Ten-eleven Translocation Genes Research Articles

Pan-cancer analyses reveal the molecular and clinical characteristics of TET family members and suggests that TET3 maybe a potential therapeutic target.

The Ten-Eleven Translocation (TET) family genes are implicated in a wide array of biological functions across various human cancers. Nonetheless, there is a scarcity of studies that comprehensively analyze the correlation between TET family members and the molecular phenotypes and clinical characteristics of different cancers. Leveraging updated public databases and employing several bioinformatics analysis methods, we assessed the expression levels, somatic variations, methylation levels, and prognostic values of TET family genes. Additionally, we explored the association between the expression of TET family genes and pathway activity, tumor microenvironment (TME), stemness score, immune subtype, clinical staging, and drug sensitivity in pan-cancer. Molecular biology and cytology experiments were conducted to validate the potential role of TET3 in tumor progression. Each TET family gene displayed distinct expression patterns across at least ten detected tumors. The frequency of Single Nucleotide Variant (SNV) in TET genes was found to be 91.24%, primarily comprising missense mutation types, with the main types of copy number variant (CNV) being heterozygous amplifications and deletions. TET1 gene exhibited high methylation levels, whereas TET2 and TET3 genes displayed hypomethylation in most cancers, which correlated closely with patient prognosis. Pathway activity analysis revealed the involvement of TET family genes in multiple signaling pathways, including cell cycle, apoptosis, DNA damage response, hormone AR, PI3K/AKT, and RTK. Furthermore, the expression levels of TET family genes were shown to impact the clinical staging of tumor patients, modulate the sensitivity of chemotherapy drugs, and thereby influence patient prognosis by participating in the regulation of the tumor microenvironment, cellular stemness potential, and immune subtype. Notably, TET3 was identified to promote cancer progression across various tumors, and its silencing was found to inhibit tumor malignancy and enhance chemotherapy sensitivity. These findings shed light on the role of TET family genes in cancer progression and offer insights for further research on TET3 as a potential therapeutic target for pan-cancer.

Ultrasensitive detection of 5-hydroxymethylcytosine in genomic DNA using a graphene-based sensor modified with biotin and gold nanoparticles

Ten-eleven translocation (TET) proteins orchestrate deoxyribonucleic acid (DNA) methylation-demethylation dynamics by oxidizing 5-methylcytosine to 5-hydroxymethylcytosine (5hmC) and are frequently inactivated in various cancers. Due to the significance of 5hmC as an epigenetic biomarker for cancer diagnosis, pathogenesis, and treatment, its rapid and precise quantification is essential. Here, we report a highly sensitive electrochemical method for quantifying genomic 5hmC using graphene sheets that were electrochemically exfoliated and functionalized with biotin and gold nanoparticles (Bt-AuNPs) through a single-step electrical method. The attachment of Bt-AuNPs to graphene enhances the specificity of 5hmC-containing DNA and augments the oxidation of 5hmC to 5-formylcytosine in DNA. When coupled to a gold electrode, the Bt-AuNP-graphene-based sensor exhibits exceptional sensitivity and specificity for detecting 5hmC, with a detection limit of 63.2 fM. Furthermore, our sensor exhibits a remarkable capacity to measure 5hmC levels across a range of biological samples, including preclinical mouse tissues with varying 5hmC levels due to either TET gene disruption or oncogenic transformation, as well as human prostate cancer cell lines. Therefore, our sensing strategy has substantial potential for cancer diagnostics and prognosis.

Loss of TET reprograms Wnt signaling through impaired demethylation to promote lung cancer development

Oncogenic imbalance of DNA methylation is well recognized in cancer development. The ten-eleven translocation (TET) family of dioxygenases, which facilitates DNA demethylation, is frequently dysregulated in cancers. How such dysregulation contributes to tumorigenesis remains poorly understood, especially in solid tumors which present infrequent mutational incidence of TET genes. Here, we identify loss-of-function mutations of TET in 7.4% of human lung adenocarcinoma (LUAD), which frequently co-occur with oncogenic KRAS mutations, and this co-occurrence is predictive of poor survival in LUAD patients. Using an autochthonous mouse model of KrasG12D -driven LUAD, we show that individual or combinational loss of Tet genes markedly promotes tumor development. In this Kras-mutant and Tet-deficient model, the premalignant lung epithelium undergoes neoplastic reprogramming of DNA methylation and transcription, with a particular impact on Wnt signaling. Among the Wnt-associated components that undergo reprogramming, multiple canonical Wnt antagonizing genes present impaired expression arising from elevated DNA methylation, triggering aberrant activation of Wnt signaling. These impairments can be largely reversed upon the restoration of TET activity. Correspondingly, genetic depletion of β-catenin, the transcriptional effector of Wnt signaling, substantially reverts the malignant progression of Tet-deficient LUAD. These findings reveal TET enzymes as critical epigenetic barriers against lung tumorigenesis and highlight the therapeutic vulnerability of TET-mutant lung cancer through targeting Wnt signaling.

