TET Family Proteins Research Articles

Approaches to anticancer therapy based on modulation of DNA methylation

Background. DNA methylation is a crucial mechanism of epigenetic regulation of transcription. Disturbances in DNA methylation mechanism are associated with various malignancies such as acute myeloid leukaemia, breast cancer, prostate cancer, etc. Influencing the functional status of DNA methyltransferases (DNMTs) enzymes and TET family proteins (TETs), which regulate DNA methylation and demethylation, is the basis of epigenetic anticancer therapy approach. In this review, we have considered the challenges and prospects of nucleoside and non-nucleoside inhibitors of DNMTs as well as TETs inhibitors. The results of clinical trials on the efficacy of DNMTs inhibitors used individually and as part of combination chemotherapy conducted over the last 15 years are also evaluated. Material and Methods. Sources were searched in PubMed, ScienceDirect, Web of Science, eLibrary, CyberLeninka. More than 700 publications were used in the analysis, but the review included mainly works of the last 10 years. A number of articles published earlier than 2015 were used for historical reference. Results. The review provides information on current advances in the development and study of epigenetically active compounds whose action is aimed at the regulation of DNA methylation. Data on the in vitro and in vivo effects of agents considered for use in the therapy of various malignancies are presented. In addition, the data of clinical trials of the most promising epigenetic modulators are presented.

Abstract 5881: Upregulation of TET2 expression by DNA methyltransferase (DNMT) inhibitors is associated with resistance in DNMT1 knockout colorectal cancer cells

Abstract Ten-eleven-translocation 2 (TET2) is a member of the TET family proteins (TET1-3) that convert 5-methylcytosine to 5-hydroxymethylcytosine (5-hmC) in DNA and causes site-specific DNA demethylation. The effects of DNMT1 on TET2 phenotype following DNMT inhibitor treatment is unclear in human cancer. In colorectal cancer HCT116 cells with DNMT1 gene intact (DNMT1+/+) and deletion (DNMT1−/−) status, we found that TET2 expression was robustly increased in DNMT1−/− cells after treatment with 0.5 µM and 5 µM decitabine for 72 h, 96 h, and 120 h. The augmentation in TET2 expression was accompanied by a dose-dependent re-expression of p16ink4A in DNMT1−/− cells. Particularly, TET2 upregulation was associated with resistance to DNMT inhibitors, showing a lack of induction of DNA damage, cell-cycle arrest and apoptosis after drug exposure in DNMT1−/− compared to DNMT1+/+ cells. The treatment with 4’-thio-5-aza-2’-deoxycytidine (aza-T-dCyd) only up-regulated TET2 and re-expressed p16ink4A at the lower dose of 0.5 µM, which was consistent with our previous findings that high dose of aza-T-dCyd at 5 µM predominantly eliminates the cancer cells with severe DNA damage by undergoing apoptosis and cell death (Scientific Reports, 13:5964, 2023). Moreover, both decitabine and aza-T-dCyd had minimal effects on TET2 upregulation and p16ink4A re-expression in cells with intact DNMT1 gene. The data suggest that the absence of DNMT1 gene made the cells prone to TET2 upregulation upon DNMT inhibitor challenges. Future study is needed to investigate the interactive relationship between DNMT1 and TET2 as well as demethylation, and its impact on cancer treatment with DNMT inhibitors. Citation Format: Angelo B. Laranjeira, Dat Nguyen, Lorraine C. Pelosof, James H. Doroshow, Sherry X. Yang. Upregulation of TET2 expression by DNA methyltransferase (DNMT) inhibitors is associated with resistance in DNMT1 knockout colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5881.

MDM2 Antagonist Nutlin-3 Stimulates Global DNA Hydroxymethylation by Enhancing p53-TET1 Signaling Axis.

