Tet2 Loss Research Articles

Abstract 2934: TET2 deficiency accelerates leukemogenesis in the NHD13 mouse model of MDS

Abstract Myelodysplastic syndromes (MDS) frequently progresses to acute myeloid leukemia (AML). Mechanisms underlying MDS transformation remain unclear. We showed that TET2 deficiency enhances MDS maintenance (PMID: 30146412). Besides frequent mutated in MDS/AML, TET2 levels were decreased during MDS progression (PMID: 23827711). Recently, we analyzed a dataset (GSE19429) and found lower TET2 levels correlated with shortened survival of patients with high-risk MDS. We thus assessed TET2 function in MDS leukemia transformation by deleting TET2 in a NHD13 knock-in mouse model, in which ~30% of mice develop AML. Therefore, we crossed conditional TET2-KO mice with NHD13 transgenic mice. NHD13/TET2-KO (NT) mice developed leukemia with shorter survival than did NHD13/TET2-WT (ctrl) mice (median days, NT: 219 vs. ctrl: 366, n=12, p<0.01). Half of the NT mice developed AML in 30 weeks, while ctrl mice did not. At 20th weeks (pre-leukemia stage), NT mice exhibited splenomegaly and hyper-cellular bone marrow (BM). Flow cytometry analysis of NT BM cells revealed increased lin-c-kit+sca-1- (LK) BM subset (LK%, NT: 3.89% vs. ctrl: 1.96%, n=12, p<0.01) and decreased apoptosis (AV+%, NT: 2.85% vs. ctrl: 7.59%, n=12, p<0.001). To search for acquired genetic lesions due to TET2-KO, we performed whole-exome sequencing of c-kit+ cells from pre-leukemic NT vs. ctrl mice. We focused on mutations newly acquired or those with increased variant allele frequency (VAF, fold change>2) in NT mice and ranked them based on their association with AML prognosis using PRECOG (http://precog.stanford.edu). We selected top three candidates SETD2, ARIH2, TET3 for further analysis. ChIP-qPCR revealed increased 5hmC modification at the mutant loci of these genes, consistent with a finding that the genomic regions with increased 5hmC modification are more subjective to occur mutations (PMID: 28440315). TET3 and SETD2, as tumor suppressors, exhibit frameshift-deletion. ARIH2 represents a single-nucleotide variant (ARIH2K440N). To test if ARIH2 loss-of-function accelerates proliferation, we transduced NHD13 c-kit+ cells with shARIH2 and performed CFC analysis. ARIH2 knockdown increased colony number relative to ctrl (shARIH2: 163±3.7 vs. shCtrl: 95±1.7, n=3, p<0.01). To assess ARIH2K440N function we constructed the corresponding human mutation ARIH2K441N as well as ARIH2R484I, seen in a patient with MDS (PMID: 19620960). We genetically deleted endogenous ARIH2 of K562 cells, then transfected with exogenous ARIH2-WT or ARIH2 mutants respectively. Unlike ARIH2-WT, ectopic expression of ARIH2K441N or ARIH2R484I blocked FGFR1 ubiquitination and degradation, activating STAT5, AKT and ERK1/2 signaling, suggesting that TET2 loss leads to secondly hits, which activate proliferation/survival signaling. Taken together, our finding provides a rationale for enhancing TET2 function to block MDS-malignant transformation. Citation Format: Feiteng Huang, Jie Sun, Wei Chen, Lei Zhang, Xin He, Haojie Dong, Yuhui Wu, Hanying Wang, Zheng Li, Victor Huang, Samer Khaled, Ling Li. TET2 deficiency accelerates leukemogenesis in the NHD13 mouse model of MDS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2934.

Spatial transcriptome profiling by MERFISH reveals fetal liver hematopoietic stem cell niche architecture

The hematopoietic stem cell (HSC) niche has been extensively studied in bone marrow, yet a more systematic investigation into the microenvironment regulation of hematopoiesis in fetal liver is necessary. Here we investigate the spatial organization and transcriptional profile of individual cells in both wild type (WT) and Tet2−/− fetal livers, by multiplexed error robust fluorescence in situ hybridization. We find that specific pairs of fetal liver cell types are preferentially positioned next to each other. Ligand-receptor signaling molecule pairs such as Kitl and Kit are enriched in neighboring cell types. The majority of HSCs are in direct contact with endothelial cells (ECs) in both WT and Tet2−/− fetal livers. Loss of Tet2 increases the number of HSCs, and upregulates Wnt and Notch signaling genes in the HSC niche. Two subtypes of ECs, arterial ECs and sinusoidal ECs, and other cell types contribute distinct signaling molecules to the HSC niche. Collectively, this study provides a comprehensive picture and bioinformatic foundation for HSC spatial regulation in fetal liver.

Aberrant Alternative Splicing in U2af1/Tet2 Double Mutant Mice Contributes to Major Hematological Phenotypes.

Mutations in splicing factors are recurrent somatic alterations identified in myelodysplastic syndromes (MDS) and they frequently coincide with mutations in epigenetic factors. About 25% of patients present concurrent mutations in such pathways, suggesting a cooperative role in the pathogenesis of MDS. We focused on the splicing factor U2AF1 involved in the recognition of the 3′ splice site during pre-mRNA splicing. Using a CRISPR/Cas9 system, we created heterozygous mice with a carboxy-terminal truncated U2af1 allele (U2af1mut/+), studied the U2af1mut/+ hematopoietic system, and did not observe any gross differences in both young (12–13 weeks) and old (23 months) U2af1mut/+ mice, except for a reduction in size of approximately 20%. However, hematopoietic stem/progenitor cells lacked reconstitution capacity in transplantation assays and displayed an aberrant RNA splicing by RNA sequencing. We also evaluated U2af1mut/+ in conjunction with Tet2-deficiency. Novel double mutant U2af1mut/+ Tet2−/− mice showed increased monogranulocytic precursors. Hematopoietic stem/progenitor cells were also enhanced and presented functional and transcriptomic alterations. Nonetheless, U2af1mut/+ Tet2−/− mice did not succumb to MDS disease over a 6-month observation period. Collectively, our data suggest that cooperation between mutant U2af1 and Tet2 loss is not sufficient for MDS initiation in mice.

