-hydroxymethyl cytosine (5hmC). In this study, we investigated the role of TET2 in IL-10 + B cell generation in HCC and its prospects for clinical application. TET2 activation in B cells triggered by oxidative stress from the HCC microenvironment promoted IL-10 expression, whereas adoptive transfer of Tet2 -deficient B cells suppressed HCC progression. The aryl hydrocarbon receptor is required for TET2 to hydroxylate Il10 . In addition, high levels of IL-10, TET2, and 5hmc in B cells indicate poor prognosis in patients with HCC. Moreover, we determined TET2 activity using 5hmc in B cells to evaluate the efficacy of anti-programmed death 1 (anti-PD-1) therapy. Notably, TET2 inhibition in B cells facilitates antitumor immunity to improve anti-PD-1 therapy for HCC. Our findings propose a TET2-dependent epigenetic intervention targeting IL-10 + B cell generation during HCC progression and identify the inhibition of TET2 activity as a promising combination therapy with immune checkpoint inhibitors for HCC.

TET1 promotes RXRα expression and adipogenesis through DNA demethylation

Adipose tissue is important for systemic metabolic homeostasis in response to environmental changes, and adipogenesis involves dynamic transcriptional regulation. Ten-eleven translocation (TET) enzymes (TET1, 2 and 3) oxidize the 5-methylcytosine (5mC) in DNA to 5-hydroxylmethylcytosine (5hmC), which associates with transcriptional activation. Step by step, 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) are further generated by TETs and the cytosine can be restored through base-excision repair. It is still unclear how DNA demethylation is involved in adipogenesis. Through a phenotypic screen, we found TET inhibition decreased adipocyte differentiation from mesenchymal stem cells (MSCs). Comparing with the undifferentiated MSCs, the differentiated adipocytes exhibited much higher levels of 5hmC and slightly increased 5fC and 5caC. Higher 5hmC was associated with better differentiation at single-cell level by image analysis. TET1 is upregulated in differentiation and depletion of it significantly impaired the gain of 5hmC. Furthermore, Tet1 depletion significantly hampered the adipocyte differentiation. Using RNA-seq, 5mC and 5hmC-DNA immunoprecipitation, we found that Tet1 knockout led to lower expression of genes associated with lipid metabolism and fat cell differentiation. Genes with loss of 5mC or gain of 5hmC in adipocytes include Lipe, Bmp4 and Rxra, etc. RXRα agonist partially rescued the inhibitory effect of Tet1 knockout for adipogenesis. So, Rxra is one of the critical TET1 modulated genes. Together, TET1-mediated active DNA demethylation plays an important role in adipogenesis.

Abstract PO-059: SOX2-mediated 5hmC dysregulation in GBM Stem Cells

Abstract Primary brain tumors are among the most devastating forms of cancer and glioblastoma (GBM) represents the most aggressive and lethal form of the disease. We now know that GBM contain small subsets of cells that display tumor-propagating stem-like phenotypes (i.e. glioma stem cells or GSCs) that act as critical determinants of GBM resistance to current treatments and tumor recurrence for which there is no proven therapy. Altered patterns of DNA methylation are widely reported in human GBM. However, substantial knowledge gaps remain in our understanding of the molecular mechanisms responsible for this epigenetic dysregulation, its downstream consequences and role in the GBM tumor propagating phenotype. Understanding and ultimately targeting the epigenetic mechanisms that induce and maintain these tumor-propagating cell subsets is critical to improving GBM therapy and patient outcomes. DNA methylation generally occurs in cytosine-guanine (CpG) sequences and is established by DNMTs, which catalyze the conversion of cytosine to 5-methylcytosine (5mC). DNA methylation is a reversible process and is partially mediated by the ten-eleven translocation (TET) family of enzymes which function as deoxygenases to catalyze the conversion of 5mC to 5-hydroxymethylcytosine (5hmC). Levels of 5hmC closely track with the differentiation state of cells with terminally differentiated cells containing high levels of 5hmC and less differentiated cells having very low levels. Multiple studies found negative correlations between 5hmC levels and glioma grade and loss of 5hmC correlates with poor prognosis of GBM patients, strongly suggesting that these enzymes activate tumor suppressing mechanisms. Our preliminary data shows that exogenous SOX2 represses the TET2 demethylase and decreases 5hmC, the enzymatic product catalyzed by TET proteins, in GSCs. TET2 repression using 2 independent shRNA hairpins efficiently decreases 5hmc levels and significantly enhanced self-renewal capacity and tumor growth capacity of low-passage GSC isolates. We also show that SOX2 induces expression of miR-10b-5p, a known onco-miR predicted to target TET2, and inhibiting this miRNA partially rescues the reduction in 5hmC expression observed in GSCs expressing transgenic SOX2, thus implicating miR-10b-5p as a critical mediator of SOX2-induced onco-methylation, GSC induction and glioma malignancy. Our preliminary results support a mechanism in which SOX2 represses TET2 leading to 5hmC loss by directly activating miR-10b-5p transcription and predicts that targeting this novel mechanism of epigenetic dysregulation by inhibiting miR-10b-5p in vivo will can lead to pre-clinical GBM therapeutics. Citation Format: Hernando Lopez-Bertoni. SOX2-mediated 5hmC dysregulation in GBM Stem Cells [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-059.