DNA hydroxymethylation is involved in many biological processes, including nuclear reprogramming, embryonic development, and tumor suppression. In this study, we report that an anticancer agent, nutlin-3, selectively stimulates global DNA hydroxymethylation in TP53 wild-type cancer cells as manifested by the elevation of 5-hydroxymethylcytosine (5hmC) in genomic DNA. In contrast, nutlin 3 fails to enhance DNA hydroxymethylation in TP53-mutated cancer cells. Consistently, nutlin-3 as a MDM2 antagonist only activates wild-type but not mutated TP53. Furthermore, nutlin-3 does not alter the expression of TET1 but slightly reduces the expression of TET2 and TET3 proteins. These TET family proteins are responsible for converting 5-methylcytosine (5mC) to 5hmC. Interestingly, TET1 knockdown could significantly block the nutlin-3-induced DNA hydroxymethylation as well as TP53 and P21 activation. Immunoprecipitation analysis supports that p53 strongly interacts with TET1 proteins. These results suggest that nutlin-3 activates TP53 and promotes p53-TET1 interaction. As positive feedback, the p53-TET1 interaction further enhances p53 activation and promotes apoptosis. Collectively, we demonstrate that nutlin-3 stimulates DNA hydroxymethylation and apoptosis via a positive feedback mechanism.

IDH2 regulates U2AF1 expression and hydroxymethylation in MDS patients

ABSTRACT The expression of some genes regulated by their DNA methylation is involved in pathogenesis and disease progression of myelodysplastic syndrome (MDS), which is characterised by abnormal differentiation and development of myeloid cells. Therefore, it is significant for us to work on investigating what factors regulate U2AF1 expression and hydroxymethylation in MDS patients. However, the members of TET protein family can change 5-methylcytosine (5mC) into 5-hydroxymethylcytosine5-methyl cytosine (5hmC). In general, 5mC and 5hmC levels maintain dynamic equilibrium, and their imbalance is associated with the onset and progression of some tumors. In this study, the expression and 5mC and 5hmC levels of U2AF1 gene decreased significantly after the treatment by decitabine in Mutz-1 cells. The decreased degree of 5hmC is far greater than that of 5mC. IDH2 expression decreased significantly followed by U2AF1 5hmC levels. However, the expression of other hydroxymethylation-related genes such as IDH1, TET1 and TET2 also decreased, but the difference did not achieve significance. Compared with IDH2 or U2AF1 wild-type MDS patients, U2AF1 expression and 5hmC level in patients with these two gene mutations were both significantly reduced.

Differential expression of Tet family genes and their potential role in regulating skeletal muscle development of Siniperca chuatsi

DNA methylation and demethylation are crucial epigenetic modification and regulation for animal development, and their dynamic changes may affect skeletal muscle development. The ten-eleven translocation (Tet) family proteins are demethylases which are involved in the dynamic changes of DNA methylation. However, the expression pattern of Tet family genes and their role in myogenesis in fish remains unclear. In this study, the temporal and spatial expression profiles of Tet1, Tet2 and Tet3 were assayed with RT-qPCR techniques in Chinese perch, Siniperca chuatsi. The obtained data showed that three Tet family genes were differentially expressed at different development stages. Tet1 was expressed low at blastula stage, but highly expressed at gastrula stage, then remained low until hatching. The expressions of Tet2 and Tet3 were significantly increased at late gastrula and kept high expression before hatching stage. At the spatial level, the Tet1 expression was highest in gill tissue, moderate level in brain and slow muscle. Tet2 was similar to that of Tet1 except that it was expressed at a lower level in slow muscle, and Tet3 exhibited a higher expression level in gill and brain, a moderate level in fast muscle. Cosinor analysis turned out that the expression of Tet1 and Tet2 displayed a significant daily rhythm in fast muscle, but Tet3 did not show daily rhythmicity. Inhibiting the activity of Tet1/2 proteins by injecting Bobcat339 significantly reduced the expression of MyoD and MRF4, but not MyoG and Myf5, by which leads to the increase of the number of satellite cells and proliferating myoblasts. Together, the results suggest that Tet1/2 may target to MyoD and MRF4 resulted in DNA demethylation and promoting their expression, thus stimulating myoblast differentiation.

Aberrations of DNA methylation in cancer

DNA methylation is a chromatin modification that plays an important role in the epigenetic regulation of gene expression. Changes in DNA methylation patterns are characteristic of many malignant neoplasms. DNA methylation is occurred by DNA methyltransferases (DNMTs), while demethylation is mediated by TET family proteins. Mutations and changes in the expression profile of these enzymes lead to DNA hypo- and hypermethylation and have a strong impact on carcinogenesis. In this review, we considered the key aspects of the mechanisms of regulation of DNA methylation and demethylation, and also analyzed the role of DNA methyltransferases and TET family proteins in the pathogenesis of various malignant neoplasms.During the preparation of the review, we used the following biomedical literature information bases: Scopus (504), PubMed (553), Web of Science (1568), eLibrary (190). To obtain full-text documents, the electronic resources of PubMed Central (PMC), Science Direct, Research Gate, CyberLeninka were used. To analyze the mutational profile of epigenetic regulatory enzymes, we used the cBioportal portal (https://www.cbioportal.org / ), data from The AACR Project GENIE Consortium (https://www.mycancergenome.org / ), COSMIC, Clinvar, and The Cancer Genome Atlas (TCGA).

