TET Deficiency Is Associated with Accumulation of G-Quadruplex and R-Loop Structures during Oncogenesis

The three members of TET family of Fe(II) and alpha-ketoglutarate-dependent dioxygenases mediate DNA demethylation by sequentially oxidizing 5-methylcytosine (5mC) to 5-hydroxymethyl- (5hmC), 5-formyl- (5fC) and 5-carboxyl-cytosine (5caC). TET enzymes are required for normal development, and loss of TET function due to mutations, metabolic perturbations and hypoxia, among other mechanisms, occurs frequently in many hematological malignancies and solid tumors. Recent studies have identified mutations in TET proteins (TET2, most commonly) and metabolic enzymes which regulate TET catalytic activity in a large cohort of patients with Diffuse Large B-cell Lymphoma (DLBCL). However, the clinical significance of these mutations in DLBCL and the molecular mechanisms through which TET proteins suppress development of malignancies in general, are not fully-understood. To investigate the role of TET loss-of-function in the pathogenesis of DLBCL, we generated mice with B-cell-specific deletion of TET2 and TET3, the major TET homologs expressed in mature B cells. TET deficiency in B cells perturbed mature B cell homeostasis resulting in spontaneous development of Germinal Center-derived B cell lymphomas. Moreover, B cells with TET deficiency demonstrated increased genomic instability, a feature previously associated with TET loss-of-function in other hematopoietic lineages. Transcriptional profiling of TET-deficient expanded B cells revealed altered expression of genes and proteins involved in modulating the levels of secondary DNA structures, G-quadruplexes and DNA:RNA hybrids (R-loops) which have been linked to genomic instability and transcriptional perturbations in many different cancers. Using previously described methods and newer approaches, we observed a substantial increase in the levels of G-quadruplex and R-loop structures in TET-deficient B cells compared with control B cells. The increase in G-quadruplex and R-loop structures was evident in naïve, activated and GC B cells following acute TET deletion as well as in TET-deficient myeloid cells and T cells. Genome-wide mapping studies and high-throughput genome-wide translocation sequencing (HTGTS) showed a strong correlation of increased G-quadruplex and R-loop structures with increased DNA DSBs in switch regions of immunoglobulin heavy chain locus in TET-deficient B cells. Deletion of the DNA methyltransferase DNMT1 in TET-deficient B cells prevented the expansion of germinal center B cells, diminished the accumulation of G-quadruplexes and R-loops, and caused a notable delay in lymphoma development, consistent with the opposing functions of DNMT and TET enzymes in DNA methylation and demethylation. CRISPR-mediated depletion of nucleases and helicases that regulate G-quadruplexes and R-loops decreased the viability of TET-deficient B cells. Our studies suggest a molecular mechanism by which TET loss-of-function might predispose to development of B cell-derived and other malignancies, and highlight novel therapeutic avenues that could be further explored. Disclosures Rao: Cambridge Epigenetix: Membership on an entity's Board of Directors or advisory committees.

EXTH-15. MULTI-FACETED INHIBITION OF TET PATHWAY WITH CELL-PERMEABLE 2HG AND BOBCAT 339 REDUCES PROLIFERATION AND INDUCES APOPTOSIS IN DIPG

Diffuse Intrinsic Pontine Glioma (DIPG) is one of the worst forms of pediatric brain tumors, with a 100% mortality-rate. A prominent mutation observed in them is a lysine to methionine change in histone3 which causes trapping of the repressive-chromatin-complex, leading to genome-level hypomethylation, affecting global epigenetic-regulation. Ten-Eleven Translocation (TET) proteins are alpha-ketoglutarate (α-KG) dependent dioxygenases that mediate the conversion of 5-methylcytosine to 5-hydroxymethylcytosine (5hmC), a key-step in removing DNA methylation-marks. We previously identified increased levels of 5hmC and TETs in DIPG. In IDH mutant glioma, the R132H-IDH produces an oncometabolite, 2-hydroxyglutarate (2HG), instead of α-KG. 2HG competitively inhibits TET function, resulting in a hypermethylated genome. Bobcat339, a cytosine-analog targets TETs by blocking the cytosine binding-site on TETs. We hypothesized that cell-permeable 2HG and Bobcat339 would synergize to block TET activity in DIPG, restore epigenetic-balance by increasing genomic methylation, and induce cell-death. 2HG induced apoptosis in DIPG cells at concentrations ranging from 100-300uM, as measured by cleaved-PARP western and cleaved-caspase3 immunofluorescence (2-7fold increase in CC3, depending on cell line). Cell-permeable 2HG also decreased proliferation, measured by phospho-RB western in DIPG cells (30-50% reduction in pRB band intensity, depending on cell line). Similarly, Bobcat339 suppressed proliferation at concentrations from 10-50uM, measured by BrdU incorporation (25% reduction in BrdU+ at 50uM, p= 0.02 compared to control). Bobcat339 induced apoptosis, measured by cPARP western and CC3 immunofluorescence (4-10fold more of CC3+ compared to control p< 0.0004). In combination, cell-permeable 2HG (100-200uM) and Bobcat339 (10-20uM) combined to suppress cell-viability, measured by CellTiterBlue assay, producing ZIP scores of ~20 in DIPG cells (indicating high-level synergy). In combination, 2HG and Bobcat339 increased apoptosis in DIPG (3-fold increase in cleaved-PARP band intensity in JHH-DIPG1 cells treated with 20uM Bobcat339 + 100uM 2HG, compared to single-treated cells). Our results may lead to new approaches that target TET pathway in DIPG tumors.

