TET2 Activity Research Articles

TET2 promotes tumor antigen presentation and T cell IFN-γ, which is enhanced by vitamin C.

Immune evasion by tumors is promoted by low T cell infiltration, ineffective T cell activity directed against the tumor, and reduced tumor antigen presentation. The TET2 DNA dioxygenase gene is frequently mutated in hematopoietic malignancies and loss of TET enzymatic activity is found in a variety of solid tumors. We showed previously that vitamin C (VC), a cofactor of TET2, enhances tumor-associated T cell recruitment and checkpoint inhibitor therapy responses in a TET2-dependent manner. Using single-cell RNA sequencing (scRNA-seq) analysis performed on B16-OVA melanoma tumors, we have shown here that an additional function for TET2 in tumors is to promote expression of certain antigen presentation machinery genes, which is potently enhanced by VC. Consistently, VC promoted antigen presentation in cell-based and tumor assays in a TET2-dependent manner. Quantifying intercellular signaling from the scRNA-seq dataset showed that T cell-derived IFN-γ-induced signaling within the tumor and tumor microenvironment requires tumor-associated TET2 expression, which is enhanced by VC treatment. Analysis of patient tumor samples indicated that TET activity directly correlates with antigen presentation gene expression and with patient outcomes. Our results demonstrate the importance of tumor-associated TET2 activity as a critical mediator of tumor immunity, which is augmented by high-dose VC therapy.

Using NMR to Monitor TET-Dependent Methylcytosine Dioxygenase Activity and Regulation.

The active removal of DNA methylation marks is governed by the ten-eleven translocation (TET) family of enzymes (TET1-3), which iteratively oxidize 5-methycytosine (5mC) into 5-hydroxymethycytosine (5hmC), and then 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). TET proteins are frequently mutated in myeloid malignancies or inactivated in solid tumors. These methylcytosine dioxygenases are α-ketoglutarate (αKG)-dependent and are, therefore, sensitive to metabolic homeostasis. For example, TET2 is activated by vitamin C (VC) and inhibited by specific oncometabolites. However, understanding the regulation of the TET2 enzyme by different metabolites and its activity remains challenging because of limitations in the methods used to simultaneously monitor TET2 substrates, products, and cofactors during catalysis. Here, we measure TET2-dependent activity in real time using NMR. Additionally, we demonstrate that in vitro activity of TET2 is highly dependent on the presence of VC in our system and is potently inhibited by an intermediate metabolite of the TCA cycle, oxaloacetate (OAA). Despite these opposing effects on TET2 activity, the binding sites of VC and OAA on TET2 are shared with αKG. Overall, our work suggests that NMR can be effectively used to monitor TET2 catalysis and illustrates how TET activity is regulated by metabolic and cellular conditions at each oxidation step.

TET2-mediated tumor cGAS triggers endothelial STING activation to regulate vasculature remodeling and anti-tumor immunity in liver cancer

Induction of tumor vascular normalization is a crucial measure to enhance immunotherapy efficacy. cGAS-STING pathway is vital for anti-tumor immunity, but its role in tumor vasculature is unclear. Herein, using preclinical liver cancer models in Cgas/Sting-deficient male mice, we report that the interdependence between tumor cGAS and host STING mediates vascular normalization and anti-tumor immune response. Mechanistically, TET2 mediated IL-2/STAT5A signaling epigenetically upregulates tumor cGAS expression and produces cGAMP. Subsequently, cGAMP is transported via LRRC8C channels to activate STING in endothelial cells, enhancing recruitment and transendothelial migration of lymphocytes. In vivo studies in male mice also reveal that administration of vitamin C, a promising anti-cancer agent, stimulates TET2 activity, induces tumor vascular normalization and enhances the efficacy of anti-PD-L1 therapy alone or in combination with IL-2. Our findings elucidate a crosstalk between tumor and vascular endothelial cells in the tumor immune microenvironment, providing strategies to enhance the efficacy of combinational immunotherapy for liver cancer.

Development of a rapid mass spectrometric method for the analysis of ten-eleven translocation enzymes.

In DNA, methylation at the fifth position of cytosine (5mC) by DNA methyltransferases is essential for eukaryotic gene regulation. Methylation patterns are dynamically controlled by epigenetic machinery. Erasure of 5mC by Fe2+ and 2-ketoglutarate (2KG) dependent dioxygenases in the ten-eleven translocation family (TET1-3), plays a key role in nuclear processes. Through the event of active demethylation, TET proteins iteratively oxidize 5mC to 5-hydroxymethyl cytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5caC), each of which has been implicated in numerous diseases when aberrantly generated. A wide range of biochemical assays have been developed to characterize TET activity, many of which require multi-step processing to detect and quantify the 5mC oxidized products. Herein, we describe the development and optimization of a sensitive MALDI mass spectrometry-based technique that directly measures TET activity and eliminates tedious processing steps. Employing optimized assay conditions, we report the steady-state activity of wild type TET2 enzymes to furnish 5hmC, 5fC and 5caC. We next determine IC50 values of several small-molecule inhibitors of TETs. The utility of this assay is further demonstrated by analyzing the activity of V1395A which is an activating mutant of TET2 that primarily generates 5caC. Lastly, we describe the development of a secondary assay that utilizes bisulfite chemistry to further examine the activity of wildtype TET2 and V1395A in a base-resolution manner. The combined results demonstrate that the activity of TET proteins can be gauged, and their products accurately quantified using our methods.