Spliceosomal Dysfunction Is a Critical Mediator of IDH2 Mutant Leukemogenesis

Mutations affecting RNA splicing factors are commonly found in leukemias and mutations in SF3B1, SRSF2, and U2AF1 impart specific changes to global splicing. We analyzed RNA-seq data from acute myeloid leukemia (AML) patients from The Cancer Genome Atlas (TCGA) to evaluate for spliceosomal alterations characteristic of mutant SRSF2, SF3B1, and U2AF1 . Although only 1 patient with an SRSF2 mutation was reported in the TCGA AML publication, we identified 19 additional patients to have the characteristic splicing changes of mutant SRSF2 . Mutational analysis of the RNA-seq data identified the SRSF2 proline 95 hotspot mutation in each of these 19 patients (19/178, 11%), making SRSF2 amongst the most commonly mutated genes in the AML TCGA dataset. Interestingly, 47% of SRSF2 mutant patients also had a co-existing IDH2 mutation (and conversely, 50% of IDH2 mutant patients had a co-existing SRSF2 mutation). These data are similar to recent observation that SRSF2 mutations were the most common accompanying mutation in IDH2 mutant myeloid leukemia patients treated on the phase I clinical trial of AG-221.We next sought to understand the functional contribution of SRSF2 mutations to co-existing IDH2 mutations. Overexpression of IDH2 R140Q and IDH2 R172K in Vav -cre Srsf2 P95H or Vav- cre Srsf2 wildtype(WT) backgrounds revealed a clear collaborative effect of mutant IDH2 and mutant Srsf2 (Figure A-B). As early as 4 weeks post-transplant, the peripheral blood of Srsf2 mutant mice transplanted with IDH2 R140Q or IDH2 R172K forms had a substantially greater percentage of GFP+ cells than seen in an Srsf2 WT background. Moreover, these mice exhibited significant myeloid skewing, macrocytic anemia, and thrombocytopenia of greater magnitude than seen with mutant IDH2 expressed in the SRSF2 WT background. IDH2 / Srsf2 double-mutant mice developed a lethal, mixed myelodysplastic/myeloproliferative neoplasm that was serially transplantable (Figure B). Effects from overexpression of mutant IDH2 were recapitulated in mice expressing Idh2 R140Q and Srsf2 P95H from endogenous loci(Mx1 -cre Idh2R140Q/+Srsf2P95H/+ double knock-in mice) (Figure C), providing the first model where spliceosomal gene mutations promote leukemogenesis in vivo .Given prior data identifying inhibition of TET2 as a mediator of IDH2 -mutant leukemogenesis, we evaluated if loss of TET2 might promote transformation of SRSF2 mutant cells. However, deletion of TET2 in an SRSF2 mutant background (Mx1 -cre Tet2fl/flSrsf2P95H/+) was insufficient to promote transformation (Figure D). We next analyzed RNA-seq data from the TCGA, Leucegene, and our own unpublished AML cohort using a novel analysis method to detect unannotated as well as complex splicing events that might explain the basis for the collaboration of mutant IDH2 and SRSF2 . This revealed that IDH2/SRSF2 double-mutant AML had increased aberrant splicing events compared to SRSF2 single-mutant AML and also that IDH2 -mutations are associated with a small but reproducible change in RNA splicing (Figure E). The vast majority of splicing changes affected by SRSF2 and/or IDH2 mutations were a change in cassette exon splicing with SRSF2 mutations promoting inclusion of C-rich containing mRNA sequences, regardless of the presence of concomitant IDH2 mutations (Figure F). One of the most robust mis-splicing events in IDH2/SRSF2 mutant AML was an intron retention event in INTS3 resulting in its reduced mRNA and protein expression (Figure G-H). INTS3 encodes a component of the integrator complex that participates in small nuclear RNA (snRNA) processing. Consistent with this, IDH2 / SRSF2 double-mutant cells exhibited alterations in processing of U2 and U4 snRNAs similar to that seen with direct INTS3 downregulation. Biologically, attenuation of INTS3 expression caused a blockade of myeloid differentiation in HL-60 cells upon ATRA treatment, an effect which was further enhanced in an IDH2 mutant background.These data uncover an important role for spliceosomal gene mutations in the pathogenesis of IDH2 mutant leukemogenesis and identify perturbations in the integrator complex as a novel mediator driving the transformation of IDH2 and SRSF2 mutant cells. The frequent coexistence of IDH2 and SRSF2 mutations underscores the enormous therapeutic potential for modulation of splicing in the ~50% of IDH2 -mutant leukemia patients who also harbor a spliceosomal gene mutation. [Display omitted] DisclosuresDe Botton:Servier: Honoraria; Agios: Honoraria, Research Funding; Celgene: Honoraria; Novartis: Honoraria; Pfizer: Honoraria. Stein:Pfizer: Consultancy, Other: Travel expenses; Novartis: Consultancy, Research Funding; Agios Pharmaceuticals, Inc.: Consultancy, Research Funding; Celgene Corporation: Consultancy, Other: Travel expenses, Research Funding; Seattle Genetics: Research Funding; GSK: Other: Advisory Board, Research Funding; Constellation Pharma: Research Funding. Peng:H3 Biomedicine Inc.: Employment. Buonamici:H3 Biomedicine Inc.: Employment. Palacino:H3 Biomedicine Inc.: Employment. Smith:H3 Biomedicine Inc.: Employment. Thompson:Merck: Membership on an entity's Board of Directors or advisory committees; Charles River Laboratories: Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Other: Founder. Levine:Roche: Research Funding; Roche: Research Funding; Qiagen: Equity Ownership; Qiagen: Equity Ownership; Celgene: Research Funding; Celgene: Research Funding.