Stage-specific regulation of DNA methylation by TET enzymes during human cardiac differentiation.

Changes in DNA methylation are associated with normal cardiogenesis, whereas altered methylation patterns can occur in congenital heart disease. Ten-eleven translocation (TET) enzymes oxidize 5-methylcytosine (5mC) and promote locus-specific DNA demethylation. Here, we characterize stage-specific methylation dynamics and the function of TETs during human cardiomyocyte differentiation. Human embryonic stem cells (hESCs) in which all three TET genes are inactivated fail to generate cardiomyocytes (CMs), with altered mesoderm patterning and defective cardiac progenitor specification. Genome-wide methylation analysis shows TET knockout causes promoter hypermethylation of genes encoding WNT inhibitors, leading to hyperactivated WNT signaling and defects in cardiac mesoderm patterning. TET activity is also needed to maintain hypomethylated status and expression of NKX2-5 for subsequent cardiac progenitor specification. Finally, loss of TETs causes a set of cardiac structural genes to fail to be demethylated at the cardiac progenitor stage. Our data demonstrate key roles for TET proteins in controlling methylation dynamics at sequential steps during human cardiac development.

Vitamin C Enhances PARPi Efficacy for the Treatment of AML

Poly-ADP-ribose polymerase inhibitors (PARPi) are currently in clinical trial to determine their therapeutic efficacy for the treatment of acute myeloid leukemia (AML). We have shown that vitamin C (VitC), an essential micronutrient and co-factor of Ten-Eleven translocation (TET) proteins, enhances AML sensitivity to PARPi, potentially due to an increased dependency on base-excision repair (BER) enzymes needed to remove TET-catalyzed oxidized methylcytosine bases via active DNA demethylation. TET2 is the most frequently mutated TET gene in patients with AML, and vitamin C treatment can mimic genetic restoration of TET2 function, leading to DNA demethylation, differentiation, and leukemia cell death. Whether vitamin C efficacy in combination with PARPi depends on the level of TET2 functional alleles is not yet known and may stratify whether TET2 wild-type or mutant patients should be targeted by vitamin C adjuvant therapy.We have generated primary murine AML-ETO9a+ and MLL-AF9+ leukemia models with Tet2 +/+, Tet2 +/- and Tet2 -/- alleles to determine the Tet2-dependent efficacy of PARPi treatment when combined with vitamin C. Furthermore, we have performed CRISPR gene knockout and drug library screening in human AML cell lines in combination with vitamin C treatment, and tested a panel of 10 AML cell lines with titrating concentrations of PARPi (Olaparib, Talazoparib, Veliparib and Rucaparib) alone or in combination with vitamin C (L-ascorbic acid) mimicking physiological to pharmacological in vivo doses. Primary murine AML cells and human cell lines were assayed for colony-forming capacity, differentiation, cell cycling, viability and effects on DNA methylation, levels of oxidized 5-mC and gene expression upon combination treatment in vitro and in vivo. TET2 mutant PDX and primary murine AMLs treated in vivo with L-ascorbate (4g/kg) and Olaparib (50mg/kg) by daily IP injection were also monitored for disease burden, cellular differentiation and survival.Vitamin C is known to drive the TET-catalyzed iterative oxidation of 5-methylcytosine (5-mC) leading to the formation of 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC). We show that VitC-PARPi combination treatment causes an accumulation of oxidized 5-mC intermediates in the AML genome that correlates with increased yH2AX formation in mid-S phase and cell cycle stalling. Vitamin C reduces the IC 50 of Olaparib and Talazoparib by greater than 10-fold in human AML cells lines and primary murine leukemia cells, and treatment in combination promotes myeloid differentiation and blocks colony-forming capacity greater than either alone. In both our in vitro and in vivo studies, Tet2 +/- AML cells exhibit increased sensitivity to vitamin C treatment alone or in combination with PARPi compared to either Tet2 +/+ or Tet2 -/- cells, suggesting that patients with TET2 haploinsufficiency, which represents the majority of TET2 mutant cases, could benefit the most from combined treatment.Our findings confirm that vitamin C can act synergistically with PARPi to block AML cell viability, reduce colony-forming capacity, and decrease leukemia burden in PDX and primary murine leukemia models in a TET2 allelic dose-dependent manner. The combinatorial effect works at clinically relevant concentrations of PARPi, and low-pharmacological doses of vitamin C. These studies suggest that vitamin C can be used as a non-toxic therapeutic adjuvant to PARPi therapy for the treatment of AML. DisclosuresNeel: Northern Biologics, LTD: Current equity holder in publicly-traded company, Other: Co- Founder; SAB: Other: Co-Founder; Navire Pharma: Consultancy, Current equity holder in publicly-traded company; Jengu Therapeutics: Consultancy, Current equity holder in publicly-traded company, Other: Co-Founder; Arvinas, Inc: Consultancy, Current equity holder in publicly-traded company; Recursion Pharma: Current equity holder in publicly-traded company.

