TET2 Expression Research Articles

Abstract 10248: Regulation of the Methylation and Expression Levels of the Bmpr2 Gene by Sin3a as a Novel Therapeutic Mechanism in Pulmonary Arterial Hypertension

Background: Epigenetic mechanisms are critical in the pathogenesis of pulmonary arterial hypertension (PAH). Previous studies have suggested that hypermethylation of the Bone Morphogenetic Protein Receptor Type 2 (BMPR2) promoter is associated with BMPR2 downregulation and progression of PAH. Here, we investigated for the first time the role of Switch-Independent 3a (SIN3a), a transcriptional regulator, in the epigenetic mechanisms underlying hypermethylation of BMPR2 in the pathogenesis of PAH. Methods: We used lung samples from PAH patients and non-PAH controls, preclinical mouse and rat PAH models, and human pulmonary arterial smooth muscle cells (hPASMC). Expression of SIN3a was modulated using a lentiviral vector or a siRNA in vitro and a specific Adeno-Associated Virus serotype 1 (AAV1) or a lentivirus encoding for human SIN3a in vivo . Results: SIN3a is a known transcriptional regulator; however, its role in cardiovascular diseases, especially PAH, is unknown. Interestingly, we detected a dysregulation of SIN3 expression in patients and in rodent models, which is strongly associated with decreased BMPR2 expression. SIN3a is known to regulate epigenetic changes. Therefore, we tested its role in the regulation of BMPR2 and found that BMPR2 is regulated by SIN3a. Interestingly, SIN3a overexpression inhibited hPASMC proliferation and upregulated BMPR2 expression by preventing the methylation of the BMPR2 promoter region. RNA sequencing analysis suggested that SIN3a downregulated the expression of DNA and histone methyltransferases such as DNMT1 and EZH2 while promoting the expression of the DNA demethylase TET1. Mechanistically, SIN3a promoted BMPR2 expression by decreasing CTCF binding to the BMPR2 promoter. Finally, we identified intratracheal delivery of AAV1.hSIN3a to be a beneficial therapeutic approach in PAH- by attenuating pulmonary vascular and RV remodeling, decreasing RVSP and mPAP pressure, and restoring BMPR2 expression in rodent models of PAH. Conclusions: Altogether, our study unveiled the protective/beneficial role of SIN3a in pulmonary hypertension. We also identified a novel and distinct molecular mechanism by which SIN3a regulates BMPR2 in hPASMC.

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

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

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

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

Minocycline Attenuates Excessive DNA Damage Response and Reduces Ectopic Calcification in Pseudoxanthoma Elasticum

Pseudoxanthoma elasticum (PXE) is a hereditary ectopic calcification disorder affecting the skin, eyes, and blood vessels. Recently, the DNA damage response (DDR), in particular PARP1, was shown to be involved in aberrant mineralization, raising the hypothesis that excessive DDR/PARP1 signaling also contributes to PXE pathogenesis. Using dermal fibroblasts of patients with PXE and healthy controls, (lesional) skin tissue, and abcc6a‒/‒ zebrafish, we performed expression analysis of DDR/PARP1 targets with QRT-PCR, western blot, immunohistochemistry, and enzyme activity assays before and after treatment with the PARP1 inhibitor minocycline. PARP1 and the ATM‒p21‒p53 axis was found to be significantly increased in PXE. In addition, PARP1 downstream targets IL-6, signal transducer and activator of transcription 1/3, TET1, and RUNX2 were upregulated, whereas the RUNX2 antagonist microRNA-204 was decreased. In PXE fibroblasts, DDR/PARP1 signaling increased with advancing ectopic calcification. Minocycline treatment attenuated DDR/PARP1 overexpression and reduced aberrant mineralization in PXE fibroblasts and abcc6a‒/‒ zebrafish. In summary, we showed the involvement of excessive DDR/PARP1 signaling in PXE pathophysiology, identifying a signal transducer and activator of transcription‒driven cascade resulting in increased expression of the epigenetic modifier TET1 and procalcifying transcription factor RUNX2. Minocycline attenuated this deleterious molecular mechanism and reduced ectopic calcification both invitro and invivo, fueling the exciting prospect of a therapeutic compound for PXE.