Leukemogenic Second Hits in CEBPA-Mutated AML Result in Diverse Effects on Haematopoiesis

Acute myeloid leukaemia (AML) is thought to develop through the stepwise acquisition of genetic aberrations within a haematopoietic stem or progenitor cell (HSPC) resulting in clonal expansion. N terminal mutations of the myeloid transcription factor CEBPA are not only a frequent event in sporadic AML, but they are also inherited in the germ line, conveying familial AML predisposition and thus can be assured to be an initiating event in leukaemogenesis. CEBPA-mutated AML also shows a remarkably conserved landscape of secondary mutations. Around a third of cases harbour mutations in the epigenetic regulator tet methylcytosine dioxygenase 2 (TET2), a gene frequently implicated in the pathogenesis of myeloid malignancy. A further third of cases are found to have mutations in the transcription factor, GATA2, moreover this latter association appears to be almost exclusive to CEBPA-mutated cases in AML.We have used a zebrafish model to interrogate the effects of these signature mutation patterns on haematopoiesis in juvenile and adult fish. A CEBPA N terminal mutant line (CebpaNterm) was generated using TALEN pairs, with a 5bp deletion leading to a frameshift between the first and second reading frame of this single exon gene, emulating that seen in both familial and sporadic AML cases. In homozygosity cebpaNTerm leads to complete absence of mature myeloid cells until 4 weeks of age. However, at 6 weeks of age all surviving cebpaNterm/Nterm fish had developed granulated myeloid cells on assessment of whole kidney marrow (WKM), with a dosage dependent increase in numbers with loss of wild-type (WT) Cebpa. By contrast in the peripheral blood (PB), a dose dependent reduction in both thrombocyte and myeloid cell numbers was observed with loss of WT Cebpa, but no evidence of anaemia. No homozygotes have survived past 2 months, due to susceptibility to infection and development of leukaemia with primitive features.To assess the effects of Tet2 loss in combination with cebpaNterm/+ we utilised a Tet2 null mutant line (tet2zdf20) crossed to cebpaNterm/+. Tet2zdf20/+ showed on haematopoietic lineages at 3 and 5 months of age as previously described {Gjini:2015bs}. However, when combined with heterozygosity for cebpaNterm, loss of Tet2 showed a cumulative deficit in both HSPC and myeloid cell numbers. Further evidence of cooperation between the two mutations was seen by assessment of thrombocyte number in PB, with platelet numbers steadily trending to their nadir at the maximum allelic loss i.e. cebpaWT/Nterm ; tet2zdf20/zdf20 noting that cebpaNterm/Nterm do not survive to this age. Whilst no anaemia was observed at 3 months in any genotype, by 5 months cebpaWT/Nterm ; tet2zdf20/+ fish showed a significant reduction in erythroid progenitors, suggesting an anticipatory effect of cebpa mutation on the tet2zdf20/+ myelodysplastic phenotype not usually observed until 8 months of age.Combination of cebpaNterm/+ with a gata2a (one of 2 zebrafish GATA2 orthologues) mutant carrying a truncating mutation prior to the zinc finger domains (gata2aum27) revealed a different picture. On assessment at 5 months gata2aum27/+ showed a reduction in both HSPC and myeloid cell numbers in the WKM, while cebpaNterm/WT showed an increase. In keeping with each individual mutation's influence on cell numbers, cebpaNterm/WT; gata2aum27/+ compound heterozygotes appeared showed near WT levels in both lineages. No abnormalities in erythroid progenitors or peripheral anaemia was evident in any genotype.In summary, we have described diverse haematopoietic effects of two known secondary mutations in cebpaNterm mutated AML. Mutations in Tet2 cooperate with cebpaNterm resulting in reduced cellularity across lineages. In the case of Gata2a, the addition of heterozygous cebpaNterm mutation appears to rescue a myeloid progenitor defect present in gata2aum27/+. Finally, in contrast to cebpaNterm/Nterm, we did not observe any cases of frank leukaemia in cebpaNterm combined with Gata2a or Tet2 loss. We speculate this is because the most common second hit in cebpaNterm, is a cebpa mutation in the carboxy terminal of the second cebpa allele, and this may be requisite for the development of AML. We have now generated a zebrafish with C terminal Cebpa mutation in order to test this hypothesis. DisclosuresNo relevant conflicts of interest to declare.

Clinical Evidence That Deficiency of the Methylcytosine Dioxygenase TET2 Gene Promotes the Clonal Expansion and Therapeutic Efficacy of CD19-Directed T Cells