Multiple Mechanisms of Tigecycline Resistance in Enterobacteriaceae from a Pig Farm, China

As a last-resort antimicrobial agent to treat serious infections, the emergence and spread of tigecycline resistance in Enterobacteriaceae and Acinetobacter have raised global concerns. Multiple mechanisms mediate tigecycline resistance in Enterobacteriaceae , such as the monooxygenase Tet(X), mutations in Tet proteins, and overexpression of efflux pumps.

Ten-eleven translocation 1 mediated-DNA hydroxymethylation is required for myelination and remyelination in the mouse brain

Ten-eleven translocation (TET) proteins, the dioxygenase for DNA hydroxymethylation, are important players in nervous system development and diseases. However, their role in myelination and remyelination after injury remains elusive. Here, we identify a genome-wide and locus-specific DNA hydroxymethylation landscape shift during differentiation of oligodendrocyte-progenitor cells (OPC). Ablation of Tet1 results in stage-dependent defects in oligodendrocyte (OL) development and myelination in the mouse brain. The mice lacking Tet1 in the oligodendrocyte lineage develop behavioral deficiency. We also show that TET1 is required for remyelination in adulthood. Transcriptomic, genomic occupancy, and 5-hydroxymethylcytosine (5hmC) profiling reveal a critical TET1-regulated epigenetic program for oligodendrocyte differentiation that includes genes associated with myelination, cell division, and calcium transport. Tet1-deficient OPCs exhibit reduced calcium activity, increasing calcium activity rescues the differentiation defects in vitro. Deletion of a TET1-5hmC target gene, Itpr2, impairs the onset of OPC differentiation. Together, our results suggest that stage-specific TET1-mediated epigenetic programming and intracellular signaling are important for proper myelination and remyelination in mice.

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.

The Potential Equivalents of TET2 Mutations

TET2 is a dioxygenase dependent on Fe2+ and a-ketoglutarate which oxidizes 5-methylcytosine (5meC) to 5-hydroxymethylcytosine (5hmeC). TET proteins successively oxidize 5mC to yield 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Among these oxidized methylcytosines, 5fC and 5caC are directly excised by thymine DNA glycosylase (TDG) and ultimately replaced with unmethylated cytosine. Mutations in TET2 have been shown to lead to a hypermethylated state of the genome and to be responsible for the initiation of the oncogenetic process, especially in myeloid and lymphoid malignancies. Nonetheless, this was also shown to be the case in other cancers. In AML, TET2 mutations have been observed to be mutually exclusive with IDH1, IDH2, and WT1 mutations, all of them showing a similar impact on the transcription profile of the affected cell. Because of this, it is possible that TET2/IDH1/2/WT1 mutated AML could be considered as having similar characteristics between each other. Nonetheless, other genes also interact with TET2 and influence its effect, thus making it possible that other signatures exist that would mimic the effect of TET2 mutations. Thus, in this review, we searched the literature for the genes that were observed to interact with TET2 and classified them in the following manner: transcription alteration, miRs, direct interaction, posttranslational changes, and substrate reduction. What we propose in the present review is the potential extension of the TET2/IDH1/2/WT1 entity with the addition of certain expression signatures that would be able to induce a similar phenotype with that induced by TET2 mutations. Nonetheless, we recommend that this approach be taken on a disease by disease basis.

Molecular mechanism for vitamin C-derived C5-glyceryl-methylcytosine DNA modification catalyzed by algal TET homologue CMD1

C5-glyceryl-methylcytosine (5gmC) is a novel DNA modification catalyzed by algal TET homologue CMD1 using vitamin C (VC) as co-substrate. Here, we report the structures of CMD1 in apo form and in complexes with VC or/and dsDNA. CMD1 exhibits comparable binding affinities for DNAs of different lengths, structures, and 5mC levels, and displays a moderate substrate preference for 5mCpG-containing DNA. CMD1 adopts the typical DSBH fold of Fe2+/2-OG-dependent dioxygenases. The lactone form of VC binds to the active site and mono-coordinates the Fe2+ in a manner different from 2-OG. The dsDNA binds to a positively charged cleft of CMD1 and the 5mC/C is inserted into the active site and recognized by CMD1 in a similar manner as the TET proteins. The functions of key residues are validated by mutagenesis and activity assay. Our structural and biochemical data together reveal the molecular mechanism for the VC-derived 5gmC DNA modification by CMD1.

TET-Mediated Epigenetic Regulation in Immune Cell Development and Disease

TET proteins oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and further oxidation products in DNA. The oxidized methylcytosines (oxi-mCs) facilitate DNA demethylation and are also novel epigenetic marks. TET loss-of-function is strongly associated with cancer; TET2 loss-of-function mutations are frequently observed in hematological malignancies that are resistant to conventional therapies. Importantly, TET proteins govern cell fate decisions during development of various cell types by activating a cell-specific gene expression program. In this review, we seek to provide a conceptual framework of the mechanisms that fine tune TET activity. Then, we specifically focus on the multifaceted roles of TET proteins in regulating gene expression in immune cell development, function, and disease.