Multi-Omic Profiling of Macrophages Lacking Tet2 or Dnmt3a Reveals Mechanisms of Hyper-Inflammation in Clonal Hematopoiesis

Introduction Clonal Hematopoiesis of Indeterminate Potential (CHIP) is a source of age-associated inflammation, caused by somatic mutations in bone marrow stem cells that promote clonal expansion. The functional impact of CHIP mutations on macrophages derived from these stem cells is linked to increased cardiovascular disease risk among other diseases. Interestingly, CHIP-associated mutations frequently occur in genes with opposing functions: DNMT3A and TET2, enzymes responsible for DNA methylation and demethylation, respectively. While it is known that DNMT3A KO and TET2 KO in macrophages lead to hyperinflammatory responses, the underlying mechanism remains poorly understood how opposing mutations lead to a similar phenotype. Methods To evaluate how DNMT3A and TET2 contribute to inflammatory responses in the context of CHIP, we exposed bone marrow derived macrophages from Dnmt3a KO, Tet2 KO, and C57BL/6 Vav1-Cre wild type (WT) control mice to 0.5 ng/mL of the inflammatory stimuli lipopolysaccharide (LPS). We then measured gene responses with RNA-seq replicates across 12 time points from 0 to 24 hours, paired with single cell RNA-seq (scRNA-seq), ATAC-seq, and 50x-coverage whole genome enzymatic methyl-seq (EM-seq) for multiple time points. Results Although the composition and kinetics of the LPS-induced expression program were similar in all genotypes, Dnmt3a KO and Tet2 KO had significantly greater changes in gene expression and chromatin accessibility by 2 hours compared to WT, particularly for a large subset of inflammatory response genes, like Il1b, Il6, Cxcl1, Tnfsf9, and Il23a. The promoter regions of the 278 genes significantly overexpressed (adjusted p < 0.05) for both KOs at 2 hours compared to WT were enriched with binding sites of the inflammatory pioneer factor AP-1 (p = 0.001). Inflammatory signals were observed even before stimulation, suggesting altered DNA methylation in each KO predisposed cells to hyperinflammatory states. Bulk RNA-seq found the gene set ‘IFNA response’ from MSigDB Hallmarks collection to be most enriched at baseline in both KOs compared to WT (FDR < 0.0007). At baseline Dnmt3a KO and Tet2 KO had 1,566 and 441 over-expressed genes, respectively (adjusted p < 0.05). scRNA-seq similarly showed a baseline inflammatory population enriched with type 1 IFN response gene expression that contained more cells with measurable baseline expression of inflammatory genes for both KOs. ATAC-seq identified highly significant enrichment of AP-1 binding sites at baseline and at 2 hours in the 2,270 regions more accessible in Dnmt3a KO compared to WT (p = 1x10 -40), as well as the 2,148 regions less accessible in Tet2 KO compared to WT (p = 1x10 -41). Although LPS stimulation did not lead to sites with altered CpG methylation using a genome-wide significance threshold, methylation globally differed between genotypes at baseline especially for promoter and enhancer regions, and this differential methylation directly explained some of the hyper-responsive chromatin accessibility and gene expression. For example, the promoter region of Cxcl1 for both KOs was significantly hypomethylated and more accessible at baseline, with these chromatin accessibility and associated expression differences becoming even larger at 2 hours of LPS stimulation. In general, DNA methylation and chromatin accessibility were rarely concordantly altered in both KOs at regions near genes with differential expression. For adjusted p-value < 0.05, Dnmt3a KO had 236,062 differentially hypomethylated CpG segments and 1,023 hypermethylated, while Tet2 KO had 7,488 hypomethylated and 4,482 hypermethylated segments. Conclusions Our integrative multi-omic analysis demonstrates that Dnmt3a and Tet2 modulate DNA methylation to limit inflammatory gene expression in healthy macrophages. We find loss of Dnmt3a or Tet2 activity in macrophages alters baseline DNA methylation at different regions, separately influencing the transcription factor binding landscape of AP-1, and inducing similar baseline over-expression of type 1 interferon response genes. Upon stimulation, these differences lead to higher inflammatory gene expression, possibly through more cooperation with transcription factors NF-κB and IRFs. Targeting AP-1 and type 1 interferon may be a therapeutic avenue for blocking the pathogenic role of CHIP for many inflammatory diseases of aging.

Classification of Acute Myeloid Leukemia by Cell-Free DNA 5-Hydroxymethylcytosine.