Adoptive transfer of autologous chimeric antigen receptor (CAR) T cells that target tumor antigens can induce remissions in patients with hematologic malignancies. A major obstacle to successful clinical responses in some patient populations has been infusion with ineffective cell populations that are terminally differentiated, exhausted, poorly proliferative and short-lived. Understanding and manipulating the mechanistic pathways that sustain T cell potency will potentially unlock durable responses to tumors. Here we present an unusual case that helps clarify determinants of CAR T cell persistence. A 78-year-old individual with advanced chronic lymphocytic leukemia (CLL) was infused with CAR T cells that target CD19 (CTL019). CAR T cell expansion was delayed and not evident until 50 days following infusion and anti-tumor activity was observed in the peripheral blood, lymph nodes as well as bone marrow, accompanied by complete remission that has been sustained for over 4.5 years. Retrospective analyses revealed that at the peak of in vivo CAR T cell activity, over 94% percent of CD8+ CTL019 cells originated from a single clone in which lentiviral vector-mediated insertion of the CAR transgene led to monoallelic loss of TET2 . Accordingly, examination of polyadenylated TET2 RNA populations showed the appearance of new chimeric RNAs that spliced from TET2 exon 9 into the vector and terminated, truncating the encoded protein. Further analysis revealed that this patient had a germline polymorphism in the other allele of TET2 . This was a missense variant at amino acid 1879 (exon 11), converting the wildtype residue, glutamic acid, to glutamine (protein change: pE1879Q; cDNA change: c.5635G>C; allele frequency of approximately 50%). TET2 deficient CTL019 cells exhibited reduced cytosine hydroxymethylation and a chromatin accessibility profile measured by ATAC-Seq consistent with altered T cell differentiation. Furthermore, at the peak of in vivo engraftment, CAR T cells from this subject showed an early (central) memory phenotype which differs from CTL019 cells of other complete responding patients that are instead characterized by late (effector) memory differentiation at the height of their response. Experimental knock-down of TET2 in healthy donor CAR T cells altered their differentiation and augmented CTL019 effector function in a manner that was consistent with the effects of TET2 deficiency observed in this subject. Our findings suggest that the profound clonal expansion of a single TET2 deficient CAR T cell resulted in a complete and durable remission of CLL, highlighting the potential for epigenetic modulation of ineffective patient T cells to generate curative T cell products. DisclosuresFraietta:Novartis: Patents & Royalties: patents, Research Funding. Morrissette:Novartis: Consultancy. Liu:Novartis: Patents & Royalties. Young:Novartis: Research Funding. Levine:Novartis Pharmaceuticals Corporation: Patents & Royalties, Research Funding; Tmunity Therapeutics: Equity Ownership, Research Funding; GE Healthcare: Consultancy; Brammer Bio: Consultancy. Zhao:Novartis: Patents & Royalties. Kalos:Novartis: Research Funding. Porter:Incyte: Honoraria; Servier: Honoraria, Other: Travel reimbursement; Genentech/Roche: Employment, Other: Family member employment, stock ownship - family member; Immunovative Therapies: Other: Member DSMB; Novartis: Honoraria, Patents & Royalties, Research Funding. Lacey:Novartis: Research Funding; Genentech: Honoraria. June:Tmunity Therapeutics: Equity Ownership, Research Funding; WIRB/Copernicus Group: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celldex: Honoraria, Membership on an entity's Board of Directors or advisory committees; Immune Design: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Novartis: Patents & Royalties, Research Funding. Melenhorst:Novartis: Research Funding.

TET2 Regulates CD4+ T Follicular Helper Differentiation

DNA methylation is a key epigenetic mechanism that controls T cell differentiation. The ten-eleven translocation (TET) family of methylcytosine dioxygenases converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and other oxidized methylcytosines. TET2 mutations have been identified in angioimmunoblastic T cell lymphomas (AITL), which are derived from CD4+ T follicular helper (TFH) cells. CD4+ TFH cells are a T cell subset that provides essential help for the germinal center reaction and class switching. Here we demonstrate that TET2 regulates murine CD4+ TFH differentiation in vivo . At steady-state, mice with a T cell specific deletion of TET2 (TET2cKO) have intact thymic and peripheral T cell populations. Following acute viral infection with LCMV-Armstrong, TET2 loss promotes CD4+ TFH cell expansion. Examination of the Peyer's patches (a site of TFH differentiation in response to gut microbes) of wild-type and TET2cKO mice demonstrate an increase in the absolute number of the CD4+ TFH (CXCR5+PD1+) population in TET2cKO mice. Furthermore, there is an increase in the frequency and absolute number of germinal center B cells (characterized by PNA+GL7+ B cells) and an increase in IgA+ B cells in the Peyer's patches of TET2cKO mice compared to wild-type mice. Ongoing studies are examining the mechanism by which TET2 controls CD4+ TFH expansion. Our data indicate that TET2 is an important regulator of CD4+ TFH cells. Understanding the role of TET2 in normal CD4+ TFH differentiation and its contribution to T cell lymphomagenesis will lead to important insights into the epigenetic and molecular mechanisms underlying AITL pathogenesis DisclosuresNo relevant conflicts of interest to declare.

A Non-Human Primate CRISPR/Cas9 Model of Clonal Hematopoiesis of Indeterminate Potential Demonstrates Expansion of TET2-Disrupted Clones

Recent large-scale genetic screening of human blood cells have identified that somatic mutations associated with clonal expansions commonly arise with aging, even in the absence of cytopenias, myelodysplasia, or leukemia. Loss of function or dominant negative mutations in genes encoding epigenetic modifier enzymes such as DNMT3, TET2, and ASXL1 are most common in this “clonal hematopoiesis of indeterminate potential (CHIP)”. However, a causal relationship between these mutations and clonal expansions or progression to myelodysplasia or leukemia is unclear, as it has been difficult to extrapolate from murine models due to the differences in lifespan, aging phenotypes and hematopoietic cell transformation susceptibility. We utilized CRISPR/Cas9 gene editing of hematopoietic stem and progenitor cells (HSPC) in the rhesus macaque model to study the relationship between mutations in these genes, and clonal expansion and progression to abnormal hematopoiesis.We optimized CRISPR/Cas9 gene-editing strategies to introduce loss-of-function mutations in rhesus macaque CD34+ HSPC and applied this methodology to macaque autologous HSPC transplantation. We initially focused on the DNMT3A gene, which has been reported as most frequently mutated in CHIP. Macaque CD34+ HSPC were split into three equal fractions, transduced with one of three lentiviral vectors carrying either a gRNA for the control AAVS1 locus, DNMT3A exon 3, or DNMT3 exon 19 (the location of the “hotspot” putative dominant negative mutation in CHIP), and then electroporated with Cas9 mRNA. These edited cells were then transplanted into the autologous macaque following ablative total body irradiation. A second macaque also received autologous CD34+ cells electroporated with either AAVS1- or DNMT3A-targeting Cas9 ribonucleoprotein (RNP) complexes. Both animals engrafted promptly, with detection of low level (0-4%) indels at the on-target sites for AAVS1 and DNMT3A for up to 12 months post transplantation, but so far, we have not detected clonal expansion of DNMT3A-mutated HSPC in these two animals.We next delivered Cas9 and a gRNA pool targeting DNMT3A, TET2, and ASXL1 (the top three mutated and expanded genes in CHIP) (80%) or AAVS1 (20%) into CD34+ HSPC, followed by autologous transplantation (Fig 1A). After engraftment, the level of indels at each target site in granulocytes was 2% or less. However, during the 12 months post-transplant, there was a gradual and marked expansion of the granulocyte fraction containing indels predicted to knock out TET2 function (Fig 1B). Of the 17 different indel signatures identified at 12 months, only two were over the limit of detection in the infusion product or at 1 month post-transplant (Fig 1C), suggesting that the expanding TET2 mutant clones originated from long-term HSPC rather than short-term progenitors or that rare, initially mutated clones were sufficient to initiate clonal expansions. Finally, TET2 mutation frequency in bone marrow CD34+ cells was more than 2-fold higher (~20%) than in mature granulocytes (8%) at 12 months post-transplant (Fig 1C), suggesting a possible block in differentiation or a defect in release from the marrow for TET2 mutated cells.In conclusion, we have successfully created a non-human primate model of CHIP utilizing autologous transplantation of CRISPR/Cas9-edited HSPC, demonstrating gradual expansion of TET2-mutated clones. In contrast, simple indel formation at the DNMT3A locus was not sufficient to result in clonal expansion in the three animals studied to date, and a strategy utilizing HDR to specifically knock-in the hotspot base pair recurrently mutated in CHIP will be required to further investigate the relationship of DNMT3A to CHIP. Taken together, these approaches should improve our understanding of the effects of TET2 loss of function on the development of CHIP and its link to hematologic and cardiovascular disease, as well as provide a model for the testing of preventative interventions for clonal expansion and progression to frank malignancy. [Display omitted] DisclosuresDunbar:Novartis/GSK to institute: Research Funding. Winkler:Novartis/GSK to institute: Research Funding.