PROSER1 mediates TET2 O-GlcNAcylation to regulate DNA demethylation on UTX-dependent enhancers and CpG islands.

DNA methylation at enhancers and CpG islands usually leads to gene repression, which is counteracted by DNA demethylation through the TET protein family. However, how TET enzymes are recruited and regulated at these genomic loci is not fully understood. Here, we identify TET2, the glycosyltransferase OGT and a previously undescribed proline and serine rich protein, PROSER1 as interactors of UTX, a component of the enhancer-associated MLL3/4 complexes. We find that PROSER1 mediates the interaction between OGT and TET2, thus promoting TET2 O-GlcNAcylation and protein stability. In addition, PROSER1, UTX, TET1/2, and OGT colocalize on many genomic elements genome-wide. Loss of PROSER1 results in lower enrichment of UTX, TET1/2, and OGT at enhancers and CpG islands, with a concomitant increase in DNA methylation and transcriptional down-regulation of associated target genes and increased DNA hypermethylation encroachment at H3K4me1-predisposed CpG islands. Furthermore, we provide evidence that PROSER1 acts as a more general regulator of OGT activity by controlling O-GlcNAcylation of multiple other chromatin signaling pathways. Taken together, this study describes for the first time a regulator of TET2 O-GlcNAcylation and its implications in mediating DNA demethylation at UTX-dependent enhancers and CpG islands and supports an important role for PROSER1 in regulating the function of various chromatin-associated proteins via OGT-mediated O-GlcNAcylation.

The roles of TET family proteins in development and stem cells.

Ten-eleven translocation (TET) methylcytosine dioxygenases are enzymes that catalyze the demethylation of 5-methylcytosine on DNA. Through global and site-specific demethylation, they regulate cell fate decisions during development and in embryonic stem cells by maintaining pluripotency or by regulating differentiation. In this Primer, we provide an updated overview of TET functions in development and stem cells. We discuss the catalytic and non-catalytic activities of TETs, and their roles as epigenetic regulators of both DNA and RNA hydroxymethylation, highlighting how TET proteins function in regulating gene expression at both the transcriptional and post-transcriptional levels.

Comprehensive DNA 5-Hydroxymethylation Landscapes in Myelodysplastic Syndromes (MDS)

Background: Epigenetic regulators as well as epigenetic marks play an essential role in regulating normal hematopoiesis. Epigenetic alterations are one of the early events reflecting changes in cell identify during the transition of a pre-leukemic condition into a malignant phenotype. Capturing epigenetic alterations at early stages of pre-leukemic conditions could benefit the diagnosis and understanding the disease progression during hematopoietic malignant transformation. DNA 5-hydroxymethylation (5hmC) is the one of the major oxidation products of 5-methycytosine (5mC) mediated by the TET protein family dioxygenase. 5hmC has been reported to be involve in the regulation of chromatin accessibility and gene transcription. The dynamic changes of 5hmC have been reported as a hallmark in myeloid malignancies. In our study, we applied an improved 5hmC profiling method (sCMSIP) developed in our lab to systematically profile 5hmC and evaluate the 5hmC dynamics in patients with myeloid malignancies. Methods: In order to profile 5hmC using low amount of input DNA isolated from patient bone-marrow aspirates, we improved our previously developed anti-CMSIP method to capture 5hmC enriched regions using ultra-low amount of genomic DNA (10-20 ng gDNA). Cytosine-5-methylenesulfonatem (CMS) is the 5hmC derivatives upon sodium bisulfite treatment. A home-made anti-CMS antibody was developed to specifically recognize CMS genome-wide after sodium bisulfite treatment. The traditional anti-CMSIP has been widely used to study the genome-wide 5hmC distribution in various biological systems, including mESC, HSC and immune cells. We further optimized the immunoprecipitation procedure to profile genome-wide 5hmC with ultra-low amount of genomic DNA (sensitive CMSIP, sCMSIP). Using this improved CMS-IP method, we performed genome-wide 5hmC analysis in ~100 individuals including healthy donors (n = 10), patients with MDS (n = 62), AML (n = 4) and CMML (n = 13). In parallel, we performed RRBS (DNA methylation) and RNA-seq (transcriptome) analysis on matched samples. We then performed integrative bioinformatics analyses to unveil the potential diagnostic and prognostic values of 5hmC in myeloid neoplasms. Results and Conclusions: Based on our analysis, we found distinct epigenetic landscapes and transcriptomes between healthy donor and patients with myeloid malignancies. Interestingly, we could further separate patients with MDS into three clusters by comparing their 5hmC landscapes with normal, AML and CMML individuals, suggesting that 5hmC might be a sensitive epigenetic mark to reflect the disease status of MDS. Furthermore, we identified a positive correlation between 5hmC distribution and the blast ratio in paired patients before and after hypomethylating agent (HMA) treatment. These findings suggest that 5hmC might be used as a prognostic marker for HMA treatment. Detailed analyses further elucidate the impaired 5hmC enrichment in various key transcription factors that are essential for HSC function and myeloid lineage specification. Disclosures Garcia-Manero: Amphivena: Consultancy, Research Funding; Helsinn: Research Funding; Novartis: Research Funding; AbbVie: Research Funding; Celgene: Consultancy, Research Funding; Astex: Consultancy, Research Funding; Onconova: Research Funding; H3 Biomedicine: Research Funding; Merck: Research Funding.