Fetal growth restriction impairs hippocampal neurogenesis and cognition via Tet1 in offspring.

Fetal growth restriction (FGR) increases the risk for impaired cognitive function later in life. However, the precise mechanisms remain elusive. Using dexamethasone-induced FGR and protein restriction-influenced FGR mouse models, we observe learning and memory deficits in adult FGR offspring. FGR induces decreased hippocampal neurogenesis from the early post-natal period to adulthood by reducing the proliferation of neural stem cells (NSCs). We further find a persistent decrease of Tet1 expression in hippocampal NSCs of FGR mice. Mechanistically, Tet1 downregulation results in hypermethylation of the Dll3 and Notch1 promoters and inhibition of Notch signaling, leading to reduced NSC proliferation. Overexpression of Tet1 activates Notch signaling, offsets the decline in neurogenesis, and enhances learning and memory abilities in FGR offspring. Our data indicate that a long-term decrease in Tet1/Notch signaling in hippocampal NSCs contributes to impaired neurogenesis following FGR and could serve as potential targets for the intervention of FGR-related cognitive disorders.

Gestational arsenic exposure induces anxiety-like behaviors in adult offspring by reducing DNA hydroxymethylation in the developing brain

Several studies found that reduction of 5-hydroxymethylcytosine (5hmC), a marker of DNA hydroxymethylation highly enriched in developing brain, is associated with anxiety-like behaviors. This study aimed to investigate whether gestational arsenic (As) exposure induces anxiety-like behaviors in adult offspring by reducing DNA hydroxymethylation in the developing brain. The dams drank ultrapure water containing NaAsO2 (15 mg/L) throughout pregnancy. Anxiety-like behaviors were evaluated and developing brain 5hmC was detected. Results showed that anxiety-like behaviors were observed in As-exposed adult offspring. In addition, 5hmC content was reduced in As-exposed fetal brain. Despite no difference on Tet1, Tet2 and Tet3 expression, TET activity was suppressed in As-exposed fetal brain. Mechanistically, alpha-ketoglutarate (α-KG), a cofactor for TET dioxygenases, was reduced and Idh2, a key enzymatic gene for mitochondrial α-KG synthesis, was downregulated in As-exposed fetal brain. Of interest, ascorbic acid, a cofactor for TET dioxygenases, reversed As-induced suppression of TET activity. Moreover, ascorbic acid attenuated As-induced reduction of 5hmC in fetal brain. In addition, ascorbic acid alleviated As-induced anxiety-like behaviors in adult offspring. Taken together, these results suggest that gestational As exposure induces anxiety-like behaviors in adult offspring, possibly at part, by inhibiting DNA hydroxymethylation in developing brain.

TET2 modulates proliferation and migration of vascular smooth muscle cells via LEMD2/NOX1/NOX4 signaling pathway

Abstract Background Ten-eleven translocation 2 (TET2), a widely reported DNA hydroxymethylation enzyme, is involved in active DNA demethylation. TET2 may play an critical role in numerous cellular processes by regulating the level of DNA hydroxymethylation and altering gene expression. TET2 expression was proved to be down-regulated in aorta of spontaneously hypertensive rats (SHR) compared to Wistar-Kyoto rats (WKY) as well as in VSMCs subjected to15% cyclic stretch compared to 5% cyclic stretch. However, whether TET2 regulates vascular smooth muscle cells (VSMCs) proliferation and migration and its underlying mechanisms remains unclear. Purpose The present study aims to investigate whether TET2 affects VSMC proliferation and migration and its possible underlying mechanisms. Methods TET2 knockdown rat VSMC was established using crispr/Cas9 method. Different expression genes were identified by next generation sequencing (NGS) between TET2 knockdown VSMC and control VSMCs while differentially hydroxymethylated promoter region were identified by hydroxymethylcytosine DNA immunoprecipitation (hMeDIP) sequencing. Expression of mRNA and proteins was detected by qRT-PCR and western blot respectively. The proliferation and migration of VSMCs were assessed by EdU assay, flowcytometry assay, and wound healing assay. LEMD2 overexpression and knockdown stable VSMC lines were established through Lentiviral infection. Results The EdU assay and wound healing assay show that knockdown of TET2 enhanced proliferation and migration of VSMCs. NGS identified 123 differentially expressed genes (91 up-regulated and 32 down-regulated) between TET2 knockdown VSMCs and control VSMCs while hMeDIP sequencing identified 166 genes with differentially hydroxymethylated promoter region (68 up-regulated and 98 down-regulated) between TET2 knockdown VSMCs and control VSMCs. Through comparing these sequencing results, we identified a gene named as LEMD2 simultaneously present in both sequencing results. Expression of LEMD2 at mRNA and protein level was significantly increased in TET2 knockdown VSMCs compared to control VSMCs. Then we successfully established LEMD2 overexpression and knockdown stable VSMC lines and found that overexpression of LEMD2 enhanced proliferation and migration of VSMCs while knockdown of LEMD2 inhibited proliferation and migration of VSMCs. Furthermore, overexpression of LEMD2 up-regulated the expression of NOX1 and down-regulated the expression of NOX4 while knockdown of LEMD2 show the opposite effect. Conclusion The present study confirmed that TET2 modulates VSMC proliferation and migration via LEMD2/NOX1/NOX4. The ROS level may be involved in VSMC proliferation and migration. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National natural science foundation of China