Epigenetic abnormality is a hallmark of acute myeloid leukemia (AML), and aberrant 5-hydroxymethylcytosine (5hmC) levels are commonly observed in AML patients. As epigenetic subgroups of AML correlate with different clinical outcomes, we investigated whether plasma cell-free DNA (cfDNA) 5hmC could categorize AML patients into subtypes. We profiled the genome-wide landscape of 5hmC in plasma cfDNA from 54 AML patients. Using an unbiased clustering approach, we found that 5hmC levels in genomic regions with a histone mark H3K4me3 classified AML samples into three distinct clusters that were significantly associated with leukemia burden and survival. Cluster 3 showed the highest leukemia burden, the shortest overall survival of patients, and the lowest 5hmC levels in the TET2 promoter. 5hmC levels in the TET2 promoter could represent TET2 activity resulting from mutations in DNA demethylation genes and other factors. The novel genes and key signaling pathways associated with aberrant 5hmC patterns could add to our understanding of DNA hydroxymethylation and highlight the potential therapeutic targets in AML. Our results identify a novel 5hmC-based AML classification system and further underscore cfDNA 5hmC as a highly sensitive marker for AML.

Crucial role of iron in epigenetic rewriting during adipocyte differentiation mediated by JMJD1A and TET2 activity.

Iron metabolism is closely associated with the pathogenesis of obesity. However, the mechanism of the iron-dependent regulation of adipocyte differentiation remains unclear. Here, we show that iron is essential for rewriting of epigenetic marks during adipocyte differentiation. Iron supply through lysosome-mediated ferritinophagy was found to be crucial during the early stage of adipocyte differentiation, and iron deficiency during this period suppressed subsequent terminal differentiation. This was associated with demethylation of both repressive histone marks and DNA in the genomic regions of adipocyte differentiation-associated genes, includingPparg, which encodes PPARγ, the master regulator of adipocyte differentiation. In addition, we identified several epigenetic demethylases to be responsible for iron-dependent adipocyte differentiation, with the histone demethylase jumonji domain-containing 1A and the DNA demethylase ten-eleven translocation 2 as the major enzymes. The interrelationship between repressive histone marks and DNA methylation was indicated by an integrated genome-wide association analysis, and was also supported by the findings that both histone and DNA demethylation were suppressed by either the inhibition of lysosomal ferritin flux or the knockdown of iron chaperone poly(rC)-binding protein 2. In summary, epigenetic regulations through iron-dependent control of epigenetic enzyme activities play an important role in the organized gene expression mechanisms of adipogenesis.

Vitamin C boosts DNA demethylation in TET2 germline mutation carriers

BackgroundAccurate regulation of DNA methylation is necessary for normal cells to differentiate, develop and function. TET2 catalyzes stepwise DNA demethylation in hematopoietic cells. Mutations in the TET2 gene predispose to hematological malignancies by causing DNA methylation overload and aberrant epigenomic landscape. Studies on mice and cell lines show that the function of TET2 is boosted by vitamin C. Thus, by strengthening the demethylation activity of TET2, vitamin C could play a role in the prevention of hematological malignancies in individuals with TET2 dysfunction. We recently identified a family with lymphoma predisposition where a heterozygous truncating germline mutation in TET2 segregated with nodular lymphocyte-predominant Hodgkin lymphoma. The mutation carriers displayed a hypermethylation pattern that was absent in the family members without the mutation.MethodsIn a clinical trial of 1 year, we investigated the effects of oral 1 g/day vitamin C supplementation on DNA methylation by analyzing genome-wide DNA methylation and gene expression patterns from the family members.ResultsWe show that vitamin C reinforces the DNA demethylation cascade, reduces the proportion of hypermethylated loci and diminishes gene expression differences between TET2 mutation carriers and control individuals.ConclusionsThese results suggest that vitamin C supplementation increases DNA methylation turnover and provide a basis for further work to examine the potential benefits of vitamin C supplementation in individuals with germline and somatic TET2 mutations.Trial registration: This trial was registered at EudraCT with reference number of 2018-000155-41 (01.04.2019).Graphical

Oxidized mC modulates synthetic lethality to PARP inhibitors for the treatment of leukemia

TET2 haploinsufficiency is a driving event in myeloid cancers and is associated with a worse prognosis in patients with acute myeloid leukemia (AML). Enhancing residual TET2 activity using vitamin C increases oxidized 5-methylcytosine (mC) formation and promotes active DNA demethylation via base excision repair (BER), which slows leukemia progression. We utilize genetic and compound library screening approaches to identify rational combination treatment strategies to improve use of vitamin C as an adjuvant therapy for AML. In addition to increasing the efficacy of several US Food and Drug Administration (FDA)-approved drugs, vitamin C treatment with poly-ADP-ribosyl polymerase inhibitors (PARPis) elicits a strong synergistic effect to block AML self-renewal in murine and human AML models. Vitamin-C-mediated TET activation combined with PARPis causes enrichment of chromatin-bound PARP1 at oxidized mCs and γH2AX accumulation during mid-S phase, leading to cell cycle stalling and differentiation. Given that most AML subtypes maintain residual TET2 expression, vitamin C could elicit broad efficacy as a PARPi therapeutic adjuvant.