Tumour suppressor TET2 safeguards enhancers from aberrant DNA methylation and epigenetic reprogramming in ERα-positive breast cancer cells

ABSTRACT Aberrant DNA methylation is an epigenetic hallmark of malignant tumours. The DNA methylation level is regulated by not only DNA methyltransferases (DNMTs) but also Ten-Eleven Translocation (TET) family proteins. However, the exact role of TET genes in breast cancer remains controversial. Here, we uncover that the ERα-positive breast cancer patients with high TET2 mRNA expression had better overall survival rates. Consistently, knockout of TET2 promotes the tumorigenesis of ERα-positive MCF7 breast cancer cells. Mechanistically, TET2 loss leads to aberrant DNA methylation (gain of 5mC) at a large proportion of enhancers, accompanied by significant reduction in H3K4me1 and H3K27ac enrichment. By analysing the epigenetically reprogrammed enhancers, we identify oestrogen responsive element (ERE) as one of the enriched motifs of transcriptional factors. Importantly, TET2 loss impairs 17beta-oestradiol (E2)-induced transcription of the epigenetically reprogrammed EREs-associated genes through attenuating the binding of ERα. Taken together, these findings shed light on our understanding of the epigenetic mechanisms underlying the enhancer reprogramming during breast cancer pathogenesis.

JAK2 and TET2 Mutation in Polycythemia Vera

The Ten-Eleven Translocation-2 (TET2) gene, located on chromosome 4q24, has been implicated in hematological malignancies. The TET2 gene shows mutations in variable myeloid malignancies with the involvement of 15% of myeloproliferative neoplasms (MPNs). The inactivation of the TET2 gene in both mice and humans has shown a high degree of deregulation of the hematopoiesis process leading to hematological malignancies. Polycythemia vera (PV), an MPN characterized by increased red blood cell mass, has been associated with the TET2 gene. Furthermore, TET2 genes have been found to facilitate Janus kinase-2 and signal transducer activator of transcription 5, as well as modulate the epigenetic composition of genomic DNA. However, little is known about the role of TET2 mutations in patients with PV. Several studies have been conducted to further assess the significant role of TET2 gene function in various disease processes and prognoses to enhance the management and care of these patients.

Single-Cell Transcriptomics of Human TET2 Knockout CD4 T-Cells and Their Clonal Evolution

Angioimmunoblastic T-cell lymphoma (AITL), the most frequent subtype of peripheral T-cell lymphoma (PTCL), is a neoplasm with characteristics of mature T follicular helper (TFH) cells. We and others have identified frequent (~75%) inactivating mutations in the TET2 (Ten-Eleven Translocation-2) gene in AITL. TET2 belongs to a 3 member family of TET dioxygenases that catalyze DNA demethylation by oxidation of 5-methyl-cytosine (5-mC) to 5-hydroxymethyl-cytosine (5-hmC) and further oxidative cytosine products. Thus, loss of function (LOF) of TET2 will cause aberrant genome hypermethylation and reduction in 5-hmC. Studies of the variant allele fraction (VAF) of TET2 mutants suggest that this mutation is a founding abnormality in AITL. However, how TET2 loss promotes the development of AITL is still unclear. To study LOF of TET2 in CD4 T-cell lymphomagenesis without the noise generated by other mutations in an established lymphoma, we generated a human TET2 knock-out (KO) CD4 T-cell model using CRISPR/Cas9 technology, which allows us to perform functional genomic studies by directly editing genes at their genomic loci. Whole transcriptome sequencing and single-cell transcriptome sequencing were used to study the cell evolution after KO. We generated multiple TET2 KO primary CD4 T-cell models using two different CRISPR/Cas9 methods. The first approach used the plasmid PX458-a, which expresses green fluorescent protein (GFP) fused Cas9 and guide RNA-a targeting TET2 exon 6, to electroporate CD4 T-cell from healthy donor F25. The second approach used homologous DNA repair (HDR) mediated knock-in (KI) of tandem GFP gene and a SV40 transcription stop signal to terminate TET2 expression at exon 3. Cas9/sgRNA-e RNP complex, along with a long DNA template (about 1.6 kb), was electroporated into CD4 T-cells from two healthy donors, F25 and M40. GFP-positive cells were sorted by FACS after electroporation and were considered to be edited cells. Edited CD4 T-cells were cultured in vitro with 50 U/ml IL-2, and stimulated regularly (every 7~10 days) with 1:1 ratio of anti-CD3/CD28 T activator beads. TET2 KO in these cells was confirmed by qRT-PCR, Sanger sequencing and Western blotting. Compared with wild-type (WT) CD4 T-cells under the same culture conditions, a lower level of 5-hmC in TET2 KO cells was observed, indicating successful editing of TET2. Compared to WT cells, KO cells had a higher growth rate, due to a lower apoptosis rate and a higher proliferation rate, by Annexin V staining, EdU staining, and MTS experiments. The growth of KO cells or WT cells was still dependent on IL-2 and T activator beads stimulation. All batches of KO cells, generated by different guide RNAs or from different donors, showed a much longer life span than WT cells, which usually lived for 3~4 months, but KO cells can keep proliferating longer than one year. We also performed TCR analysis on these cell samples. Both WT and KO cells demonstrated oligoclonality when examined at Day 40 (40D, early stage) and TET2 KO cells showed a dominant clone by Day 90 (90D, late stage). We performed single-cell transcriptome analysis on M40 KO vs. WT cells, at 40D and 90D. KO90D cells had a low TCR diversity with the dominant population representing ~88% of cells (TRAV9-2,TRBV5-1). From single-cell transcriptome analysis, cell clustering profiles were very distinctive in these 4 cell populations analyzed (Figure 1A) and these clusters had unique gene expression profiles (Figure 1B). Cluster 6 was prominent in KO90D but almost absent in WT90D, whereas the reverse was true for clusters 1 and 5. From pathway analysis, KO90D cells showed a higher expression of signatures associated with proliferation, cell cycle and chemokine signaling and lower histidine and tryptophan metabolism signatures. Sanger sequencing showed a 79 bp indel in addition to the GFP KI allele in KO90D cells, demonstrated the homozygous deletion of TET2 on these cells. Similar results were observed in F25 TET2 KO cells by plasmid PX458-a. This indicated the selection of homozygously deleted TET2 cells in long-term culture. However, clonal evolution is highly dynamic and a minor clone in KO40D cells may become the dominant clone in KO90D cells. Comparison of the 5-mC and 5-hmC profiles between KO and WT cells are being conducted to elucidate epigenetic alterations that are associated with the functional alterations and predisposition to AITL lymphomagenesis. Figure Disclosures No relevant conflicts of interest to declare.