Analysis of Gene Expression Patterns of Epigenetic Enzymes Dnmt3a, Tet1 and Ogt in Murine Chondrogenic Models.

We investigated the gene expression pattern of selected enzymes involved in DNA methylation and the effects of the DNA methylation inhibitor 5-azacytidine during in vitro and in vivo cartilage formation. Based on the data of a PCR array performed on chondrifying BMP2-overexpressing C3H10T1/2 cells, the relative expressions of Tet1 (tet methylcytosine dioxygenase 1), Dnmt3a (DNA methyltransferase 3), and Ogt (O-linked N-acetylglucosamine transferase) were further examined with RT-qPCR in murine cell line-based and primary chondrifying micromass cultures. We found very strong but gradually decreasing expression of Tet1 throughout the entire course of in vitro cartilage differentiation along with strong signals in the cartilaginous embryonic skeleton using specific RNA probes for in situ hybridization on frozen sections of 15-day-old mouse embryos. Dnmt3a and Ogt expressions did not show significant changes with RT-qPCR and gave weak in situ hybridization signals. The DNA methylation inhibitor 5-azacytidine reduced cartilage-specific gene expression and cartilage formation when applied during the early stages of chondrogenesis. In contrast, it had a stimulatory effect when added to differentiated chondrocytes, and quantitative methylation-specific PCR proved that the DNA methylation pattern of key chondrogenic marker genes was altered by the treatment. Our results indicate that the DNA demethylation inducing Tet1 plays a significant role during chondrogenesis, and inhibition of DNA methylation exerts distinct effects in different phases of in vitro cartilage formation.

Nickel chloride regulates ANGPTL4 via the HIF-1α-mediated TET1 expression in lung cells

Angiopoietin-like protein 4 (ANGPTL4) is a hypoxia-induced gene, and its high expression is associated with poor prognosis and promotion of tumour progression in several cancers. Some studies reported that ANGPTL4 is affected by epigenetic regulation. Our previous results demonstrated that ANGPTL4 is highly expressed in most lung cancer cell lines than in normal cell lines and is upregulated by HIF-1α accumulation under NiCl2 exposure. The accurate role of ANGPTL4 and its methylation status caused by nickel in the lung carcinogenesis is not fully explored yet. In this study, we found that ANGPTL4 and HIF-1α in lung adenocarcinoma (LUAD) tissues were significantly upregulated compared with those in normal tissues in The Cancer Genome Atlas (TCGA) cohort (p < 0.001). The ANGPTL4 expression was statistically correlated to advanced stage (p = 0.019) and N value (p = 0.002). The Kaplan–Meier analysis revealed that ANGPTL4 and HIF-1α expression levels were independently associated with the 5-year survival of patients with LUAD in TCGA database and immunohistochemistry staining. In vitro experiments indicated that ANGPTL4 was upregulated by the demethylation agent. The methylation-specific PCR and bisulfite sequencing assessed the methylation status of the ANGPTL4 promoter, and results showed that NiCl2-treated cells had low ANGPTL4 methylation status. We further demonstrated that the DNA demethylase, TET1, was significantly increased under NiCl2 exposure. The knockdown of TET1 expression repressed the NiCl2-induced ANGPTL4. We also showed that nickel-induced TET1 was stimulated by HIF-1α. Our work established ANGPTL4 as a potential oncogene that contributes to lung cancer progression and nickel-elicited carcinogenesis.