Acute Deletion of Tet2 or Tet3 Transiently Dysregulate Murine Erythropoiesis

The majority of palindromic CpG dinucleotides in mammalian DNA are symmetrically methylated on cytosine (5mC). Erythroid differentiation is associated with replication-dependent genome-wide loss of ~30-50% of all 5mC (1). One potential mechanism of demethylation involves Tet dioxygneases, which oxidize 5 methylcytosine (5mC) to 5-hydroxy-methylcytosine (5hmC), thereby interfering with maintenance methylation by DNMT1 (2). Tet2 and Tet3 are the two major Tet proteins expressed in hematopoietic cells. Tet2 is one of the most commonly mutated genes in hematological malignancies. Germline deletion of Tet2 results in increased hematopoietic stem cell self-renewal, erythroid and myeloid hyperplasia and myeloid and lymphoid malignancies (3, 4). Germline deletion of Tet3 is embryonic lethal (5). Here we investigated the potential role of Tet2 and Tet3 in the global loss of DNA methylation during erythroid differentiation, using mouse genetic models.We bred Tet2fl/fl and Tet3fl/fl mice onto the Rosa26-CreERT2 background, enabling acute deletion in vivo of either Tet2, Tet3 or both genes in adult and fetal mice. Within 4 to 10 days of deletion, we noted a rapid decline in total bone marrow cellularity in all genotypes; bone-marrow erythroblasts decreased to 15% of control value in Tet2-deleted (Tet2del) mice (p<0.0001), and to 43% (p=0.05) and 20% (p<0.0001) of controls in the Tet3 del and Tet2/Tet3 double deleted mice (DKO), respectively. These findings were associated with decreased reticulocyte counts in peripheral blood, and a decline in hematocrit, in Tet2del (36% vs. 46% in controls, p<0.004) and the DKO (31%, p=0.03). Bone-marrow CFU-e progenitors, but not BFU-e progenitors, also decreased in number. These losses partially improved over the next 2 to 4 weeks in mice deleted for either Tet2 or Tet3, but not in the DKO mice, in which anemia persisted. Megakaryocyte CFU-Mk progenitors increase in the DKO but are unaffected in the single deletions. Lymphoid lineage cells (CD4+, CD8+ and CD19+) showed similar transient losses, but the numbers of granylocytic and monocytic progenitors in the bone marrow was not affected. Similarly, fetal liver cell number decreased in all genotypes within 4 days of deletion, compared with controls, in all genotypes, and most severely in the DKO.We used three approaches to determine the potential effect of Tet proteins on erythroid DNA demethylation: Mass spectrometry, to measure global levels of 5mC and 5hmC in fetal liver erythroblasts; 5hMe-DIP-seq (5hyrdoxymethyl-DNA immune precipitation- sequencing), to determine the genomic distribution of hydroxymethylation in erythroblasts; and pyrosequencing for DNA methylation/hydroxymethylation at specific genomic loci including repetitive LINE_1 elements. We found that 5hmC is highly enriched in previously identified erythroid enhancers. Tet2 accelerates, but is not essential, for DNA demethylation of specific erythroid enhancers. Neither Tet2 nor Tet3 are required for global DNA demethylation.Taken together, we conclude that Tet2 and Tet3 are required during erythropoiesis, where they play partially redundant roles, possibly contributing to the changing chromatin environment during activation of erythroid terminal differentiation. Their acute deletion leads to transient loss of erythroblast viability and to anemia, but this is followed by a process of adaptation and chronically compensated erythropoiesis. These results raise the possibility that the adaptation required for the rapid partial recovery in the erythroid and lymphoid lineages following acute Tet2 loss may contribute, over the long term, to the development of myeloid and lymphoid malignancies.1. J. R. Shearstone et al., Global DNA demethylation during mouse erythropoiesis in vivo. Science334, 799-802 (2011).2. M. Ko et al., Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2. Nature468, 839-843 (2010).3. M. Ko et al., TET proteins and 5-methylcytosine oxidation in hematological cancers. Immunol Rev263, 6-21 (2015).4. J. An et al., Acute loss of TET function results in aggressive myeloid cancer in mice. Nature communications6, 10071 (2015).5. T. P. Gu et al., The role of Tet3 DNA dioxygenase in epigenetic reprogramming by oocytes. Nature477, 606-610 (2011). DisclosuresNo relevant conflicts of interest to declare.