Effect of Perfluorooctanoic Acid on the Epigenetic and Tight Junction Genes of the Mouse Intestine.

Perfluorooctanoic acid (PFOA) has been implicated in various toxicities including neurotoxicity, genotoxicity, nephrotoxicity, epigenetic toxicity, immunotoxicity, reproductive toxicity, and hepatotoxicity. However, information on the accumulation of PFOA in the intestine and its toxic effects on intestinal epigenetics and tight junction (TJ) genes is sparse. CD1 mice were dosed with PFOA (1, 5, 10, or 20 mg/kg/day) for 10 days, and its accumulation and induced alterations in the expression of epigenetic and tight junction genes in the small intestine and colon were evaluated using LC–MS and qPCR techniques. PFOA reduced the expression levels of DNA methyltransferases (Dnmt1, Dnmt3a, Dnmt3b) primarily in the small intestine whereas, in the colon, a decrease was observed only at high concentrations. Moreover, ten-eleven translocation genes (Tet2 and Tet3) expression was dysregulated in the small intestine, whereas in the colon Tets remained unaffected. The tight junction genes Claudins (Cldn), Occludin (Ocln), and Tight Junction Protein (Tjp) were also heavily altered in the small intestine. TJs responded differently across the gut, in proportion to PFOA dosing. Our study reveals that PFOA triggers DNA methylation changes and alters the expression of genes essential for maintaining the physical barrier of intestine, with more profound effects in the small intestine compared to the colon.

Low expression of TET2 gene in pediatric acute lymphoblastic leukemia is associated with poor clinical outcome.

TET2, a member of the Ten-Eleven translocation gene family, catalyzes the conversion of 5-methylcytosine to 5-hydroxymethylcytosine in DNA. Low expression of TET2 has been reported as a prognostic factor for several types of malignancies in adult patients. However, there have been few data on the effect of TET2 mRNA level on the prognosis of children with ALL so far. In this study, TET2 expression of samples cryopreserved in the liquid nitrogen from January 1, 2007 through December 31, 2011 was retrospectively analyzed in 136 newly diagnosed ALL patients by real-time polymerase chain reaction (PCR) assay. The patients' samples were divided into two groups by the median value of patients group and divided into TET2 low and TET2 high groups. A total of 136 childhood ALL patients demonstrated lower TET2 expression than control group (P=.038). TET2 mRNA expression levels were correlated with the disease status. In addition, patients with low TET2 expression had lower platelet counts and lower CR rates. Survival analysis showed that low TET2 expression in children with ALL was associated with lower 5-year overall survival (OS) (63% vs 88%, P=.011) and event-free survival (EFS) (60% vs 85%, P=.003). Multivariate analysis revealed that low TET2 expression was an independent poor prognostic factor of OS and EFS. Low expression of TET2 in children with ALL is associated with poor prognosis and can be used as a molecular prognostic marker for risk group stratification.