GENE-38. IDH1MUT INDUCES N6-METHYLADENOSINE (m6A) RNA HYPERMETHYLATION VIA D-2-HG IN GLIOMA

Abstract INTRODUCTION IDH1 MUT confers slower tumor growth rates and improved prognoses for glioma patients, but the precise mechanisms underlying these clinically relevant benefits remain largely unknown. IDH1MUT gliomas produce high levels of D-2-HG, a putative oncometabolite that inhibits a range of α-ketoglutarate-dependent dioxygenases. D-2-HG-mediated inhibition of TET-family proteins involved in DNA demethylation is known to be a key driver leading to the oncogenic DNA hypermethylation phenotype (known as G-CIMP) seen in IDH1MUT gliomas. Recent evidence indicates that D-2-HG also functionally inhibits FTO and AlkBH5, two dioxygenases responsible for demethylation of RNA N6-methyladenosine (m6A) sites. This study sought to determine if D-2-HG mediates an increase in m6A content in IDH1MUT gliomas. METHODS Total RNA was isolated from patient tumor samples, patient-derived gliomaspheres, human embryonic kidney cell (HEK293T), normal human astrocyte (NHA), and U87 glioma model systems expressing either IDH1MUT or IDH1WT. Relative abundance of m6A modifications was determined both quantitatively and qualitatively using colorimetric ELISA-like assays and m6A-antibody dot blots, respectively. RESULTS Quantification of m6A abundance in IDH1MUT patient tumor samples and patient-derived gliomaspheres revealed an increase in m6A content compared to IDH1WT samples. Forced expression of IDH1MUT in HEK293T, NHA, and U87 cells increased intracellular D-2-HG content and global m6A abundance in purified RNA. Additionally, D-2-HG treatment of IDH1WT cell lines increased m6A abundance, including in IDH1WT gliomaspheres. Conversely, inhibition of D-2-HG generation in IDH1MUT cell lines decreased m6A abundance. Data will also be presented suggesting that increased m6A abundance is associated with decreased cellular proliferation. CONCLUSIONS These results indicate that increased intracellular D-2-HG arising in the context of IDH1MUT mediates increases in RNA m6A methylation in glioma. The association with m6A hypermethylation and reduced growth suggests that RNA methylation provides a novel therapeutic target.