Modulation of TET2 Activity with Ascorbic Acid in Myeloid Neoplasia: Diagnostic and Therapeutic Implications

TET2 mutations (TET2MT) are ubiquitous in myeloid neoplasia, but are most frequent in MDS and MDS/MPN. TET2 is an iron-catalyzed dioxygenase critical in active DNA demethylation that progressively oxidizes 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5CaC). As 5hmC residues are not recognized by DNMT3A, their methylation pattern is not copied onto the daughter DNA strand, leading to net DNA demethylation: TET2 can also therefore be considered a demethylase.Ascorbic acid (AA) is required as a prosthetic group of TET-mediated cytosine demethylation. AA may increase the residual function/throughput of intact TET2 in cases with heterozygous TET2MT and restore hydroxymethylation, thus reversing the expected epigenetic phenotype. We hypothesized that in heterozygous TET2MT neoplasms, AA alone or in combination may constitute a rational targeted therapy to restore TET2 function.We first assessed the effects of AA on TET2 in cell-free experiments. We combined purified wild-type TET2 (TET2WT) with a synthetic poly mC substrate and 0-4 mM AA, and measured levels of 5mC, 5hmC, 5fC, and 5CaC via 2D-UPLC-MS/MS profiling. AA increased the ratio of 5hmC, 5CaC, and 5fC/5mC in a dose-dependent fashion (peak effect 4.5 to 9). To model heterozygous TET2MT seen in myeloid neoplasia, we combined TET2WT with a catalytically-dead TET2S1898F in a 1:1 ratio. AA again increased the ratio of 5hmC, 5fC, and 5CaC to 5mC (peak effect 3 to 6, p<.0001); AA had no effect on TET2S1898F alone.To determine if AA TET2 upregulation could be reproduced in human cells, K562 and MOLM-13 AML lines were treated for 24 hours with 0-1000 uM of AA before subjecting them to 2D-UPLC-MS/MS analysis. AA increased 5hmC levels in K562 cells by 1.8-fold (500 uM, p=.04) and 1.7-fold (1000 uM, p=.15), and increased 5fC levels by 2.1-fold (500 uM, p <0.0001) and 3.1-fold (1000 uM, p=.035). Similarly, AA increased 5hmC levels in MOLM13 cells by 1.8-fold (500 uM, p=.01) and 2-fold (1000 uM, p<.0001), and increased 5fC levels by 3.6-fold (500 uM, p<.0001; 1000 uM, p=.01).We then studied the effect of TET2 loss on leukemogenesis in dual Tet2+/- ; Gulo -/- knockout mice unable to produce endogenous AA, making them ideal to determine the effect of exogenous AA on in vivo TET2 function. Bone marrow samples isolated from Tet2+/- ; Gulo -/- mice had a 1.7-fold reduction in 5hmC (p<.0001), a 2-fold reduction in 5fC (p=.0009), and a 3.4-fold reduction in 5CaC (p<.0001). Treatment of Tet2+/- ; Gulo -/- mice with 3.3 g/L ascorbic acid (in the drinking water for 6 months) partially rescued these DNA oxidation products in their bone marrow cells, with significantly increased levels of 5fC (p = .02) and 5CaC (p =.04) compared to untreated Tet2+/- ; Gulo -/- mice. Compared to TET2WT mice, Tet2+/- ; Gulo -/- mice had only a 1.2-fold reduction in 5hmC (p = 0.2), a 1.15-fold reduction in 5fC (p = .004), and a 1.7-fold reduction in 5CaC (p =.3).Similar to the TET2 knockout mice results, 8/8 of TET2MT patient samples profiled had reduced levels of at least one TET2-dependent DNA oxidation product. Intriguingly, 24-hour treatment with 250 uM AA treatment had no significant effect on any TET2 MT bone marrow samples; in TET2 WT samples, 250 uM AA increased 5fC levels by 2-fold (p =.014) and 5CaC levels by 3-fold (p =.0025), though it nonsignificantly reduced 5hmC levels by 80% (p =.069).In our study, AA significantly increased the levels of TET2-dependent DNA oxidation products in purified TET2, Tet2+/- ; Gulo -/- knockout mice, TET2WT human cell lines, and TET2WT patient samples. Therapeutic AA may therefore play a role in treating myeloid neoplasia with IDH1 or IDH2 mutations, which have structurally-intact but functionally hypoactive TET2 from loss of the IDH-catalyzed TET2 substrate alpha-ketoglutarate. Given that AA was unable to restore TET2 activity in samples from TET2MT patients, AA may not be beneficial in TET2MT myeloid neoplasia.Our study also tells a cautionary tale about using 5hmC levels alone to estimate TET2 activity. 5hmC levels were normal in 3/8 (37.5%) of patients with pathogenic TET2MT; simultaneous measurement of multiple TET2-dependent products is therefore important to accurately assess in-vivo TET2 activity and determine who might benefit from therapeutic AA intervention. DisclosuresGreenberg:Acetylon Pharmaceuticals Inc.: Patents & Royalties: Treatment of Protein Degradation Disorders; Oisin Biotechnologies: Consultancy. Makishima:Yasuda Medical Foundation: Research Funding. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Speaker Fees; Apellis Pharmaceuticals: Consultancy; Ra Pharma: Consultancy.

Ten-eleven translocation 2 modulates allergic inflammation by 5-hydroxymethylcytosine remodeling of immunologic pathways.

Allergic rhinitis (AR) is an allergen-specific immunoglobulin E-mediated inflammatory disease. Both genetic and environmental factors could play a role in the pathophysiology of AR. 5-methylcytosine (5mC) can be converted to 5-hydroxymethylcytosine (5hmC) by the ten-eleven translocation (Tet) family of proteins as part of active deoxyribonucleic acid (DNA) demethylation pathway. 5hmC plays an important role in the regulation of gene expression and differentiation in immune cells. Here, we show that loss of Tet protein 2 (Tet2) could impact the severity of AR in the ovalbumin-induced mouse model. Genome-wide 5hmC profiling of both wild-type and Tet2 KO mice in response to AR revealed that the loss of Tet2 could lead to 5hmC alteration at specific immune response genes. Both partial loss and complete loss of Tet2 alters the 5hmC dynamic remodeling for the adaptive immune pathway as well as cytokines. Thus, our results reveal a new role of Tet2 in immunology, and Tet2 may serve as a promising target in regulating the level of immune response.