Epigenetic downregulation of TET3 reduces genome-wide 5hmC levels and promotes glioblastoma tumorigenesis.

Loss of 5-hydroxymethylcytosine (5hmC) has been associated with mutations of the ten-eleven translocation (TET) enzymes in several types of cancer. However, tumors with wild-type TET genes can also display low 5hmC levels, suggesting that other mechanisms involved in gene regulation might be implicated in the decline of this epigenetic mark. Here we show that DNA hypermethylation and loss of DNA hydroxymethylation, as well as a marked reduction of activating histone marks in the TET3 gene, impair TET3 expression and lead to a genome-wide reduction in 5hmC levels in glioma samples and cancer cell lines. Epigenetic drugs increased expression of TET3 in glioblastoma cells and ectopic overexpression of TET3 impaired in vitro cell growth and markedly reduced tumor formation in immunodeficient mice models. TET3 overexpression partially restored the genome-wide patterns of 5hmC characteristic of control brain samples in glioblastoma cell lines, while elevated TET3 mRNA levels were correlated with better prognosis in glioma samples. Our results suggest that epigenetic repression of TET3 might promote glioblastoma tumorigenesis through the genome-wide alteration of 5hmC.

Role of ten-eleven translocation proteins and 5-hydroxymethylcytosine in hepatocellular carcinoma.

In mammals, methylation of the 5th position of cytosine (5mC) seems to be a major epigenetic modification of DNA. This process can be reversed (resulting in cytosine) with high efficiency by dioxygenases of the ten‐eleven translocation (TET) family, which perform oxidation of 5mC to 5‐hydroxymethylcytosine (5hmC), 5‐formylcytosine and 5‐carboxylcytosine. It has been demonstrated that these 5mC oxidation derivatives are in a dynamic state and have pivotal regulatory functions. Here, we comprehensively summarized the recent research progress in the understanding of the physiological functions of the TET proteins and their mechanisms of regulation of DNA methylation and transcription. Among the three TET genes, TET1 and TET2 expression levels have frequently been shown to be low in hepatocellular carcinoma (HCC) tissues and received most attention. The modulation of TET1 also correlates with microRNAs in a post‐transcriptional regulatory process. Additionally, recent studies revealed that global genomic 5hmC levels are down‐regulated in HCC tissues and cell lines. Combined with the reported results, identification of 5hmC signatures in HCC tissues and in circulating cell‐free DNA will certainly contribute to early detection and should help to design therapeutic strategies against HCC. 5hmC might also be a novel prognostic biomarker of HCC. Thus, a detailed understanding of the molecular mechanisms resulting in the premalignant and aggressive transformation of TET proteins and cells with 5hmC disruption might help to develop novel epigenetic therapies for HCC.

Effect of EZH2 knockdown on preimplantation development of porcine parthenogenetic embryos

The EZH2 protein endows the polycomb repressive complex 2 (PRC2) with histone lysine methyltransferase activity that is associated with transcriptional repression. Recent investigations have documented crucial roles for EZH2 in mediating X-inactivation, stem cell pluripotency and cancer metastasis. However, there is little evidence demonstrating the maternal effect of EZH2 on porcine preimplantation development. Here, we took parthenogenetic activation embryos to eliminate the confounding paternal influence. We showed that the dynamic expression of EZH2 during early development was accompanied by changes in H3K27me3 levels. Depletion of EZH2 in MII oocytes by small interfering RNA not only impaired embryonic development at the blastocyst stage (P < 0.05), but also disrupted the equilibrium of H3K4me3 and H3K27me3 in the embryo. Interestingly, the expression of TET1, a member of Ten-Eleven Translocation gene family for converting 5-methylcytosine (5 mC) to 5-hydroxymethylcytosine (5hmC), was decreased after EZH2 knockdown, in contrast to the increase of the other two members, TET2 and TET3 (P < 0.05). These results indicate a correlation between histone methylation and DNA methylation, and between EZH2 and TET1. Along with the downregulation of TET1, the expression of the pluripotency gene NANOG was decreased (P < 0.05), which is consistent with a previous finding in mouse ES cells. Meanwhile, the abundance of OCT4 and SOX2 were also down-regulated. Moreover, EZH2 knockdown reduced the capacity of cells in the blastocysts to resist apoptosis. Taken together, our data suggest that EZH2 is integral to the developmental program of porcine parthenogenetic embryos and exerts its function by regulating pluripotency, differentiation and apoptosis.

Adverse maternal environment leads to cardiac fibrosis in adult male mice.