AML-170: High MYC Expression Predicts Poor Survival Outcomes in IDH1/2 Mutant AML Patients

<h3>Context:</h3> Clinical trials of IDH1/2 inhibitors demonstrated promising outcomes in <i>IDH1/2^mut</i> AML patients. However, a substantial fraction of patients fail to respond to IDH1/2 inhibitors in AML. Recent preclinical studies have shown MYC prevents the normal myeloid differentiation and cell death by suppressing IDH1/2-TET2-5hmC signaling. <h3>Objective:</h3> Evaluate if high MYC expression is associated with poor survival outcomes in <i>IDH1/2^mut</i> AML patients treated with ivosidenib or enasidenib. <h3>Design:</h3> We retrospectively identified AML patients who had <i>IDH1/2</i> somatic mutations based on NGS assessments. Clinical and demographic data were extracted from the medical records. Pre-treatment MYC protein levels were assessed by immunohistochemistry (IHC) staining using BM biopsy specimens. <h3>Results:</h3> A total of 28 (<i>IDH1^mut</i>, n=11; <i>IDH2^mut,</i> n=18; <i>IDH1/2^mut,</i> n=1) patients were included in the study. Median age at AML diagnosis was 66 years, and 60% of patients were male. Twelve (42%) patients had secondary AML and 9 (32%), 11 (39%), and 6 (21%) patients had favorable, intermediate, and adverse risk, respectively, at AML diagnosis. A total of 17 (60.7%) and 6 (21.4%) patients received intensive chemotherapy and hypomethylating agents as their first-line therapy. Among 28 patients, 22 (78.5%) and 6 (21.4%) patients had low and high MYC expression (≥5% by IHC staining), respectively, prior to IDH1/2 inhibitor treatment. Median number of treatments prior to IDH1/2 inhibitors was 3 (1–6) and the median duration of IDH1/2 inhibitor treatment was 3.2 (0.3–30) months (<i>IDH1^mut</i>, 3.3 [0.3–30] months; <i>IDH2^mut</i>, 2.5 [0.7–14.5] months). Treatment response was assessed in 27 patients, and 11 (40.7%) had CR/CRi (high <i>vs</i> low MYC, 20 <i>vs</i> 45.5%, p=0.6185). The median PFS was shorter in high MYC patients (1.6 <i>vs</i> 4.4 months, HR=2.348, p=0.0515). The median OS was also significantly shorter in high MYC patients (2.5 <i>vs</i> 8.5 months, HR=5.971, p<0.0001). <h3>Conclusions:</h3> High MYC expression was associated with significantly shorter PFS and OS in <i>IDH1/2^mut</i> AML patients. Consistent with our recent studies that showed MYC represses IDH1/2 and TET2 expression in AML, this study suggests that MYC may play an important role in the resistance mechanism of IDH1/2 inhibitors. Additional studies with larger cohorts are warranted to further confirm and validate these findings.

Tet2 Controls the Responses of \u03b2 cells to Inflammation in Autoimmune Diabetes

β cells may participate and contribute to their own demise during Type 1 diabetes (T1D). Here we report a role of their expression of Tet2 in regulating immune killing. Tet2 is induced in murine and human β cells with inflammation but its expression is reduced in surviving β cells. Tet2-KO mice that receive WT bone marrow transplants develop insulitis but not diabetes and islet infiltrates do not eliminate β cells even though immune cells from the mice can transfer diabetes to NOD/scid recipients. Tet2-KO recipients are protected from transfer of disease by diabetogenic immune cells.Tet2-KO β cells show reduced expression of IFNγ-induced inflammatory genes that are needed to activate diabetogenic T cells. Here we show that Tet2 regulates pathologic interactions between β cells and immune cells and controls damaging inflammatory pathways. Our data suggests that eliminating TET2 in β cells may reduce activating pathologic immune cells and killing of β cells.