Deletion of Tet proteins results in quantitative disparities during ESC differentiation partially attributable to alterations in gene expression

BackgroundThe Tet protein family (Tet1, Tet2, and Tet3) regulate DNA methylation through conversion of 5-methylcytosine to 5-hydroxymethylcytosine which can ultimately result in DNA demethylation and play a critical role during early mammalian development and pluripotency. While multiple groups have generated knockouts combining loss of different Tet proteins in murine embryonic stem cells (ESCs), differences in genetic background and approaches has made it difficult to directly compare results and discern the direct mechanism by which Tet proteins regulate the transcriptome. To address this concern, we utilized genomic editing in an isogenic pluripotent background which permitted a quantitative, flow-cytometry based measurement of pluripotency in combination with genome-wide assessment of gene expression and DNA methylation changes. Our ultimate goal was to generate a resource of large-scale datasets to permit hypothesis-generating experiments.ResultsWe demonstrate a quantitative disparity in the differentiation ability among Tet protein deletions, with Tet2 single knockout exhibiting the most severe defect, while loss of Tet1 alone or combinations of Tet genes showed a quantitatively intermediate phenotype. Using a combination of transcriptomic and epigenomic approaches we demonstrate an increase in DNA hypermethylation and a divergence of transcriptional profiles in pluripotency among Tet deletions, with loss of Tet2 having the most profound effect in undifferentiated ESCs.ConclusionsWe conclude that loss of Tet2 has the most dramatic effect both on the phenotype of ESCs and the transcriptome compared to other genotypes. While loss of Tet proteins increased DNA hypermethylation, especially in gene promoters, these changes in DNA methylation did not correlate with gene expression changes. Thus, while loss of different Tet proteins alters DNA methylation, this change does not appear to be directly responsible for transcriptome changes. Thus, loss of Tet proteins likely regulates the transcriptome epigenetically both through altering 5mC but also through additional mechanisms. Nonetheless, the transcriptome changes in pluripotent Tet2−/− ESCs compared to wild-type implies that the disparities in differentiation can be partially attributed to baseline alterations in gene expression.

TET2 Function in Hematopoietic Malignancies, Immune Regulation, and DNA Repair.

Over the last decade, investigation of Ten-Eleven Translocation 2 (TET2) gene function and TET2 mutation have become of increasing interest in the field of hematology. This heightened interest was sparked by the seminal discoveries that (1) TET2 mutation is associated with development of hematological malignancies and that (2) the TET family of proteins is critical in promoting DNA demethylation and immune homeostasis. Since then, additional studies have begun to unravel the question “Does TET2 have additional biological functions in the regulation of hematopoiesis?” Here, we present a mini-review focused on the current understanding of TET2 in hematopoiesis, hematological malignancies, and immune regulation. Importantly, we highlight the critical function that TET2 facilitates in maintaining the stability of the genome. Based on our review of the literature, we provide a new hypothesis that loss of TET2 may lead to dysregulation of the DNA repair response, augment genome instability, and subsequently sensitize myeloid leukemia cells to PARP inhibitor treatment.

New tools for cell reprogramming and conversion: Possible applications to livestock.

Somatic cell nuclear transfer and iPS are both forms of radical cell reprogramming able to transform a fully differentiated cell type into a totipotent or pluripotent cell. Both processes, however, are hampered by low efficiency and, in the case of iPS, the application to livestock species is uncertain.Epigenetic manipulation has recently emerged as an efficient and robust alternative method for cell reprogramming. It is based upon the use of small molecules that are able to modify the levels of DNA methylation with 5-azacitidyne as one of the most widely used. Among a number of advantages, it includes the fact that it can be applied to domestic species including pig, dog and cat.Treated cells undergo a widespread demethylation which is followed by a renewed methylation pattern induced by specific chemical stimuli that lead to the desired phenotype. A detailed study of the mechanisms of epigenetic manipulation revealed that cell plasticity is achieved through the combined action of a reduced DNA methyl transferase activity with an active demethylation driven by the TET protein family. Surprisingly the same combination of molecular processes leads to the transformation of fibroblasts into iPS and regulate the epigenetic changes that take place during early development and, hence, during reprogramming following SCNT.Finally, it has recently emerged that mechanic stimuli in the form of a 3D cell rearrangement can significantly enhance the efficiency of epigenetic reprogramming as well as of maintenance of pluripotency. Interestingly these mechanic stimuli act on the same mechanisms both in epigenetic cell conversion with 5-Aza-CR and in iPS.We suggest that the balanced combination of epigenetic erasing, 3D cell rearrangement and chemical induction can go a long way to obtain ad hoc cell types that can fully exploit the current exiting development brought by gene editing and animal cloning in livestock production.

Chronic ethanol-mediated hepatocyte apoptosis links to decreased TET1 and 5-hydroxymethylcytosine formation.