Cardiac fibrosis is a cardinal feature of multiple types of cardiovascular disease, which lead to heart failure. Multiple studies connect adverse maternal environment (AME) with cardiac fibrosis. AME does not always result in fibrosis, though. An additional "insult", such as an adult Western diet (WD), is frequently necessary. The additive effects of AME and adult WD on cardiac fibrosis is not well-understood. AME can also alter DNA methylation. DNA methyltransferase (DNMT) and ten-eleven translocation (TET) are methylation modifying genes that regulate DNA methylation, but it is unknown if AME changes cardiac gene expression of DNMT and TET. We sought to use a model of AME and adult WD to investigate the development of cardiac fibrosis and cardiac mRNA expression of DNMT and TET genes. We exposed dams to WD or control diet (CD) 5 weeks before pregnancy and through lactation. We added environmental stressors during the last third of pregnancy to dams on WD to create AME. Dams on CD experienced no added stressors to create control maternal environment (CME). Male offspring were weaned at Postnatal Week 3 (W3) and placed on WD or CD to create four groups: CME-CD, CME-WD, AME-CD, and AME-WD. AME-WD increased cardiac fibrosis in adulthood (p < .05), whereas AME-CD and CME-WD did not. TET1-3 and DNMT3a mRNA levels decreased in AME versus CME offspring (p < .01). AME increases susceptibility to cardiac fibrosis in adult male mice. Early-life changes to TET expression may mediate susceptibility to fibrosis, but further testing is needed.

Diverse Landscape of TET2 Variants in MDS and AML

INTRODUCTION The TET2 (Ten‐Eleven Translocation 2) gene, located at chromosome 4q24.1, belongs to TET family of proteins that possesses the capacity of catalyzing the conversion of 5‐methylcytosine into 5‐hydroxymethylcytosine. It is considered to be a putative tumor suppressor gene during cancer initiation and development. TET2 mutations are extensively studied and reported in a variety of human hematological malignancies including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), suggesting a crucial role of TET2 in the pathogenesis of blood cancers. The landscape of rare TET2 variants which may be of potential clinical relevance is not fully described. The landscape of TET2 variants from a large cohort of MDS and AML patients was explored. METHODS We analyzed data from bone marrow samples of 2634 patients with AML or MDS who presented at MD Anderson between the years 2012-2016. Seven hundred seventy seven patients were found to have TET2 variants that were not classifiable as known mutations. Two hundred sixty-three patients were diagnosed with MDS, 144 with secondary AML transformed from MDS, and 370 with AML without prior history of MDS. The coding regions of TET2 gene was analyzed by next-generation sequencing in the molecular diagnostics laboratory. Non-mutation variants were classified into 2 groups: 1) those reported as germline polymorphisms (GP) in population studies, literature or matched tumor-normal analysis on patients at MDACC; and 2) variants for which a germline versus somatic origin could not be determined unequivocally (variants of uncertain origin, VUO). Very common GP variants (defined as those with a population frequency of over 20% in laboratory sample cohort) were not evaluated. Variants9 predicted frequency in the population according to gnomAD Exomes 2.0.2, and classification according to American College of Medical Genetics and Genomics (ACMG) guidelines for interpretation of sequence variants, were assessed using the VarSome platform by Saphetor. RESULTS Two-hundred and twenty-eight unique variants were identified. The amino acid substitutions from missense variants were widely distributed throughout TET2. Fifty-four unique variants were classified as GP and several of these recurrent variants were found to be present in our population at a significantly higher frequency (p CONCLUSIONS The landscape of rare TET2 variants in a large group of MDS and AML patients was frequent, diverse, and included both GP and VUO. GP variants were more likely benign by ACMG classification and found in both MDS and AML patients. A large number of singleton VUO were found among MDS patients, and many were predicted to be pathogenic or had uncertain significance. VUO9s also contained many fs and nonsense variants. These result support the idea that rare pathogenic variants in TET2 are currently underrecognized as contributory, although they are likely to help drive leukemogenesis. Greater understanding of the importance of individual TET2 variants and additional classification metrics are needed. Disclosures DiNardo:Karyopharm: Honoraria; Abbvie: Honoraria; Agios: Consultancy; Medimmune: Honoraria; Celgene: Honoraria; Bayer: Honoraria. Daver:Karyopharm: Research Funding; Incyte: Consultancy; Novartis: Consultancy; ImmunoGen: Consultancy; Sunesis: Consultancy; Pfizer: Consultancy; Daiichi-Sankyo: Research Funding; Kiromic: Research Funding; Alexion: Consultancy; Otsuka: Consultancy; Sunesis: Research Funding; Incyte: Research Funding; ARIAD: Research Funding; BMS: Research Funding; Karyopharm: Consultancy; Novartis: Research Funding; Pfizer: Research Funding. Kadia:Celgene: Research Funding; Takeda: Consultancy; BMS: Research Funding; Abbvie: Consultancy; Novartis: Consultancy; Novartis: Consultancy; Amgen: Consultancy, Research Funding; Jazz: Consultancy, Research Funding; Abbvie: Consultancy; Pfizer: Consultancy, Research Funding; Takeda: Consultancy; Amgen: Consultancy, Research Funding; BMS: Research Funding; Pfizer: Consultancy, Research Funding; Jazz: Consultancy, Research Funding; Celgene: Research Funding. Konopleva:Stemline Therapeutics: Research Funding; abbvie: Research Funding; cellectis: Research Funding; Immunogen: Research Funding. Andreeff:Amgen: Consultancy, Research Funding; Astra Zeneca: Research Funding; Jazz Pharma: Consultancy; Oncolyze: Equity Ownership; Aptose: Equity Ownership, Membership on an entity9s Board of Directors or advisory committees; Oncoceutics: Equity Ownership, Membership on an entity9s Board of Directors or advisory committees; SentiBio: Equity Ownership; United Therapeutics: Patents & Royalties: GD2 inhibition in breast cancer ; Reata: Equity Ownership; Celgene: Consultancy; Eutropics: Equity Ownership, Membership on an entity9s Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Patents & Royalties: MDM2 inhibitor activity patent, Research Funding.