Investigation of the Association Between TET2 Expression and Response to CAPE

Investigation of the Association Between TET2 Expression and Response to CAPE

Stress modulates Ahi1-dependent nuclear localization of ten-eleven translocation protein 2.

Major depression disorder is one of the most common psychiatric diseases. Recent evidence supports that environmental stress affects gene expression and promotes the pathological process of depression through epigenetic mechanisms. Three ten-eleven translocation (Tet) enzymes are epigenetic regulators of gene expression that promote 5-hydroxymethylcytosine (5hmC) modification of genes. Here, we show that the loss of Tet2 can induce depression-like phenotypes in mice. Paradoxically, using the paradigms of chronic stress, such as chronic mild stress and chronic social defeat stress, we found that depressive behaviors were associated with increased Tet2 expression but decreased global 5hmC level in hippocampus. We examined the genome-wide 5hmC profile in the hippocampus of Tet2 knockout mice and identified 651 dynamically hydroxymethylated regions, some of which overlapped with known depression-associated loci. We further showed that chronic stress could induce the abnormal nuclear translocation of Tet2 protein from cytosol. Through Tet2 immunoprecipitation and mass spectrum analyses, we identified a cellular trafficking protein, Abelson helper integration site-1 (Ahi1), which could interact with Tet2 protein. Ahi1 knockout or knockdown caused the accumulation of Tet2 in cytosol. The reduction of Ahi1 protein under chronic stress explained the abnormal Ahi1-dependent nuclear translocation of Tet2. These findings together provide the evidence for a critical role of modulating Tet2 nuclear translocation in regulating stress response.

Abstract 1427: Sensitizing HL60 acute myeloid leukemia cells to decitabine with pterostilbene

Abstract Introduction. Decitabine (DAC), is a DNA hypomethylating agent that has been shown to be potent towards leukemia cells at low doses. However, many patients develop resistance to the drug due to dysregulation of genes associated with responsiveness to DAC-induced DNA demethylation. To this regard, it has been reported that downregulation of TET2, one of the demethylating enzymes, increases sensitivity to DAC in acute myeloid leukemia (AML) cells. Remarkably, dietary bioactive compounds such as pterostilbene (PTS), with a stilbenoid ring structure, have previously been shown to mediate changes in gene expression through regulation of the epigenetic machinery in cancer models. Thus, we sought to test the impact of treatment of leukemia cells with PTS to augment expression of genes involved in the responsiveness of low-dose DAC in AML cells. Methods. HL60, an AML cell line, was either treated in combination with PTS (1.0 µM) and DAC (200 nM) or pre-treated with different concentrations of PTS (0 µM, 0.5 µM, 1.0 µM, 2 µM) for four days followed by DAC treatment (100 nM) for three days. Cell growth was assessed via trypan blue dye exclusion test and comparisons were made between combination treatment and pre-treatment with PTS. qPCR was performed in triplicates for each assay to assess expression of selected genes. Results. Treatment of HL60 cells with 200 nM DAC alone led to a 60% decrease in cell growth compared to control (treated with ethanol as a vehicle). After the combined treatment with PTS, cell growth decreased by further 27% compared to DAC alone. In order to decipher if this decrease in cell growth was due to the effect of PTS sensitizing cells to DAC, the cells were first pre-treated with PTS for four days and subsequently treated with DAC for three days. Compared to control (ethanol pre-treated cells), growth of cells treated with 100 nM DAC alone was decreased by about 37%, while growth of cells pre-treated with 0.5 µM, 1.0 µM, and 2.0 µM PTS followed by 100 nM DAC exposure was further reduced by 13%, 14.5%, and 30%, respectively. This suggests that PTS-pre-treated cells are more sensitive to DAC. Interestingly, DAC alone at 200 nM led to an increase in expression of TET2 (p&amp;lt;0.01) while PTS at 1.0 µM alone led a decrease (p&amp;lt;0.01) compared to control. However, when PTS and DAC were combined, TET2 levels increased significantly (p&amp;lt;0.01) compared to both control and DAC alone, despite its better ability to inhibit growth. In order to explore this further, we will proceed with investigating the effects of pre-treatment with PTS on TET2 expression in HL60 cells. Conclusion. Treatment of HL60 cells with PTS results in improved sensitivity of cells to low doses of DAC, possibly due to modulating TET2 expression. Citation Format: Cayla Boycott, Barbara Stefanska. Sensitizing HL60 acute myeloid leukemia cells to decitabine with pterostilbene [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 1427.