DNA hydroxymethylation is associated with disease severity and persists at enhancers of oncogenic regions in multiple myeloma

BackgroundMultiple myeloma (MM) is a heterogeneous plasma cell malignancy that remains challenging to cure. Global hypomethylation correlates with an aggressive phenotype of the disease, while hypermethylation is observed at particular regions of myeloma such as B cell-specific enhancers. The recently discovered active epigenetic mark 5-hydroxymethylCytosine (5hmC) may also play a role in tumor biology; however, little is known about its level and distribution in myeloma. In this study, we investigated the global level and the genomic localization of 5hmC in myeloma cells from 40 newly diagnosed patients, including paired relapses, and of control individuals.ResultsCompared to normal plasma cells, we found global 5hmC levels to be lower in myeloma (P < 0.001). Higher levels of 5hmC were found in lower grades of the International Staging System prognostic index (P < 0.05) and tend to associate with a longer overall survival (P < 0.1). From the hydroxymethylome data, we observed that the remaining 5hmC is organized in large domains overlapping with active chromatin marks and chromatin opening. We discovered that 5hmC strongly persists at key oncogenic genes such as CCND1, CCND2 and MMSET and characterized domains that are specifically hydroxymethylated in myeloma subgroups. Novel 5hmC-enriched domains were found at putative enhancers of CCND2 and MYC in newly diagnosed patients.Conclusions5hmC level is associated with clinical aspects of MM. Mapping 5hmC at a genome-wide level provides insights into the disease biology directly from genomic DNA, which makes it a potent mark to study epigenetics on large patient cohorts.

DNA Methylation and Demethylation Are Regulated by Functional DNA Methyltransferases and DnTET Enzymes in Diuraphis noxia.

Aphids are economically important insect pests of crops worldwide. Despite resistant varieties being available, resistance is continuously challenged and eventually broken down, posing a threat to food security. In the current study, the epigenome of two related Russian wheat aphid (Diuraphis noxia, Kurdjumov) biotypes (i.e., SA1 and SAM) that differ in virulence was investigated to elucidate its role in virulence in this species. Whole genome bisulfite sequencing covered a total of 6,846,597,083 cytosine bases for SA1 and 7,397,965,699 cytosine bases for SAM, respectively, of which a total of 70,861,462 bases (SA1) and 74, 073,939 bases (SAM) were methylated, representing 1.126 ± 0.321% (SA1) and 1.105 ± 0.295% (SAM) methylation in their genomes. The sequence reads were analyzed for contexts of DNA methylation and the results revealed that RWA has methylation in all contexts (CpG, CHG and CHH), with the majority of methylation within the CpG context (± 5.19%), while the other contexts show much lower levels of methylation (CHG − ± 0.27%; CHH − ± 0.34%). The top strand was slightly (0.02%) more methylated than the bottom strand. Of the 35,493 genes that mapped, we also analyzed the contexts of methylation of each of these and found that the CpG methylation was much higher in genic regions than in intergenic regions. The CHG and CHH levels did not differ between genic and intergenic regions. The exonic regions of genes were more methylated (±0.56%) than the intronic regions. We also measured the 5mC and 5hmC levels between the aphid biotypes, and found little difference in 5mC levels between the biotypes, but much higher levels of 5hmC in the virulent SAM. RWA had two homologs of each of the DNA methyltransferases 1 (DNMT1a and DNMT1b) and DNMT3s (DNMT3a and DNMT3b), but only a single DNMT2, with only the expression of DNMT3 that differed significantly between the two RWA biotypes. RWA has a single ortholog of Ten eleven translocase (DnTET) in the genome. Feeding studies show that the more virulent RWA biotype SAM upregulate DnDNMT3 and DnTET in response to wheat expressing antibiosis and antixenosis.