Epigenetic drug screen identified IOX1 as an inhibitor of Th17-mediated inflammation through targeting TET2.

Targeting helper T cells, especially Th17cells, has become a plausible therapy for many autoimmune diseases. Using an invitro culture system, we screened an epigenetics compound library for inhibitors of IFN-γ and IL-17 expression in murine Th1 and Th17 cultures. This identified IOX1 as an effective suppressor of IL-17 expression in both murine and human CD4+ T cells. Furthermore, we found that IOX1 suppresses Il17a expression directly by targeting TET2 activity on its promoter in Th17cells. Using established pre-clinical models of intraocular inflammation, treatment with IOX1 invivo reduced the migration/infiltration of Th17cells into the site of inflammation and tissue damage. These results provide evidence of the strong potential for IOX1 as a viable therapy for inflammatory diseases, in particular of the eye. This study was supported by the National Key Research and Development Program of China 2021YFA1101200 (2021YFA1101204) to LW and XW; the National Natural Science Foundation of China 81900844 to XH and 82171041 to LW; the China Postdoctoral Science Foundation 2021M700776 and the Scientific Research Project of Guangdong Provincial Bureau of Traditional Chinese Medicine 20221373 to YZ; and the National Institute for Health Research (NIHR) Biomedical Research Centre at Moorfields Eye Hospital NHS (National Health Service) Foundation Trust and University College London Institute of Ophthalmology, UK (DAC, LPS, PJPL, MS, ADD and RWJL). The views expressed are those of the authors and not necessarily those of the NIHR or the UK's Department of Health and Social Care.

Phase II Study Assessing Safety and Preliminary Efficacy of High Dose Intravenous Ascorbic Acid in Patients with TET2 Mutant Clonal Cytopenias of Undetermined Significance

Background: Clonal cytopenia(s) of undetermined significance (CCUS) is defined as persistent cytopenias arising in the context of myeloid neoplasm (MN)-associated somatic mutations (MT) in hematopoietic stem cells. Patients (PTs) with TET2 mutant (TET2MT) CCUS have a high probability of progression to MN. To date, no FDA-approved treatment for CCUS exist, with PTs often having similar cytopenias and transfusion needs as those with myelodysplastic syndromes (MDS). High dose intravenous ascorbic acid (HI-AA) demonstrates anti-cancer activity via two mechanisms: (1) hydrogen peroxide-induced oxidative stress and (2) DNA demethylation mediated by TET activation (cofactor for TET1/2/3). TET2 is an AA and oxoglutarate-dependent dioxygenase that catalyzes the conversion of 5-methylcytosine to 5-hydroxymethylcytosine (5hmC), providing the rationale to study HI-AA in TET2MT CCUS. Methods: This is an investigator-initiated, single-arm, single-institution, phase II trial assessing the safety and preliminary efficacy of HI-AA for PTs with TET2MT CCUS (NCT03418038). PTs³ 18 years with TET2MT CCUS (≥1 TET2MT with or without additional somatic MTs) and with any of the following laboratory criteria: (1) hemoglobin (Hb) ≤10g/dL, (2) absolute neutrophil count (ANC) ≤1(10^9/L), (3) platelet count (PLT)≤100 (10^9/L) are eligible. HI-AA (1g/kg, maximum 100g) is given 3 times weekly for 12 weeks through a PICC line, with response assessments before each cycle (4 weeks), at 20 weeks, and one year. The primary endpoint is the hematologic response rates determined by the proposed MDS IWG 2018 criteria at week 20. (Platzbecker Blood 2018) and reported as hematologic improvement (HI)- erythropoietic (HI-E), platelets (HI-P), neutrophils (HI-N). Secondary endpoints include safety and adverse events (AEs). AEs were graded using NCI-CTCAE v4.03. Correlative studies include changes in TET2MT variant allele fraction (VAF), TET2 activity, DNA methylation, hydroxymethylation, and 5hmC in bone marrow cells as determined by immunohistochemistry (IHC). Results: As of July 22, 2022, 8 (89%) of 9 enrolled PTs completed 12 weeks of therapy. The mean age was 74 (SD: 5.4) years, 7 (87.5%) males. One PT (PT_X) came off study after cycle one due to a PICC line-associated thrombosis. All PT were TET2MT (44% truncating), with 8 of 9 (89%) having co-mutations (4- 2 MTs, 3- 3 MTs, and 1- 4 MTs; 2 had ≥1 TET2MT). Comutations included SRSF2 (55%), ASXL1 (22%), ZRSR2 and KRAS (11% each). HI-AA was well tolerated with the most common AEs being infusion-related polyuria (56%) and polydipsia (44%, 3 Grade 1, 1 Grade 2). One (11%) PT had constipation and headaches (both Grade 1), and 1 (11%) experienced dyspepsia. No treatment-related Grade 3 or 4 AEs or deaths were observed. Six of 8 (75%) assessable PTs had post-treatment response assessments at week 20, with 3 PTs having completed 1-year assessments. All PTs' baseline Hb was within the normal range, except for 1 (17%) (PT_5- TET2MT/SRSF2MT) PT who continued to remain red blood transfusion dependent. Five (63%) PTs were thrombocytopenic PLT <100 (10^9/L), with 2 (25%) PTs (PT_1 and PT_7) having PLT £50 (10^9/L) and 5 (83%) PTs had ANC<1 (10^9/L) with 1 (17%) (PT_8) with ANC <0.5 (10^9/L). One (17%) PT met criteria for HI-P (PT_6 had an increment to 118 (10^9/L) at week 20- TET2MT/SRSF2MT/KRASMT) and 1 (17%) PT met criteria for HI-N (PT_2, ANC baseline:1.08 vs. week 20: 1.76(10^9/L)-TET2MT/SRSF2MT). PT_1, who had a baseline PLT of 25 (10^9/L) and ANC of 0.72 (10^9/L), had disease progression to MDS-EB-1 (TET2MT/SRSF2MT) at week 20. The 3 PTs with 1 year follow-up have stable disease. Panel 1 demonstrates changes in blood parameters with treatment. Panel 2 shows IHC determined changes in 5hmC changes with two control samples: normal control and TET2MT CMML. In 5 assessable PTs, there were no changes in TET2 VAF at 20 weeks (mean VAF : baseline 37.2% vs. week 20: 36.6%, p=0.3). Additional correlative studies are ongoing. Conclusion: We report safety and preliminary efficacy analysis for the first clinical trial dedicated to TET2MT CCUS. The early results of this pilot demonstrate that HI-AA is safe and has potential for biological and clinical effects. At last follow-up, 2 (33%) PTs met criteria for HI (HI-P-1 and HI-N-1), 3 PTs (50%) had stable disease, and 1 (17%) PT progressed to MDS. Future studies assessing potential synergy of adding HI-AA to standard of care therapies for TET2MT clonal processes are planned. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