Abstract 1526: Single-step precision genome engineering platform enables versatile generation of personalized (neoTCR) adoptive cell therapy T cells with supplementary anti-tumor attributes

Abstract Personalized autologous TCR-T cell therapies targeting neoE-specific neoepitopes (NeoE) derived from tumor-exclusive mutations are a compelling approach for the treatment of patients with solid tumors. Using the ultra-sensitive imPACT Isolation Technology®, antigen-experienced neoE-specific CD8 T cells are captured from the blood of patients with solid cancers followed by cloning of the cognate neoepitope-specific HLA class I-restricted T cell receptors (HLA-I neoTCRs). Using DNA-mediated (non-viral) precision genome engineering technology, fresh CD8 and CD4 T cells from the same patient with cancer are engineered to express the HLA-I neoTCR. We have further innovated this platform of precision genome engineering to confer neoTCR-T cells with supplementary gene edits to address the array of potential sources for tumor immune evasion. Two examples of the versatility of this single step gene editing platform are described in this study: 1) concurrent expression of a CD8 co-receptor (CD8coR) to augment activity of CD4 T cells engineered with lower affinity HLA-I neoTCRs; and 2) simultaneous disruption of TET2 expression in T cells, which has previously been reported to positively affect T cell differentiation and persistence. The data shows that these simultaneous supplemental gene modifications were achieved with high efficiency, resulting in fully functional CD8 and CD4 neoTCR-T cells. Surface expression of CD8coR together with the neoE-targeted TCR increased T cell effector function and antigen-specific tumor cell killing. NeoTCR-T cells with concomitant deletion of TET2-expression exhibited enhanced cytotoxicity against neoantigen-expressing tumor cells. Together, these results demonstrate the applicability of this versatile precision genome engineering platform technology to yield enhanced next generation neoTCR-T cell therapies to expand the potential for clinical benefit in treating persons with solid cancers. Citation Format: Michael M. Dubreuil, Charles W. Tran, Bhamini Purandare, William Lu, James Byers, Michal Mass, Kyle Jacoby, Barbara Sennino, Alex Franzusoff, Stefanie J. Mandl. Single-step precision genome engineering platform enables versatile generation of personalized (neoTCR) adoptive cell therapy T cells with supplementary anti-tumor attributes [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 1526.

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.

DNA demethylase Tet2 suppresses cisplatin-induced acute kidney injury

Demethylase Tet2 plays a vital role in the immune response. Acute kidney injury (AKI) initiation and maintenance phases are marked by inflammatory responses and leukocyte recruitment in endothelial and tubular cell injury processes. However, the role of Tet2 in AKI is poorly defined. Our study determined the degree of renal tissue damage associated with Tet2 gene expression levels in a cisplatin-induced AKI mice model. Tet2-knockout (KO) mice with cisplatin treatment experienced severe tubular necrosis and dilatation, inflammation, and AKI markers’ expression levels than the wild-type mice. In addition, the administration of Tet2 plasmid protected Tet2-KO mice from cisplatin-induced nephrotoxicity, but not Tet2-catalytic-dead mutant. Tet2 KO was associated with a change in metabolic pathways like retinol, arachidonic acid, linolenic acid metabolism, and PPAR signaling pathway in the cisplatin-induced mice model. Tet2 expression is also downregulated in other AKI mice models and clinical samples. Thus, our results indicate that Tet2 has a renal protective effect during AKI by regulating metabolic and inflammatory responses through the PPAR signaling pathway.

RASAL2 Deficiency Attenuates Hepatic Steatosis by Promoting Hepatic VLDL Secretion via the AKT/TET1/MTTP Axis.