Characterization of global 5-hydroxymethylcytosine in pediatric posterior fossa ependymoma

Background5-Hydroxymethylcytosine (5hmC) is a novel epigenetic mark and may be involved in the mechanisms of tumorigenesis and malignant transformation. However, the role of 5hmC in ependymoma, the third most common brain tumor in children, remains unclear. The aim of this study sought to identify the characterization of 5hmC levels in pediatric posterior fossa ependymoma and to evaluate whether 5hmC levels could be a potential factor to predict clinical outcomes.ResultsOur results showed that 5hmC levels were globally decreased in posterior fossa ependymoma compared with normal cerebellum tissues (P < 0.001). Group A posterior fossa ependymomas had higher 5hmC levels than group B tumors (P = 0.007). Moreover, 5hmC levels positively correlated with Ki-67 index in posterior fossa ependymoma (r = 0.428, P = 0.003). Multivariate Cox hazards model revealed that patients with high 5hmC levels (> 0.102%) had worse PFS and OS than patients with lower 5hmC levels (< 0.102%) (PFS: HR = 3.014; 95% CI, 1.040–8.738; P = 0.042; OS: HR = 2.788; 95% CI, 0.974–7.982; P = 0.047).ConclusionsOur findings suggest that loss of 5hmC is an epigenetic hallmark for pediatric posterior fossa ependymoma. 5hmC levels may represent a potential biomarker to predict prognosis in children with posterior fossa ependymoma.

Abstract B010: SOX2-mediated 5hmC dysregulation in GBM Stem Cells

Abstract Primary brain tumors are among the most devastating forms of cancer and glioblastoma (GBM) represents the most aggressive and lethal form of the disease. We now know that GBM contain small subsets of cells that display tumor-propagating stem-like phenotypes (i.e. glioma stem cells or GSCs) that act as critical determinants of GBM resistance to current treatments and tumor recurrence for which there is no proven therapy. Altered patterns of DNA methylation are widely reported in human GBM. However, substantial knowledge gaps remain in our understanding of the molecular mechanisms responsible for this epigenetic dysregulation, its downstream consequences and role in the GBM tumor propagating phenotype. Understanding and ultimately targeting the epigenetic mechanisms that induce and maintain these tumor-propagating cell subsets is critical to improving GBM therapy and patient outcomes. DNA methylation generally occurs in cytosine-guanine (CpG) sequences and is established by DNMTs, which catalyze the conversion of cytosine to 5-methylcytosine (5mC). DNA methylation is a reversible process and is partially mediated by the ten-eleven translocation (TET) family of enzymes which function as deoxygenases to catalyze the conversion of 5mC to 5-hydroxymethylcytosine (5hmC). Levels of 5hmC closely track with the differentiation state of cells with terminally differentiated cells containing high levels of 5hmC and less differentiated cells having very low levels. Multiple studies found negative correlations between 5hmC levels and glioma grade and loss of 5hmC correlates with poor prognosis of GBM patients, strongly suggesting that these enzymes activate tumor suppressing mechanisms. Our preliminary data shows that exogenous SOX2 represses the TET2 demethylase and decreases 5hmC, the enzymatic product catalyzed by TET proteins, in GSCs. TET2 repression using 2 independent shRNA hairpins efficiently decreases 5hmc levels and significantly enhanced self-renewal capacity and tumor growth capacity of low-passage GSC isolates. We also show that SOX2 induces expression of miR-10b-5p, a known onco-miR predicted to target TET2, and inhibiting this miRNA partially rescues the reduction in 5hmC expression observed in GSCs expressing transgenic SOX2, thus implicating miR-10b-5p as a critical mediator of SOX2-induced onco-methylation, GSC induction and glioma malignancy. Our preliminary results support a mechanism in which SOX2 represses TET2 leading to 5hmC loss by directly activating miR-10b-5p transcription and predicts that targeting this novel mechanism of epigenetic dysregulation by inhibiting miR-10b-5p in vivo will can lead to pre-clinical GBM therapeutics. Citation Format: Hernando Lopez-Bertoni. SOX2-mediated 5hmC dysregulation in GBM Stem Cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B010. doi:10.1158/1535-7163.TARG-19-B010