AHR/TET2/NT5E axis downregulation is associated with the risk of systemic lupus erythematosus and its progression.

The prognosis of systemic lupus erythematosus (SLE) is unpredictable. This study aimed to examine the regulatory mechanism of the AHR/TET2/NT5E pathway during SLE progression. The AHR, TET2 and NT5E expression levels were examined in T regulatory cells (Tregs) of patients with SLE. The correlation of AHR, TET2 or NT5E expression levels with the immunosuppressive functions of Tregs was analysed. In patients with SLE, the number of CD4+ IL2RA- FOXP3+ T cell subset was positively correlated with the SLE disease activity index value and negatively correlated with the AHR and TET2 expression levels in CD4+ IL2RA+ FOXP3+ Tregs. Transcriptional profiles of 79 patients with SLE obtained from the Gene Expression Omnibus database (GSE61635 dataset) revealed a significant positive correlation between the mRNA expression levels of AHR and TET2. In silico analysis predicted that the TET2 promoter comprises an AHR-binding site. Kynurenine (KYN) promoted the binding of AHR to the TET2 promoter in Tregs of patients with SLE and Jurkat T cell lines. Furthermore, NT5E expression was significantly downregulated in Tregs of patients with SLE, which can be attributed to the dysregulation of NT5E promoter methylation status induced by downregulated TET2 activity. Furthermore, the Treg immunosuppressive activity, which is mediated through the TET2 and A2AR-adenosine pathways, in the KYN-treated group was approximately two-fold higher than that in the control group. The AHR/TET2/NT5E axis mediates the Treg immunosuppressive activity. These findings provide novel insights for the development of therapeutic approaches for SLE and related autoimmune diseases.

ROS attenuates TET2-dependent ZO-1 epigenetic expression in cerebral vascular endothelial cells

AimsTo investigate whether DNA active demethylase TET regulates the expression of tight junction proteins in endothelial cells of the blood–brain barrier (BBB).MethodsCorrelations between TET2 activity (indicated by its catalytic product 5hmC) and the expression of BBB tight junction proteins were examined in Tet2 knockout mice and post-mortem human brain tissues. In cultured endothelial cells, the impact of changes of TET activity on the expression of tight junction protein, ZO-1, was studied. BBB permeability assays were performed in Tet2 knockout mice.ResultsIt was found that the level of 5hmC decreased in brain microvascular endothelial cells of aging mice. In Tet2 knockout mice, the level of 5hmC in endothelial cells was weaker and significantly correlated with the reduced expression of tight junction protein ZO-1. In cultured endothelial cells, H2O2 significantly decreased the expression of 5hmC and ZO-1. Tet2 knock-down using siRNA significantly downregulated the expression of ZO-1 in endothelial cells. hMeChip-PCR showed that H2O2 decreased the level of 5hmC in the ZO-1 promoter region, which was rescued by N-acetyl cysteine (NAC). Consistently, Tet2 knock-down using siRNA significantly downregulated the level of 5hmC in the ZO-1 promoter region. It was also found that the level of 5hmC decreased in endothelial cells of aging human brains compared with that of adult brains, and the level of ZO-1 was positively correlated with that of 5hmC in microvascular endothelial cells.ConclusionsThese findings suggest that TET activity is essential in regulating ZO-1 expression of BBB. It might be a potential target for neuroprotection during aging and in diverse neurological conditions.