5-Hydroxymethylcytosine Loss in Conjunctival Melanoma.

Aims: Conjunctival and cutaneous melanoma partially share similar clinical and molecular backgrounds. As 5-hydroxymethylcytosine (5-hmC) loss has been demonstrated in cutaneous melanoma, we decided to assess if similar changes were occurring in conjunctival melanoma. Methods: 5-methylcytosine (5-mC), 5-hmC and TET2 were respectively identified by immunohistochemistry and RNA ISH in 40 conjunctival nevi and 37 conjunctival melanomas. Clinicopathological correlations were established. Results: 5-mC, TET2 and 5-hmC were respectively identified in 67.5%, 95% and 100% of conjunctival nevi and in 81.1%, 35.1% and 54% of conjunctival melanomas. A significant 5-hmC and TET2 loss was identified in conjunctival melanoma comparing to nevus, as well as a significant correlation between TET2 and 5-hmC expression. In the melanomas, 5-hmC expression was only significantly associated with local lymphatic invasion, but not with other clinicopathological parameters. There was a correlation between TET2 expression and the localization of the tumors. 5-mC expression was not associated with any clinicopathological parameters. Conclusions: We identified a significant 5-hmC loss in conjunctival melanoma similar to cutaneous melanoma. This loss may possibly be attributed to TET2 loss or IDH1 mutations. 5-hmC loss in conjunctival melanoma may help in the differential diagnosis between atypical conjunctival nevus and conjunctival melanoma.

Muscle regeneration controlled by a designated DNA dioxygenase

Tet dioxygenases are responsible for the active DNA demethylation. The functions of Tet proteins in muscle regeneration have not been well characterized. Here we find that Tet2, but not Tet1 and Tet3, is specifically required for muscle regeneration in vivo. Loss of Tet2 leads to severe muscle regeneration defects. Further analysis indicates that Tet2 regulates myoblast differentiation and fusion. Tet2 activates transcription of the key differentiation modulator Myogenin (MyoG) by actively demethylating its enhancer region. Re-expressing of MyoG in Tet2 KO myoblasts rescues the differentiation and fusion defects. Further mechanistic analysis reveals that Tet2 enhances MyoD binding by demethylating the flanking CpG sites of E boxes to facilitate the recruitment of active histone modifications and increase chromatin accessibility and activate its transcription. These findings shed new lights on DNA methylation and pioneer transcription factor activity regulation.

BATF3/MYC axis drives hyper-proliferation of TET2 deficient CAR T cells

Abstract A recent report described a patient presenting a dominant chimeric antigen receptor (CAR) T cell clone at the height of anti-tumor response wherein a TET2 allele was disrupted by the integration of the lentiviral 4-1BB CAR vector (Fraietta et. al. Nature 2018). The enhanced proliferative ability conferred by this chance TET2 disruption opened up the possibility of generating TET2 edited CAR T cells for an enhanced anti-tumor efficacy. We modeled and evaluated loss of TET2 in human T cells engineered to express different CAR receptors in a murine model of human acute lymphoblastic leukemia (ALL). Our results indicate that CAR design plays a critical role in determining anti-tumor efficacy and early stage T cell phenotype upon TET2 disruption. We further observe that TET2 edited CAR T cells, over time, enter a hyper-proliferative state with a near total loss of effector function. The frequency with which TET2 edited T cells achieve hyper-proliferation is dependent on the signaling properties of the CAR receptor. This state is in contrast to the canonical exhaustion state wherein loss of proliferative ability precedes loss of effector function. This hyper-proliferative state shares some transcriptional features with T cell leukemia/lymphoma with sustained upregulation of cell cycle associated factors. Exome analysis identified point mutations and chromosomal aberrations in hyper-proliferative cells, but they were not conserved across different populations. Chromatin accessibility analysis revealed that loss of TET2 sets an epigenetic state that allows for sustained elevated levels of BATF3 which in turn drives a MYC-dependent proliferative program in TET2 deficient CAR T cells.

Loss of ten-eleven translocation 2 induces cardiac hypertrophy and fibrosis through modulating ERK signaling pathway

The ten-eleven translocation (Tet) family of dioxygenases convert 5-methylcytosine to 5-hydroxymethylcytosine (5hmC). Previous studies have shown that 5hmC-mediated epigenetic modifications play essential roles in diverse biological processes and diseases. Here, we show that Tet proteins and 5hmC display dynamic features during postnatal cardiac development and that Tet2 is the predominant dioxygenase present in heart. Tet2 knockout results in abnormal cardiac function, progressive cardiac hypertrophy and fibrosis. Mechanistically, Tet2 deficiency leads to reduced hydroxymethylation in the cardiac genome and alters the cardiac transcriptome. Mechanistically, Tet2 loss leads to a decrease of Hspa1b expression, a regulator of the extracellular signal-regulated protein kinase (Erk) signaling pathway, which leads to over-activation of Erk signaling. Acute Hspa1b knock down (KD) increased the phosphorylation of Erk and induced hypertrophy of cardiomyocytes, which could be blocked by Erk signaling inhibitor. Consistently, ectopic expression of Hspa1b was able to rescue the deficits of cardiomyocytes induced by Tet2 depletion. Taken together, our study's results reveal the important roles of Tet2-mediated DNA hydroxymethylation in cardiac development and function.

TETology: Epigenetic Mastermind in Action.