Abstract 829: SOX2-mediated 5hmC dysregulation in GBM stem cells

Abstract Primary brain tumors are among the most devastating forms of cancer and glioblastoma (GBM) represents the most aggressive and lethal form of the disease. We now know that GBM contains small subsets of cells that display tumor-propagating stem-like phenotypes (i.e. glioma stem cells or GSCs) that act as critical determinants of resistance to current treatments and tumor recurrence for which there is no proven therapy. Altered patterns of DNA methylation are widely reported in human GBM. However, substantial knowledge gaps remain in our understanding of the molecular mechanisms responsible for this epigenetic dysregulation, its downstream consequences and its role in the GBM tumor-propagating phenotype. Understanding and ultimately targeting the epigenetic mechanisms that induce and maintain these tumor-propagating cell subsets is critical to improving GBM therapy and patient outcomes. DNA methylation generally occurs in cytosine-guanine (CpG) sequences and is established by DNMTs, which catalyze the conversion of cytosine to 5-methylcytosine (5mC). DNA methylation is dynamically regulated and reversible by the ten-eleven translocation (TET) family of deoxygenases that catalyze the conversion of 5mC to 5-hydroxymethylcytosine (5hmC). Levels of 5hmC closely align with the differentiation state of cells; terminally differentiated cells contain high levels of 5hmC and less differentiated cells have very low levels. Multiple studies found negative correlations between 5hmC levels and glioma grade and loss of 5hmC correlates with poor prognosis of GBM patients. This strongly suggests that tumor suppressing epigenetic mechanisms are driven by TET-dependent 5hmC generation. We now show that SOX2, a reprogramming transcription factor that induces GSCs, represses the TET2 and decreases 5hmC in multiple GSC isolates. TET2 repression using two independent shRNA hairpins efficiently decreased 5hmc levels and significantly enhanced self-renewal capacity and tumor growth capacity of low-passage GSCs. We also show that low concentrations of vitamin C, a TET enzyme cofactor, significantly increases 5hmC in low-passage GSCs. This epigenetic change induced by vitamin C associated with increased GSC death induced by temozolomide (TMZ), the main chemotherapeutic used to treat GBM. These findings support a mechanism by which SOX2 induces loss of 5hmC in GSCs by repressing TET2 and show that vitamin C can reverse this epigenetic tumor promoting pathway and potentially enhance GBM chemotherapeutics. Citation Format: Sweta Sudhir, Hernando Lopez-Bertoni, Maria Lugo-Fagundo, Bachchu Lal, John Laterra. SOX2-mediated 5hmC dysregulation in GBM stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 829.