RAS protein activator like 2 (RASAL2) is a newly discovered metabolic regulator involved in energy homeostasis and adipogenesis. However, whether RASAL2 is involved in hepatic lipid metabolism remains undetermined. This study explored the function of RASAL2 and elucidated its potential mechanisms in nonalcoholic fatty liver disease (NAFLD). NAFLD models were established either by feeding mice a high-fat diet or by incubation of hepatocytes with 1 mM free fatty acids (oleic acid:palmitic acid=2:1). Pathological changes were observed by hematoxylin and eosin staining. Lipid accumulation was assessed by Oil Red O staining, BODIPY493/503 staining, and triglyceride quantification. The in vivo secretion rate of very low-density lipoprotein was determined by intravenous injection of tyloxapol. Gene regulation was analyzed by chromatin immunoprecipitation assays and hydroxymethylated DNA immunoprecipitation combined with real-time polymerase chain reaction. RASAL2 deficiency ameliorated hepatic steatosis both in vivo and in vitro. Mechanistically, RASAL2 deficiency upregulated hepatic TET1 expression by activating the AKT signaling pathway and thereby promoted MTTP expression by DNA hydroxymethylation, leading to increased production and secretion of very low-density lipoprotein, which is the major carrier of triglycerides exported from the liver to distal tissues. Our study reports the first evidence that RASAL2 deficiency ameliorates hepatic steatosis by regulating lipid metabolism through the AKT/TET1/MTTP axis. These findings will help understand the pathogenesis of NAFLD and highlight the potency of RASAL2 as a new molecular target for NAFLD.

MRNA Expressions of Methylation Related Enzymes and Duration of Thermal Conditioning in Chicks.

DNA methylation regulates gene expression by modifying the nucleosome structure of DNA, without altering the gene sequence. It has been reported that DNA methylation reactions are catalyzed by several enzymes. In chickens, thermal conditioning treatment affects the central DNA methylation levels. The purpose of this study was to clarify the changes in DNA methylation and demethylation factors during thermal conditioning in the hypothalamus of 3-day-old chicks. Male chicks (3-days old) were exposed to 40±0.5°C as a thermal conditioning treatment for 1, 2, 6, 9, or 12 h. The control chicks were kept in a thermoneutral zone (30±0.2°C). After thermal conditioning, the mRNA levels of DNA methyltransferase (DNMT)-1, -3a, -3b, and ten-eleven translocation (TET)-1, -2, and -3 in the hypothalamus were measured by q-PCR. The mRNA levels of DNMT-3a and TET-1 were increased by thermal conditioning. Moreover, the expression level of TET-1 increased with the loading time of the thermal conditioning. The gene expressions of DNMT-1, DNMT-3b, TET-2, and TET-3 were not affected by thermal conditioning. Since DNMT-3a is a catalyst for de-novo DNA methylation and TET-1 catalyzes the oxidation of methylated cytosine, it is suggested that the thermal conditioning increased the activation of DNA methylation and demethylation factors, which occur in the hypothalamus of neonatal chicks.

Chronic alcohol exposure promotes HCC stemness and metastasis through β-catenin/miR-22-3p/TET2 axis.

Hepatocellular Carcinoma (HCC) patients usually have a high rate of relapse and metastasis. Alcohol, a risk factor for HCC, promotes the aggressiveness of HCC. However, the basic mechanism is still unclear. We used HCC cells and an orthotopic liver tumor model of HCC-LM3 cells for BALB/C nude mice to study the mechanism of alcohol-induced HCC progression. We showed that chronic alcohol exposure promoted HCC cells metastasis and pulmonary nodules formation. First, we identified miR-22-3p as an oncogene in HCC, which promoted HCC cells stemness, tumor growth, and metastasis. Further, we found that miR-22-3p directly targeted TET2 in HCC. TET2, a dioxygenase involved in cytosine demethylation, has pleiotropic roles in hematopoietic stem cells self-renewal. In clinic HCC specimen, TET2 expression was not only decreased by alcohol consumption, but also inversely correlated with miR-22-3p levels. Then, we demonstrated that TET2 depletion promoted HCC cells stemness, tumor growth and metastasis. Furthermore, we identified that β-catenin was an upstream activator of miR-22-3p. In conclusion, this study suggests that chronic alcohol exposure promotes HCC progression and β-catenin/miR-22-3p/TET2 regulatory axis plays an important role in alcohol-promoted HCC malignancy.