Hepatic metabolite profiling of polychlorinated biphenyl (PCB)-resistant and sensitive populations of Atlantic killifish (Fundulus heteroclitus)

Atlantic killifish inhabiting polluted sites along the east coast of the U.S. have evolved resistance to toxic effects of contaminants. One such contaminated site is the Acushnet River estuary, near New Bedford Harbor (NBH), Massachusetts, which is characterized by very high PCB concentrations in the sediments and in the tissues of resident killifish. Though killifish at this site appear to be thriving, the metabolic costs of survival in a highly contaminated environment are not well understood. In this study we compared the hepatic metabolite profiles of resistant (NBH) and sensitive populations (Scorton Creek (SC), Sandwich, MA) using a targeted metabolomics approach in which polar metabolites were extracted from adult fish livers and quantified. Our results revealed differences in the levels of several metabolites between fish from the two sites. The majority of these metabolites are associated with one-carbon metabolism, an important pathway that supports multiple physiological processes including DNA and protein methylation, nucleic acid biosynthesis and amino acid metabolism. We measured the gene expression of DNA methylation (DNA methyltransferase 1, dnmt1) and demethylation genes (Ten-Eleven Translocation (TET) genes) in the two populations, and observed lower levels of dnmt1 and higher levels of TET gene expression in the NBH livers, suggesting possible differences in DNA methylation profiles. Consistent with this, the two populations differed significantly in the levels of 5-methylcytosine and 5-hydroxymethylcytosine nucleotides. Overall, our results suggest that the unique hepatic metabolite signatures observed in NBH and SC reflect the adaptive mechanisms for survival in their respective habitats.

Reduced ten-eleven translocation and isocitrate dehydrogenase expression in inflammatory hidradenitis suppurativa lesions.

As environmental factors appear to predispose patients to hidradenitis suppurativa (HS), studying epigenetic modifications is of interest to further understand the pathogenesis of HS. To study the expression of DNA hydroxymethylation regulators, namely the ten-eleven translocation (TET) and isocitrate dehydrogenase (IDH) family, in the skin of HS patients. Twenty patients with HS and 12 healthy subjects were recruited. We analysed the expression of TET1, TET2, TET3, IDH1, IDH2, IDH3a, and IDH3b in lesional and perilesional HS tissue as well as tissue from healthy controls by quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). In addition, immunohistochemistry was performed for TET1, TET2, and TET3. RT-PCR analysis showed that mRNA of all the studied genes was significantly under-expressed in lesional HS skin compared to healthy skin. IDH1 and IDH2 mRNA expression was also significantly lower in perilesional HS skin compared to healthy skin, and TET3 mRNA expression was significantly lower in lesional HS skin compared to perilesional HS skin. RT-PCR analysis for TET1, TET2, and TET3 mRNA expression was confirmed by immunohistochemical analysis. Correlation analysis revealed a significant positive correlation between TET and IDH gene expression in perilesional and lesional HS skin. Our results suggest that epigenetic changes occur in HS tissue and that aberrant expression of the DNA hydroxymethylation regulators may play a role in the pathogenesis of HS. As epigenetic modifications are reversible, further research into the cause of these aberrant expression patterns is warranted in order to develop possible novel therapeutic approaches.