Abstract 4325: 5-hydroxymethylation captures the heterogeneity in keratinization and cytoskeletal rearrangement processes in head &amp; neck cancers

Abstract Infection with high-risk HPV, or human papillomavirus, has been shown to be a risk factor for head and neck squamous cell carcinoma (HNSCC) development in addition to the classical risk factors of tobacco and alcohol usage. Previous studies have identified epigenetic differences between these two classes of HNSCC, including DNA methylation, histone modifications and non-coding RNAs. 5-hydroxymethylation (5hmC) is an oxidative derivative of 5-methylcytosine (5mC) and is depleted in human cancers of many different origins. However, the roles of 5hmC in HNSCC is still unknown. Our study is the first to characterize 5hmC in HNSCC, which we profiled in 18 HPV(+) and 18 HPV(-) HNSCC tumors. Since previous studies found higher levels of 5hmC in more differentiated cells and lower levels in stem-like cells, we hypothesized an overall higher 5hmC level in HPV(-) tumors, which tend to be more differentiated in nature. Consistent with our hypothesis, results showed significant hyper-hydroxymethylation in HPV(-) tumors, and detected multiple cancer-related genes with hyper-hydroxymethylation in either HPV(+) and HPV(-) tumors. For example, CDKN2A (p16) and MTAP were the most hyper-methylated genes in HPV(+) tumors, while 5hmC levels of HPV(-) tumors were particularly high in CDH13, TIMP2, and BCAR1. We found that the two HPV(+) subtypes defined by Zhang et al., IMU (heightened immune response, more mesenchymal differentiation) and KRT (more keratinization, more likely viral integration), explained a high level of heterogeneity in 5hmC levels at promoter and enhancer regions. By testing for genes and pathways that demonstrate concordant changes in gene expression and 5hmC levels, we found that keratinocyte differentiation, cell-cell adhesion and adherens junction pathways are both up-regulated and have hyper-hydroxymethylation in HPV(-) tumors. Using predicted enhancers of epidermal keratinocytes, we found a much higher portion of hydroxymethylated regions fall in enhancer regions for HPV(-) samples compared with HPV(+) samples. The higher differential 5hmC in enhancer regions was further validated by stronger histone marks of H3K4me1 and H3K27ac in HPV(-) tumors. Since some of these enhancers can be linked to differentially expressed target genes, this result indicates that the enhancer hydryoxymethylation in HNSCC could also play an important role in downstream gene regulation. We hope that this study could partially explain the different mechanisms and cast light on potential targeted therapies in HNSCC both based on HPV status and different HPV(+) subtypes. Citation Format: Siyu Liu, Katie Zarins, Evan Fernandez, Laura Rozek, Maureen Sartor. 5-hydroxymethylation captures the heterogeneity in keratinization and cytoskeletal rearrangement processes in head &amp; neck cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4325.

Global distribution of DNA hydroxymethylation and DNA methylation in chronic lymphocytic leukemia

BackgroundChronic lymphocytic leukemia (CLL) has been a good model system to understand the functional role of 5-methylcytosine (5-mC) in cancer progression. More recently, an oxidized form of 5-mC, 5-hydroxymethylcytosine (5-hmC) has gained lot of attention as a regulatory epigenetic modification with prognostic and diagnostic implications for several cancers. However, there is no global study exploring the role of 5-hydroxymethylcytosine (5-hmC) levels in CLL. Herein, using mass spectrometry and hMeDIP-sequencing, we analysed the dynamics of 5-hmC during B cell maturation and CLL pathogenesis.ResultsWe show that naïve B-cells had higher levels of 5-hmC and 5-mC compared to non-class switched and class-switched memory B-cells. We found a significant decrease in global 5-mC levels in CLL patients (n = 15) compared to naïve and memory B cells, with no changes detected between the CLL prognostic groups. On the other hand, global 5-hmC levels of CLL patients were similar to memory B cells and reduced compared to naïve B cells. Interestingly, 5-hmC levels were increased at regulatory regions such as gene-body, CpG island shores and shelves and 5-hmC distribution over the gene-body positively correlated with degree of transcriptional activity. Importantly, CLL samples showed aberrant 5-hmC and 5-mC pattern over gene-body compared to well-defined patterns in normal B-cells. Integrated analysis of 5-hmC and RNA-sequencing from CLL datasets identified three novel oncogenic drivers that could have potential roles in CLL development and progression.ConclusionsThus, our study suggests that the global loss of 5-hmC, accompanied by its significant increase at the gene regulatory regions, constitute a novel hallmark of CLL pathogenesis. Our combined analysis of 5-mC and 5-hmC sequencing provided insights into the potential role of 5-hmC in modulating gene expression changes during CLL pathogenesis.