The 5-hydroxymethylcytosine (5hmc) is a newly identified epigenetic modification thought to be regulated by the TET family of proteins. Little information is available about how ethanol consumption may modulate 5hmC formation and alcoholic liver disease (ALD) progression. A rat ALD model was used to study 5hmC in relationship to hepatocyte apoptosis. Human ALD liver samples were also used to validate these findings. It was found that chronic ethanol feeding significantly reduced 5hmC formation in a rat ALD model. There were no significant changes in TET2 and TET3 between the control- and ethanol-fed animals. In contrast, methylcytosine dioxygenase TET1 (TET1) expression was substantially reduced in the ethanol-fed rats and was accompanied by increased hepatocyte apoptosis. Similarly, knockdown of TET1 in human hepatocyte-like cells also significantly promoted apoptosis. Down-regulation of TET1 resulted in elevated expression of the DNA damage marker, suggesting a role for 5hmc in hepatocyte DNA damage as well. Mechanistic studies revealed that inhibition of TET1 promoted apoptotic gene expression. Similarly, targeting TET1 activity by removing cosubstrate promoted apoptosis and DNA damage. Furthermore, treatment with 5-azacitidine significantly mimics these effects, suggesting that chronic ethanol consumption promotes hepatocyte apoptosis and DNA damage by diminishing TET1-mediated 5hmC formation and DNA methylation. In summary, the current study provides a novel molecular insight that TET1-mediated 5hmC is involved in hepatocyte apoptosis in ALD progression.-Ji, C., Nagaoka, K., Zou, J., Casulli, S., Lu, S., Cao, K. Y., Zhang, H., Iwagami, Y., Carlson, R. I., Brooks, K., Lawrence, J., Mueller, W., Wands, J. R., Huang, C.-K. Chronic ethanol-mediated hepatocyte apoptosis links to decreased TET1 and 5-hydroxymethylcytosine formation.

Abstract IA26: Role of mutations in epigenetic regulators in pathogenesis of myeloid malignancies

Abstract Clinical, cytogenetic, and gene-based studies have been used to inform biology and improve prognostication for patients with acute myeloid leukemia (AML), myelodysplasia (MDS), and myeloroliferative neoplasms (MPN). Most recently, a series of candidate gene and whole-genome studies have identified recurrent somatic mutations in AML patients including TET2, ASXL1, DNMT3A, and cohesin complex mutations. Moreover, these mutations can be used to improve risk stratification in AML independent of established clinical parameters. Integrating mutational data with dose-intensity revealed that high-dose daunorubicin improved survival in patients with DNMT3A/NPM1 mutations or MLL translocations relative to treatment with standard-dose daunorubicin, but not in patients wild-type for these alterations. These data provide important clinical implications of genetic alterations in AML by delineating mutation combination genotypes that predict outcome in AML and improve AML risk stratification. Of biologic importance, the TET family of proteins have been shown to place a hydroxyl mark on methylated DNA and lead to DNA demethylation. We and others have found that TET2/IDH mutations lead to loss of DNA hydroxymethylation and a hypermethylation phenotype in leukemia patients. In addition, in vitro and in vivo studies show that TET2 loss or neomorphic IDH1/2 mutations lead to impaired hematopoietic differentiation, increased stem cell self-renewal, and myeloid transformation in vivo. We have also investigated the role of mutant DNMT3A in AML pathogenesis, and shown that these mutations cooperate with other disease alleles to induce leukemic transformation and to confer chemoresistance due to impaired DNA damage sensing. We will present novel data showing how these mutations coopt the epigenetic state of hematopoietic stem/progenitor cells in order to contribute to transformation and that these mutations have biologic and prognostic relevance. Citation Format: Ross Levine. Role of mutations in epigenetic regulators in pathogenesis of myeloid malignancies [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr IA26.

TET-Mediated Sequestration of miR-26 Drives EZH2 Expression and Gastric Carcinogenesis.

DNA demethylases of the TET family function as tumor suppressors in various human cancers, but their pathogenic contributions and mechanisms of action in gastric carcinogenesis and progression remain unclear. Here, we report that TET is transcriptionally upregulated in gastric cancer, where it correlates with poor prognosis. Mechanistic investigations revealed that TET facilitated gastric carcinogenesis through a noncoding function of the 3'UTR, which interacted with miR-26. This interaction resulted in sequestration of miR-26 from its target EZH2, which released the suppression on EZH2, and thereby led to EZH2 overexpression in gastric cancer. Our findings uncover a novel noncoding function for TET family proteins in facilitating gastric carcinogenesis. Cancer Res; 77(22); 6069-82. ©2017 AACR.