A network-based analysis of the human TET Gene Family

Ten-eleven translocation (TET) proteins, a family of Fe2+- and 2-oxoglutarate-dependent dioxygenases, are involved in DNA demethylation. Three TET paralogs have been identified (TET1, TET2, and TET3) and they show different patterns of tissue-specific expression. In our previous evolutionary studies, we found that the TET1 and TET2 genes underwent positive selection more frequently than the TET3 gene, possibly due to changes in the selective constraints during their evolutionary process. In this study, we performed a network-based analysis of the mRNA expression profiles of TET knockdown and the TET-containing co-expression modules identified in early human developmental stages. Analyses based on the PPI subnetwork demonstrated that TET DEGs PPI subnetwork genes were more evolutionarily conserved than all the human-chimpanzee orthologs during evolutionary history. GO annotation of gene co-expression modules containing a TET gene ortholog revealed particular features of the potential role of TET gene family members. Our study implicated the TET1 module in fundamental aspects of cellular physiology, such as the regulation of glucose metabolism, and the TET2 module in GPCR signal transduction. The TET3 module was related to signaling pathways involved in developmental regulation. The evolutionary rate and phylogenetic age distribution analysis of network member genes also support these network-based analyses. The present study provides an integrated view of TET gene family properties and might be informative for elucidating the molecular mechanisms of their biological functions.

TET2 expression is a potential prognostic and predictive biomarker in cytogenetically normal acute myeloid leukemia.

TET2 (Ten-Eleven Translocation 2) gene is a member of TET family that can modify DNA through catalyzing the conversion of 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine (5-hmC). Although TET2 mutation has been disclosed in a variety of hematopoietic malignancies, the prognostic implication of TET2 expression in acute myeloid leukemia (AML) remains largely elusive. In this study, real-time quantitative PCR was carried out to detect the level of TET2 transcript in 134 de novo AML patients and 35 healthy donors. TET2 mRNA level was significantly down-regulated in AML patients compared with controls (p = 0.010). Among the French-American-British (FAB) subtypes, the incidence of TET2 under-expression in M0/M1 subtypes was significantly higher than in the other subtypes M2/M3/M4/M5/M6 (p = 0.017), and also markedly higher than in the other granulocyte subtypes M2/M3 (p = 0.005). TET2 low-expressed patients showed a significantly higher frequency of NPM1 mutations than TET2 high-expressed patients. Although there was no significant difference in complete remission rate between two groups (low and high TET2 expression), patients with low TET2 expression had markedly shorter overall survival (OS) in both non-M3 and cytogenetically normal AML (CN-AML) (p = 0.016 and 0.044, respectively). Furthermore, multivariate analysis confirmed the prognostic value of TET2 expression on OS among CN-AML patients (p = 0.049). Importantly, TET2 expression in complete remission (CR) time was significantly higher than newly diagnosis time (p = 0.001), and was returned to lower level when in relapse time (p < 0.001). These findings indicated that down-regulation of TET2 expression was a common event and acted as a prognostic and predictive biomarker in CN-AML patients.

Coordinate Regulation of TET2 and EBNA2 Controls the DNA Methylation State of Latent Epstein-Barr Virus.

Epstein-Barr virus (EBV) latency and its associated carcinogenesis are regulated by dynamic changes in DNA methylation of both virus and host genomes. We show here that the ten-eleven translocation 2 (TET2) gene, implicated in hydroxymethylation and active DNA demethylation, is a key regulator of EBV latency type DNA methylation patterning. EBV latency types are defined by DNA methylation patterns that restrict expression of viral latency genes. We show that TET2 mRNA and protein expression correlate with the highly demethylated EBV type III latency program permissive for expression of EBNA2, EBNA3s, and LMP transcripts. We show that short hairpin RNA (shRNA) depletion of TET2 results in a decrease in latency gene expression but can also trigger a switch to lytic gene expression. TET2 depletion results in the loss of hydroxymethylated cytosine and a corresponding increase in cytosine methylation at key regulatory regions on the viral and host genomes. This also corresponded to a loss of RBP-jκ binding and decreased histone H3K4 trimethylation at these sites. Furthermore, we show that the TET2 gene itself is regulated in a fashion similar to that of the EBV genome. Chromatin immunoprecipitation high-throughput sequencing (ChIP-seq) revealed that the TET2 gene contains EBNA2-dependent RBP-jκ and EBF1 binding sites and is subject to DNA methylation-associated transcriptional silencing similar to what is seen in EBV latency type III genomes. Finally, we provide evidence that TET2 colocalizes with EBNA2-EBF1-RBP-jκ binding sites and can interact with EBNA2 by coimmunoprecipitation. Taken together, these findings indicate that TET2 gene transcripts are regulated similarly to EBV type III latency genes and that TET2 protein is a cofactor of EBNA2 and coregulator of the EBV type III latency program and DNA methylation state.IMPORTANCE Epstein-Barr virus (EBV) latency and carcinogenesis involve the selective epigenetic modification of viral and cellular genes. Here, we show that TET2, a cellular tumor suppressor involved in active DNA demethylation, plays a central role in regulating the DNA methylation state during EBV latency. TET2 is coordinately regulated and functionally interacts with the viral oncogene EBNA2. TET2 and EBNA2 function cooperatively to demethylate genes important for EBV-driven B-cell growth transformation.