Transcription Factors Drive Tet2-Mediated Enhancer Demethylation to Reprogram Cell Fate

Here, we report DNA methylation and hydroxymethylation dynamics at nucleotide resolution using C/EBPα-enhanced reprogramming of B cells into induced pluripotent cells (iPSCs). We observed successive waves of hydroxymethylation at enhancers, concomitant with a decrease in DNA methylation, suggesting active demethylation. Consistent with this finding, ablation of the DNA demethylase Tet2almost completely abolishes reprogramming. C/EBPα, Klf4, and Tfcp2l1 each interact with Tet2 and recruit the enzyme to specific DNA sites. During reprogramming, some of these sites maintain high levels of5hmC, and enhancers and promoters of key pluripotency factors become demethylated as early as 1dayafter Yamanaka factor induction. Surprisingly, methylation changes precede chromatin opening in distinct chromatin regions, including Klf4 bound sites, revealing a pioneer factor activity associated with alternation in DNA methylation. Rapid changes in hydroxymethylation similar to those in B cells were also observed during compound-accelerated reprogramming of fibroblasts into iPSCs, highlighting the generality of our observations.

Dysregulation and prognostic potential of 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) levels in prostate cancer

BackgroundPrognostic tools for prostate cancer (PC) are inadequate and new molecular biomarkers may improve risk stratification. The epigenetic mark 5-hydroxymethylcytosine (5hmC) has recently been proposed as a novel candidate prognostic biomarker in several malignancies including PC. 5hmC is an oxidized derivative of 5-methylcytosine (5mC) and can be further oxidized to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). The present study is the first to investigate the biomarker potential in PC for all four DNA methylation marks in parallel. Thus, we determined 5mC, 5hmC, 5fC, and 5caC levels in non-malignant (NM) and PC tissue samples from a large radical prostatectomy (RP) patient cohort (n = 546) by immunohistochemical (IHC) analysis of serial sections of a tissue microarray. Possible associations between methylation marks, routine clinicopathological parameters, ERG status, and biochemical recurrence (BCR) after RP were investigated.Results5mC and 5hmC levels were significantly reduced in PC compared to NM prostate tissue samples (p ≤ 0.027) due to a global loss of both marks specifically in ERG− PCs. 5fC levels were significantly increased in ERG+ PCs (p = 0.004), whereas 5caC levels were elevated in both ERG− and ERG+ PCs compared with NM prostate tissue samples (p ≤ 0.019). Positive correlations were observed between 5mC, 5fC, and 5caC levels in both NM and PC tissues (p < 0.001), while 5hmC levels were only weakly positively correlated to 5mC in the PC subset (p = 0.030). There were no significant associations between 5mC, 5fC, or ERG status and time to BCR in this RP cohort. In contrast, high 5hmC levels were associated with BCR in ERG− PCs (p = 0.043), while high 5caC levels were associated with favorable prognosis in ERG+ PCs (p = 0.011) and were borderline significantly associated with worse prognosis in ERG− PCs (p = 0.058). Moreover, a combined high-5hmC/high-5caC score was a significant adverse predictor of post-operative BCR beyond routine clinicopathological variables in ERG− PCs (hazard ratio 3.18 (1.54–6.56), p = 0.002, multivariate Cox regression).ConclusionsThis is the first comprehensive study of 5mC, 5hmC, 5fC, and 5caC levels in PC and the first report of a significant prognostic potential for 5caC in PC.