Nickel(II) Inhibits Tet-Mediated 5-Methylcytosine Oxidation by High Affinity Displacement of the Cofactor Iron(II).

Ten-eleven translocation (Tet) family proteins are Fe(II)- and 2-oxoglutarate-dependent dioxygenases that regulate the dynamics of DNA methylation by catalyzing the oxidation of DNA 5-methylcytosine (5mC). To exert physiologically important functions, redox-active iron chelated in the catalytic center of Tet proteins directly involves the oxidation of the multiple substrates. To understand the function and interaction network of Tet dioxygenases, it is interesting to obtain high affinity and a specific inhibitor. Surprisingly, here we found that natural Ni(II) ion can bind to the Fe(II)-chelating motif (HXD) with an affinity of 7.5-fold as high as Fe(II). Consistently, we further found that Ni(II) ion can displace the cofactor Fe(II) of Tet dioxygenases and inhibit Tet-mediated 5mC oxidation activity with an estimated IC50 of 1.2 μM. Essentially, Ni(II) can be used as a high affinity and selective inhibitor to explore the function and dynamics of Tet proteins.

Automated Chemical Solid-Phase Synthesis and Deprotection of 5-Hydroxymethylcytosine-Containing RNA.

5-Hydroxymethylcytosine is an epigenetic base modification that is part of the demethylation pathway of 5-methylcytosine in DNA. 5-Methylcytosine is iteratively oxidized to 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxycytosine by enzymes of the TET protein family. Since the discovery of 5-hydroxymethylcytosine also in RNA its role in regulatory processes and metabolism remains elusive. To gain more insight into the function of RNA containing 5-hydroxymethylcytidine, innovative and interdisciplinary approaches are required. In this context, synthetic oligoribonucleotides containing 5-hyroxymethylcytidine are an inevitable tool. Therefore, in this chapter, we present the efficient synthesis of RNA oligonucleotides containing 5-hydroxymethylcytosine by solid-phase synthesis. The incorporation of the modified cytosine derivative into RNA is compatible with standard phosphoramidite-based synthesis procedures of oligoribonucleotides.