DNA Methylation Underlies the Long‐Term Association Between Periodontitis and Atherosclerotic Cardiovascular Disease

Periodontitis, one of the most common inflammatory conditions, and the leading cause of adult tooth loss, has been identified as an independent risk factor for cardiovascular disease (CVD). Long‐term studies in edentulous patients with a history of periodontitis showed persistence of increased CVD risk for years after edentulism, suggesting that clinical elimination of disease is not sufficient in decreasing periodontitis‐induced CVD risk. This is similar to other CVD risk factors, such as smoking and diabetes. We hypothesized that periodontitis induces epigenetic changes in hematopoietic stem cells in the bone marrow (BM), and such changes persist after clinical elimination of the disease and underlie the induced‐CVD risk.To simulate clinical elimination of periodontitis and persistence of the hypothesized epigenetic reprogramming, we used a BM transplant approach. Using the low density lipoprotein receptor (LDLR) KO atherosclerosis mouse model, BM donor mice were fed a high fat diet (HFD) to induce atherosclerosis, and orally inoculated with Porphyromonas gingivalis(Pg), a keystone periodontal pathogen, to induce periodontitis; a second group was sham inoculated. (We previously showed that HFD‐fed, Pginoculated mice developed more atherosclerosis than sham‐inoculated). Naïve LDLR KO mice were irradiated and transplanted with BM from one of the 2 donor groups.Recipients of BM from Pg‐inoculated donors developed significantly more atherosclerosis, accompanied by more pro‐inflammatory plasma and macrophage cytokine profiles. Using whole genome bisulfite sequencing, 375 differentially methylated regions (DMR), and a global hypomethylation in recipients of BM from Pg‐inoculated donors, were identified. Some DMRs pointed to involvement of enzymes with major roles in methylation and demethylation. In recipients of BM from Pg‐inoculated donors, methionine adenosyl transferase (MAT), which catalyzes the conversion of methionine to S‐denosylmethionine (SAM), the universal methyl‐donor, was hypermethylated. The gene for S‐adenosylhomocysteine hydrolase, which catalyzes the reversible conversion of the potent methylation inhibitor, S‐adenosylhomocysteine (SAH), to homocysteine and adenosine, was also hypermethylated. In contrast, the de‐methylation enzyme, Ten‐Eleven Translocase (TET) 2, was hypomethylated. In validation assays, we found a significant increase in activity of TET2 and a decrease in activity of DNA methyltransferases. Plasma SAH levels were significantly higher and the SAM to SAH ratio was decreased, both of which have been associated with CVD. Homocysteine, is at the intersection of the methionine cycle and the transsulphuration pathway, where it can either be re‐methylated to methionine and go through the methionine cycle, or it can synthesize cysteine through the transsulphuration pathway. Glutathione, a critical antioxidant, is the end‐product of the transsulphuration pathway. In conditions of oxidative stress, the transsulphuration pathway is favored over the methionine cycle. Periodontitis is associated with oxidative stress. These data suggest a paradigm shifting mechanism linking epigenetic changes in gene methylation with the long term association between periodontitis and atherosclerotic CVD.

Itaconate inhibits TET DNA dioxygenases to dampen inflammatory responses.

As one of the most induced genes in activated macrophages, immune-responsive gene 1 (IRG1) encodes a mitochondrial metabolic enzyme catalysing the production of itaconic acid (ITA). Although ITA has an anti-inflammatory property, the underlying mechanisms are not fully understood. Here we show that ITA is a potent inhibitor of the TET-family DNA dioxygenases. ITA binds to the same site on TET2 as the co-substrate α-ketoglutarate, inhibiting TET2 catalytic activity. Lipopolysaccharide treatment, which induces Irg1 expression and ITA accumulation, inhibits Tet activity in macrophages. Transcriptome analysis reveals that TET2 is a major target of ITA in suppressing lipopolysaccharide-induced genes, including those regulated by the NF-κB and STAT signalling pathways. In vivo, ITA decreases the levels of 5-hydroxymethylcytosine, reduces lipopolysaccharide-induced acute pulmonary oedema as well as lung and liver injury, and protects mice against lethal endotoxaemia, depending on the catalytic activity of Tet2. Our study thus identifies ITA as an immune modulatory metabolite that selectively inhibits TET enzymes to dampen the inflammatory responses.