Differential association of 5-hydroxymethylcytosine levels with clinical and histopathological characteristics in breast cancer tumors.

e23173 Background: Breast cancer is one of the top causes of cancer death worldwide. In Mexico, locally advanced breast cancer (LABC) comprises the majority of the breast cancer stages at diagnosis. Recent studies have suggested that 5-hydroxymethylcytosine (5hmC), could be a prognostic marker in breast cancer. However, the role of 5hmC on clinical and histopathologic characteristics in LABC has not been explored. Methods: From a cohort of locally advanced and advanced breast cancer patients treated at the National Cancer Institute in Mexico City with a 3-year follow-up, we measured 5hmC levels by immunodetection from fresh frozen tissue samples taken from the initial biopsy (N = 193). We determined the association between 5hmC levels and the most relevant clinical and histopathological characteristics. Results: From the full cohort analyzed; 42% were luminal A (n = 82), 33% were luminal B (N = 63), 9% were HER2-positive (N = 18), and 15% were triple-negative tumors. We found higher levels of global 5hmC in HER2 positive tumors vs. all other subtypes (p = 0.028, Kruskal-Wallis test). In luminal B tumors, 5hmC levels were associated with Ki67 (β = -0.04, 95%CI: -0.08, -0.01, p = 0.01 from multivariable model) but not in luminal A. Furthermore, we found that low 5hmC levels were associated with higher histological grade in HER2-positive tumors (p = 0.03, Kruskal-Wallis test). In subgroup analysis of LABC patients, we found higher 5hmC levels in non-ductal vs. ductal invasive tumors (β = 1.38, 95%CI: 0.049, 2.911, p = 0.043 from multivariable model). Also, lower 5hmC levels were associated with Ki67 (β = -2.53, 95%CI: -4.32, -0.74; p = 0.009 from multivariable model) and with histological type (p = 0.028, Kruskal-Wallis test) in LABC luminal B tumors. A borderline association with OS (p = 0.07, log-rank test) and RFS (p = 0.071, log-rank test) was observed in luminal A tumors in the LABC group. Conclusions: Our findings suggest that 5hmC levels are differentially associated with distinct clinical and histopathological characteristics in breast cancer, especially those linked to aggressiveness, including Ki67, histological type, and grade.

Early pathogenesis during infectious bursal disease in susceptible chickens is associated with changes in B cell genomic methylation and loss of genome integrity

We propose a model by which an increase in the genomic modification, 5-hydroxymethylcytosine (5hmC), contributes to B cell death within the chicken bursa of Fabricus (BF) infected with infectious bursal disease virus (IBDV). Our findings indicate that, following an IBDV infection, Rhode Island Red (RIR) chickens have fewer surviving B cells and higher levels of 5hmC in the BF than the more resistant 15l line of birds. Elevated genomic 5hmC levels within the RIR BF are associated with markers of immune responses: infiltrating T cells and increased expression of CD40L, FasL and iNOS. Such changes correlate with genomic fragmentation and the presence of IBDV capsid protein, VP2. To explore the effects of CD40L, the immature B cell line, DT40, was exposed to recombinant chicken CD40L that resulted in changes in nuclear 5hmC distribution. Collectively, our observations suggest that T cell infiltration exacerbates early immunopathology within the BF during an IBDV infection contributing to B cell genomic instability and death to facilitate viral egress and immunosuppression.

Low level of 5-Hydroxymethylcytosine predicts poor prognosis in non-small cell lung cancer.

The loss of 5-hydroxymethylcytosine (5-hmC) has previously been demonstrated to be implicated in the initiation and progression of various tumors. However, its role in non-small cell lung cancer (NSCLC) remains unknown. The present study aimed to determine the level of 5-hmC in NSCLC and their adjacent normal lung tissues by immunohistochemistry and dot-blot analysis; then the relationship between 5-hmC level and the clinicopathological features of NSCLC and the prognostic significance of 5-hmC level in NSCLC patients were analyzed. By employing the dot-blot analysis, a significant reduction of 5-hmC level in NSCLC tissues compared with the adjacent normal tissues was detected, which were further verified by the immunohistochemistry results on tissue microarrays. Further analyses demonstrated that 65.38% (136/208) presented with low 5-hmC level, and low 5-hmC level was significantly associated with lymph node metastasis (P<0.001), histological type (P<0.001) and large tumor size (P=0.031). Notably, the 5-year overall survival rate of patients with low 5-hmC levels were significantly lower than patients with high 5-hmC levels (P<0.001). In addition, it was demonstrated that 5-hmC level was identified as independent prognostic factor in patients' overall survival. In conclusion, downregulation of 5-hmC may serve as a useful biomarker for NSCLC prognosis evaluation.