Cytosine methylation is a well-explored epigenetic modification mediated by DNA methyltransferases (DNMTs) which are considered "methylation writers"; cytosine methylation is a reversible process. The process of removal of methyl groups from DNA remained unelucidated until the discovery of ten-eleven translocation (TET) proteins which are now considered "methylation editors." TET proteins are a family of Fe(II) and alpha-ketoglutarate-dependent 5-methyl cytosine dioxygenases-they convert 5-methyl cytosine to 5-hydroxymethyl cytosine, and to further oxidized derivatives. In humans, there are three TET paralogs with tissue-specific expression, namely TET1, TET2, and TET3. Among the TETs, TET2 is highly expressed in hematopoietic stem cells where it plays a pleiotropic role. The paralogs also differ in their structure and DNA binding. TET2 lacks the CXXC domain which mediates DNA binding in the other paralogs; thus, TET2 requires interactions with other proteins containing DNA-binding domains for effectively binding to DNA to bring about the catalysis. In addition to its role as methylation editor of DNA, TET2 also serves as methylation editor of RNA. Thus, TET2 is involved in epigenetics as well as epitranscriptomics. TET2 mutations have been found in various malignant hematological disorders like acute myeloid leukemia, and non-malignant hematological disorders like myelodysplastic syndromes. Increasing evidence shows that TET2 plays an important role in the non-hematopoietic system as well. Hepatocellular carcinoma, gastric cancer, prostate cancer, and melanoma are some non-hematological malignancies in which a role of TET2 has been implicated. Loss of TET2 is also associated with atherosclerotic vascular lesions and endometriosis. The current review elaborates on the role of structure, catalysis, physiological functions, pathological alterations, and methods to study TET2, with specific emphasis on epigenomics and epitranscriptomics.

TET2 is a component of the estrogen receptor complex and controls 5mC to 5hmC conversion at estrogen receptor cis-regulatory regions

SummaryEstrogen receptor-α (ER) drives tumor development in ER-positive (ER+) breast cancer. The transcription factor GATA3 has been closely linked to ER function, but its precise role in this setting remains unclear. Quantitative proteomics was used to assess changes to the ER complex in response to GATA3 depletion. Unexpectedly, few proteins were lost from the ER complex in the absence of GATA3, with the only major change being depletion of the dioxygenase TET2. TET2 binding constituted a near-total subset of ER binding in multiple breast cancer models, with loss of TET2 associated with reduced activation of proliferative pathways. TET2 knockdown did not appear to change global methylated cytosine (5mC) levels; however, oxidation of 5mC to 5-hydroxymethylcytosine (5hmC) was significantly reduced, and these events occurred at ER enhancers. These findings implicate TET2 in the maintenance of 5hmC at ER sites, providing a potential mechanism for TET2-mediated regulation of ER target genes.

Low shear stress-induced endothelial mesenchymal transformation via the down-regulation of TET2

Atherosclerotic cardiovascular disease is the major cause of death worldwide. Low shear stress plays key roles on the initiation and progression of atherosclerosis (As). However, its underlying mechanism remains unclear. In this study, the effect of low shear stress on endothelial mesenchymal transformation (EndMT) and its underlying mechanism were explored. Results showed that in cultured human umbilical vein endothelial cells, low shear stress down-regulated the expression of TET2 and promoted EndMT. Loss of TET2 promoted EndMT with the Wnt/β-catenin signaling pathway. The enhancement in EndMT induced by low shear stress was attenuated by TET2 overexpression. In apoE−/− mice subjected to carotid artery local ligation, the EndMT and atherosclerotic lesions induced by low shear stress was attenuated by TET2 overexpression. Taken together, low shear stress promoted EndMT through the down-regulation of TET2, indicating that intervention with EndMT or the up-regulation of TET2 might be an alternative strategy for preventing As.

AAV Joins the Rank of Genotoxic Vectors

AAV Joins the Rank of Genotoxic Vectors

Circular RNA CircMAP3K5 Acts as a MicroRNA-22-3p Sponge to Promote Resolution of Intimal Hyperplasia Via TET2-Mediated Smooth Muscle Cell Differentiation.

Aberrant expression of circular RNA contributes to human diseases. Circular RNAs regulate gene expression by sequestering specific microRNAs. In this study, we investigated whether circMAP3K5 (circular mitogen-activated protein kinase 5) could act as a competing endogenous microRNA-22-3p (miR-22-3p) sponge and regulate neointimal hyperplasia. Circular RNA profiling from genome-wide RNA sequencing data was compared between human coronary artery smooth muscle cells (SMCs) treated with or without platelet-derived growth factor. Expression levels of circMAP3K5 were assessed in human coronary arteries from autopsies on patients with dilated cardiomyopathy or coronary heart disease. The role of circMAP3K5 in intimal hyperplasia was further investigated in mice with adeno-associated virus 9-mediated circMAP3K5 transfection. SMC-specific Tet2 (ten-eleven translocation-2) knockout mice and global miR-22-3p knockout mice were used to delineate the mechanism by which circMAP3K5 attenuated neointimal hyperplasia using the femoral arterial wire injury model. RNA sequencing demonstrated that treatment with platelet-derived growth factor-BB significantly reduced expression of circMAP3K5 in human coronary artery SMCs. Wire-injured mouse femoral arteries and diseased arteries from patients with coronary heart disease (where platelet-derived growth factor-BB is increased) confirmed in vivo downregulation of circMAP3K5 associated with injury and disease. Lentivirus-mediated overexpression of circMAP3K5 inhibited the proliferation of human coronary artery SMCs. In vivo adeno-associated virus 9-mediated transfection of circMap3k5 (mouse circular Map3k5) specifically inhibited SMC proliferation in the wire-injured mouse arteries, resulting in reduced neointima formation. Using a luciferase reporter assay and RNA pull-down, circMAP3K5 (human circular MAP3K5) was found to sequester miR-22-3p, which, in turn, inhibited the expression of TET2. Both in vitro and in vivo results demonstrate that the loss of miR-22-3p recapitulated the antiproliferative effect of circMap3k5 on vascular SMCs. In SMC-specific Tet2 knockout mice, loss of Tet2 abolished the circMap3k5-mediated antiproliferative effect on vascular SMCs. We identify circMAP3K5 as a master regulator of TET2-mediated vascular SMC differentiation. Targeting the circMAP3K5/miR-22-3p/TET2 axis may provide a potential therapeutic strategy for diseases associated with intimal hyperplasia, including restenosis and atherosclerosis.