Flanking sequences influence the activity of TET1 and TET2 methylcytosine dioxygenases and affect genomic 5hmC patterns

TET dioxygenases convert 5-methylcytosine (5mC) preferentially in a CpG context into 5-hydroxymethylcytosine (5hmC) and higher oxidized forms, thereby initiating DNA demethylation, but details regarding the effects of the DNA sequences flanking the target 5mC site on TET activity are unknown. We investigated oxidation of libraries of DNA substrates containing one 5mC or 5hmC residue in randomized sequence context using single molecule readout of oxidation activity and sequence and show pronounced 20 and 70-fold flanking sequence effects on the catalytic activities of TET1 and TET2, respectively. Flanking sequence preferences were similar for TET1 and TET2 and also for 5mC and 5hmC substrates. Enhanced flanking sequence preferences were observed at non-CpG sites together with profound effects of flanking sequences on the specificity of TET2. TET flanking sequence preferences are reflected in genome-wide and local patterns of 5hmC and DNA demethylation in human and mouse cells indicating that they influence genomic DNA modification patterns in combination with locus specific targeting of TET enzymes.

The KDM5 Family of Histone Lysine Demethylases Are Targets of R-2-Hydroxyglutarate in IDH Mutant Tumors

Gain-of-function mutations in isocitrate dehydrogenase enzymes IDH1 and IDH2 occur in ∼10% of acute myeloid leukemias (AML) and >80% of gliomas. The mutant enzymes convert 2-oxoglutarate (2OG) to the oncometabolite R-2-hydroxyglutarate (R-2HG). R-2HG promotes cellular transformation by modulating the activities of 2OG-dependent dioxygenases (2OGDDs). The only functionally validated direct target of R-2HG is TET2, a 2OGDD myeloid tumor suppressor that catalyzes the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC).Interestingly, in clonal myeloid disorders the patterns of IDH and TET2 mutations are vastly different. TET2 mutations occur at similar frequencies in clonal hematopoiesis of unknown significance (CHIP), lower- and higher-grade myeloproliferative (MPN) and myelodysplastic (MDS) disorders, and primary and secondary AML. IDH mutations, on the other hand, are associated with higher-grade and blast-phase MPN and MDS and with de novo AML and are rare in CHIP and low-grade MDS. This suggests that mutant IDH promotes a more aggressive disease phenotype and that R-2HG has additional targets other than TET2 that contribute to its leukemogenic activity.To ask if the in vitro transforming activity of R-2HG directly correlates with TET2 inhibition, we treated TF-1 cells, a cytokine-dependent human AML cell line, with a dose range of cell-permeable esterified R-2HG. We found that R-2HG induces cytokine independence at concentrations that have no effect on 5hmC levels. To identify other 2OGDD myeloid tumor suppressors that could be contributing to R-2HG-mediated transformation, we performed a positive-selection CRISPR-Cas9 screen under cytokine-poor conditions in TF-1 cells. We identified three H3K4 histone lysine demethylases, KDM5A, KDM5C and KDM5D, as genes whose sgRNAs were enriched upon cytokine withdrawal. Triple knockout of KDM5A, KDM5C and KDM5D (TKO) in TF-1 cells induces robust cytokine independence. Likewise, treatment of TF-1 cells with KDM5c70, a specific inhibitor of KDM5 enzymes, strongly induces TF-1 cytokine independence. Of note, KDM5 inhibition has no effect on TET2 expression or 5hmC levels. We further found that R-2HG is a more potent inhibitor of KDM5A, KDM5C and KDM5D than of TET2. We then assessed the effect of mutant IDH1 expression, TKO, R-2HG treatment and KDM5c70 treatment on H3K4 trimethylation by ChIP-seq and found that each of these perturbations results in a significant enrichment in H3K4me3 peaks relative to controls.TET enzymes are not recurrently mutated in glioma and although there is a strong correlation between mutant IDH status and the CpG island methylator phenotype (CIMP), direct inhibition of TET2 by R-2HG has not been reproducibly demonstrated in glioma. To ask if TET2 activity is suppressed in IDH mutant glioma, we quantified 5hmC levels in a panel of primary IDH wild-type and IDH mutant glioma and AML samples by mass spectrometry. We found that, unlike in AML, in glioma there is no correlation between IDH1 mutation status and loss of 5hmC. We likewise saw no correlation between 5hmC levels and either IDH mutation status or intracellular R-2HG levels in patient derived xenograft (PDX) models of glioma. Given the lack of evidence that TET enzymes are tumor suppressor targets of R-2HG in IDH mutant glioma, we asked if mutant IDH positivity is associated with increased levels of H3K4 methylation in glioma. We performed ChIP-seq on a panel of IDH wild-type and IDH mutant glioma PDX lines and found H3K4me peaks to be highly enriched in the IDH mutant lines when compared to IDH wild-type lines. Trimethyl-H3K4 levels were likewise increased in isogenic normal human astrocyte (NHA) cells ectopically expressing mutant IDH1.Collectively, these data suggest that R-2HG inhibits KDM5 histone lysine demethylases to promote mutant IDH-mediated transformation in AML and glioma. These studies identify a novel direct target of R-2HG in IDH mutant tumors and provide a functional link between IDH mutations and dysregulated histone lysine methylation in cancer. DisclosuresNo relevant conflicts of interest to declare.