Mutational Landscape of Pediatric Acute Lymphoblastic Leukemia

Pediatric ALL is the most common childhood tumor and the leading cause of childhood cancer deaths. To gain a better understanding of the landscape of somatic mutations in ALL, we performed whole exome and targeted sequencing of 240 pediatric B-ALL patients with their matched remission samples. The significantly mutated genes fall into several common categories: RAS/receptor tyrosine kinases, epigenetic regulators, transcription factors involved in lineage commitment and p53/cell cycle pathway.RAS/receptor tyrosine kinases: the most frequently mutated genes were members of RAS signaling (NRAS, KRAS, FLT3, PTPN11). Besides the well know hotspot mutations [G12D/V/C (NRAS 13 cases, KRAS 13 cases), G13D (NRAS 14 cases, KRAS 11 cases) and Q61H/L/R/K (NRAS 15 cases, KRAS 1 case)], novel mutational sites were also identified for KRAS: A146T/P (3 cases), K117N/T (4 cases) and V14I (1 case). High frequency missense mutations of PTPN11 clustered in SH2 domain (included the canonical hotspot A72T (5 cases) and E76K/V (4 cases)) and tyrosine-phosphatase catalytic domain (G503R/V). For FLT3, well-appreciated activating hotspot mutations in the kinase domain (D835Y/Y842C) and several novel recurrent mutationswere identified.Epigenetic regulators: hotspot mutations were identified in histone H3K36 methyltransferase WHSC1. Mutation E1099K located in the SET domain, was identified in 10 patients as well as two of the 5 ALL cell lines that we sequenced (RS4;11, SEM). Stable silencing of E1099K mutant WHSC1 in RS4;11 cells by either lentiviral shRNA or CRISPR guide RNA (sgRNA) markedly reduced clonogenic growth both in vitro and in vivo, underscoring the critical role of WHSC1 in lymphoid malignancies. Two highly-related histone/non-histone acetyltransferases, CREBBP and EP300, were also prominently mutated in our cohort. Mutations of CREBBP predominantly occurred in the acetyltransferase domain, particularly in the hotspot R1446C/H. Mutations of chromatin remodeling genes (ARID1A and ARID2) have been identified in a number of cases. Silencing of ARID1A in ALL cell lines by lentiviral shRNA resulted in upregulation of the pro-growth regulator c-MYC, while forced expression of ARID1A reduced c-MYC luciferase reporter activity. In addition, silencing of ARID1A by either shRNA or CRISPR-sgRNA resulted in enhanced clonogenic growth, suggesting that ARID1A may be involved in the c-MYC pathway and modulates the ALL cell proliferation. Mutations of epigenetic regulators were also found in the polycomb complex (EZH2, EED, SUZ12), chromatin/nucleosome structure modifying proteins (CHD2, CHD3, CHD4), TET family proteins [TET1 (2 cases), TET2 (5 cases)] and histone modification proteins (HDAC1, SIRT1, BCOR, BRD8, lysine demethylase PHF2/KDM6A, histone acetyltransferase KAT6B).Transcription factors and p53/cell cycle pathway: a number of alterations of transcription factors essential for hematopoietic and lymphoid differentiation were noted including the lineage regulator PAX5 (5 missense, 3 indels) and ETV6 (6 cases, 3 were frameshift indel and 1 was a splice-site mutations). In addition, mutations were also found in other lineage transcription factors (IKZF2, IKZF3, EBF1), WT1 (6 cases, including 3 indels and 1 stop-gain mutations), RUNX family member [RUNX2 (7 cases), RUNX1 (1 case)], ERG1 (3 cases), GATA1/3 (1 case each) and CTCF. Somatic mutations of genes involved in the p53 pathway occurred in 18 patients, including TP53, ATM and the kinases that regulate p53 activities (HIPK1, HIPK2). Germline TP53 pathogenic variants were found in these 2 patients.Taken together, we extensively interrogated the mutational landscape of a large cohort of pediatric ALL samples by exome and targeted resequencing. This study provides a detailed mutational portrait of pediatric ALL and gives new insights into the molecular pathogenesis of this disease. DisclosuresKantarjian:Amgen: Research Funding; ARIAD: Research Funding; Bristol-Myers Squibb: Research Funding; Pfizer Inc: Research Funding; Delta-Fly Pharma: Research Funding; Novartis: Research Funding. Ogawa:Sumitomo Dainippon Pharma: Research Funding; Kan research institute: Consultancy, Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding.

Epigenetic upregulation of ARL4C, due to DNA hypomethylation in the 3'-untranslated region, promotes tumorigenesis of lung squamous cell carcinoma.

ADP-ribosylation factor (ARF)-like 4c (ARL4C) expression, induced by a combination of Wnt/β-catenin and EGF/Ras signaling, has been demonstrated to form epithelial morphogenesis. ARL4C overexpression, due to Wnt/β-catenin and EGF/Ras signaling alterations, was involved in tumorigenesis. It was also reported that ARL4C expression correlates with DNA hypomethylation in the 3’-untranslated region (UTR) of ARL4C gene during lymphogenesis. The current study was conducted to investigate the expression and functions of ARL4C due to DNA hypomethylation in lung and tongue cancers. Immunohistochemical analyses of tissue specimens obtained from lung and tongue squamous cell carcinoma (SCC) patients revealed that ARL4C is not observed in non-tumor regions, but is strongly expressed at high frequencies in tumor lesions. Although inhibition of Wnt/β-catenin or Ras/MAP kinase signaling did not decrease ARL4C expression in NCI-H520 lung SCC cells, ARL4C DNA was clearly hypomethylated in the 3’-UTR. Ten-eleven translocation methylcytosine dioxygenase (TET) enzyme, which mediates DNA demethylation, was highly expressed in NCI-H520 cells. Knockout of TET family proteins (TET1-3) in NCI-H520 cells reduced 5-hydroxymethylcytosine (5hmC) levels and promoted DNA methylation in the 3’-UTR, leading to the decrease in ARL4C expression and ARL4C-mediated cellular migration. In tumor lesions of ARL4C-positive lung SCC, 5hmC was frequently detected and DNA methylation in the 3’-UTR of ARL4C gene was lower than in non-tumor regions, which were consistent with the Cancer Genome Atlas dataset. These results suggest that ARL4C is expressed due to hypomethylation in the 3’-UTR for certain types of cancers and that ARL4C methylation status is involved in cancer cell function.