Myelodysplastic Syndromes: A More Global 5-Hydroxymethylcytosine Deficiency Disorder Than Suggested By the Presence of TET2 Mutations

Myelodysplastic Syndromes: A More Global 5-Hydroxymethylcytosine Deficiency Disorder Than Suggested By the Presence of TET2 Mutations

Investigating Germline Predisposition to Clonal Hematopoiesis through Perturbation of a Variant-Harboring Enhancer of TET2

Clonal hematopoiesis (CH) is the age-related expansion of hematopoietic stem and progenitor cells (HSPCs) due to acquired genetic changes and is associated with increased blood cancer risk. Despite considerable progress in understanding how specific acquired somatic mutations result in clonal expansion, we have a limited understanding of the role of germline mutations that also predispose to clonal expansion.Recent work has revealed a low frequency germline variant found exclusively in individuals of African diasporic descent that confers a 2.4-fold increased risk for CH (Bick et al., Nature, 2020). Remarkably, this variant is found within a putative enhancer of the TET2 gene, which encodes a key epigenetic modifier that catalyzes conversion of methylated cytosines to 5-hydroxymethylcytosine, thereby facilitating DNA demethylation. However, the precise role of this enhancer variant in altering TET2 activity and human hematopoiesis is poorly understood. We specifically hypothesize that this germline variant may alter TET2 activity subtly in hematopoietic stem cells (HSCs) to modify DNA methylation at a number of HSC regulatory elements and subsequently gene expression, which are likely mediated through selective changes in transcription factor (TF) activity.To explore this hypothesis, we performed deletions of the putative TET2 enhancer in adult CD34 +HSPCs using CRISPR/Cas9 editing through the introduction of Cas9 ribonucleoproteins. Following deletion of this enhancer, we observed a moderate increase in the total number of phenotypic long-term reconstituting hematopoietic stem cells (LT-HSCs; as marked by CD34 +CD45RA -CD90 +CD201 +CD133 +CD49c +) and in primary colony formation unit (CFU) assays compared to control editing (AAVS1 edited). Interestingly, the enhancer deletion did not cause an increase in the number of phenotypic short-term HSCs (CD34 +CD45RA -CD90 +CD201 +CD133 +CD49c -), HSPC proliferation, or secondary CFU plating. Assessment of the deletion stability showed selection against enhancer deleted cells during myeloid differentiation, however some cells could still be identified after more than 30 days following editing. This result suggests that the enhancer likely functions in a selective manner within HSCs. The overall phenotypes we observe suggest some overlap, but distinct presentations in comparison to concomitant TET2 coding disruption that we have also performed.Ongoing studies seek to use these promising phenotypic results to define changes in accessible chromatin and DNA methylation states in this isogenic perturbed model. This will enable insights into the specific enhancers altered through this perturbation and we plan to examine TF motif alterations at these regulatory elements. While the perturbation performed may represent a larger perturbation than is seen through the naturally-occurring variant, this provides a platform for defining how this CH predisposition arises with a larger impact perturbation. Use of base editors in a similar manner should enable more selective perturbations, as well. Together, these results will further reveal potential germline genetic and molecular origins of CH and further explain broader mechanisms of TET2 function and the importance of proper DNA methylation during human hematopoiesis that may provide clinical relevance for the potential prevention of blood cancers. DisclosuresSankaran: Cellarity: Consultancy; Forma: Consultancy; Novartis: Consultancy; Ensoma: Consultancy; Branch Biosciences: Consultancy.

Ascorbate Inhibits Proliferation and Promotes Myeloid Differentiation in TP53-Mutant Leukemia

Loss-of-function mutations in the DNA demethylase TET2 are associated with the dysregulation of hematopoietic stem cell differentiation and arise in approximately 10% of de novo acute myeloid leukemia (AML). TET2 mutations coexist with other mutations in AML, including TP53 mutations, which can indicate a particularly poor prognosis. Ascorbate can function as an epigenetic therapeutic in pathological contexts involving heterozygous TET2 mutations by restoring TET2 activity. How this response is affected when myeloid leukemia cells harbor mutations in both TET2 and TP53 is unknown. Therefore, we examined the effects of ascorbate on the SKM-1 AML cell line that has mutated TET2 and TP53. Sustained treatment with ascorbate inhibited proliferation and promoted the differentiation of these cells. Furthermore, ascorbate treatment significantly increased 5-hydroxymethylcytosine, suggesting increased TET activity as the likely mechanism. We also investigated whether ascorbate affected the cytotoxicity of Prima-1Met, a drug that reactivates some p53 mutants and is currently in clinical trials for AML. We found that the addition of ascorbate had a minimal effect on Prima-1Met–induced cytotoxicity, with small increases or decreases in cytotoxicity being observed depending on the timing of treatment. Collectively, these data suggest that ascorbate could exert a beneficial anti-proliferative effect on AML cells harboring both TET2 and TP53 mutations whilst not interfering with targeted cytotoxic therapies such as Prima-1Met.