Targeting DNA methyltransferases for cancer therapy.

DNA methyltransferases (DNMTs) play a crucial role in genomic DNA methylation. In mammals, DNMTs regulate the dynamic patterns of DNA methylation in embryonic and adult cells. Abnormal functions of DNMTs are often indicative of cancers, including overall hypomethylation and partial hypermethylation of tumor suppressor genes (TSG), which accelerate the malignancy of tumors, worsen the condition of patients, and significantly exacerbate the difficulty of cancer treatment. Currently, nucleoside DNMT inhibitors such as Azacytidine and Decitabine have been approved by the FDA and EMA for the treatment of acute myeloid leukemia (AML), chronic myelomonocytic leukemia (CMML), and myelodysplastic syndrome (MDS). Therefore, targeting DNMTs is a very promising anti-tumor strategy. This review mainly summarizes the therapeutic effects of DNMT inhibitors on cancers. It aims to provide more possibilities for the treatment of cancers by discovering more DNMT inhibitors with high activity, high selectivity, and good drug-like properties in the future.

KDM1A, a potent and selective target, for the treatment of DNMT3A-deficient non-small cell lung cancer.

BACKGROUND: DNMT3A is a crucial epigenetic regulation enzyme. However, due to its heterogeneous nature and frequent mutation in various cancers, the role of DNMT3A remains controversial. Here, we determine the role of DNMT3A in non-small cell lung cancer (NSCLC) to identify potential treatment strategies. METHODS: To investigate the role of loss-of-function mutations of DNMT3A in NSCLC, CRISPR/Cas9 was used to induce DNMT3A-inactivating mutations. Epigenetic inhibitor library was screened to find the synthetic lethal partner of DNMT3A. Both pharmacological inhibitors and gene manipulation were used to evaluate the synthetic lethal efficacy of DNMT3A/KDM1A in vitro and in vivo. Lastly, MS-PCR, ChIP-qPCR, dual luciferase reporter gene assay and clinical sample analysis were applied to elucidate the regulation mechanism of synthetic lethal interaction. RESULTS: We identified DNMT3A is a tumour suppressor gene in NSCLC and KDM1A as a synthetic lethal partner of DNMT3A deletion. Both chemical KDM1A inhibitors and gene manipulation can selectively reduce the viability of DNMT3A-KO cells through inducing cell apoptosis in vitro and in vivo. We clarified that the synthetic lethality is not only limited to the death mode, but also involved into tumour metastasis. Mechanistically, DNMT3A deficiency induces KDM1A upregulation through reducing the methylation status of the KDM1A promoter and analysis of clinical samples indicated that DNMT3A expression was negatively correlated with KDM1A level. CONCLUSION: Our results provide new insight into the role of DNMT3A in NSCLC and elucidate the mechanism of synthetic lethal interaction between KDM1A and DNMT3A, which might represent a promising approach for treating patients with DNMT3A-deficient tumours.

Non-nucleoside inhibitors of DNMT1 and DNMT3 for targeted cancer therapy.

DNA methylation can deactivate tumor suppressor genes thus causing cancers. Two DNA methylation inhibitors have been approved by the Food and Drug Administration (FDA) and have entered clinical use. However, these inhibitors are nucleoside analogues that can be incorporated into DNA or RNA and induce significant side effects. DNMT1 and DNMT3 are key enzymes involved in DNA methylation. In the acute myeloid leukemia model, a non-nucleoside DNMT1-specific inhibitor has shown lower toxicity and improved pharmacokinetics compared to traditional nucleoside drugs. DNMT3 is also implicated in certain specific cancers. Thus, developing non-nucleoside inhibitors for DNMT1 or DNMT3 can help in understanding their roles in carcinogenesis and provide targeted treatment options in certain cancers. Although no non-nucleoside inhibitors have yet entered clinical trials, in this review, we focus on DNMT1 or DNMT3 selective inhibitors. For DNMT1 selective inhibitors, we have compiled information on the repurposed drugs, derivative compounds and selective inhibitors identified through virtual screening. Additionally, we have outlined potential targets for DNMT1, including protein-protein complex, RNA mimics and aptamers. Compared to DNMT1, research on DNMT3-specific inhibitors has been less extensive. In this context, our exploration has identified a limited number of molecular inhibitors, and we have proposed specific long non-coding RNAs (lncRNAs) as potential contributors to the selective inhibition of DNMT3. This collective effort aims to offer valuable insights into the development of non-nucleoside inhibitors that selectively target DNMT1 or DNMT3.

Prediction of the binding mechanism of a selective DNA methyltransferase 3A inhibitor by molecular simulation.

DNA methylation is an epigenetic mechanism that introduces a methyl group at the C5 position of cytosine. This reaction is catalyzed by DNA methyltransferases (DNMTs) and is essential for the regulation of gene transcription. The DNMT1 and DNMT3A or -3B family proteins are known targets for the inhibition of DNA hypermethylation in cancer cells. A selective non-nucleoside DNMT3A inhibitor was developed that mimics S-adenosyl-l-methionine and deoxycytidine; however, the mechanism of selectivity is unclear because the inhibitor-protein complex structure determination is absent. Therefore, we performed docking and molecular dynamics simulations to predict the structure of the complex formed by the association between DNMT3A and the selective inhibitor. Our simulations, binding free energy decomposition analysis, structural isoform comparison, and residue scanning showed that Arg688 of DNMT3A is involved in the interaction with this inhibitor, as evidenced by its significant contribution to the binding free energy. The presence of Asn1192 at the corresponding residues in DNMT1 results in a loss of affinity for the inhibitor, suggesting that the interactions mediated by Arg688 in DNMT3A are essential for selectivity. Our findings can be applied in the design of DNMT-selective inhibitors and methylation-specific drug optimization procedures.

Dual inhibitors of DNMT and HDAC remodels the immune microenvironment of colorectal cancer and enhances the efficacy of anti-PD-L1 therapy.

Colorectal cancer is the second most prevalent and deadly cancer worldwide. The emergence of immune checkpoint therapy has provided a revolutionary strategy for the treatment of solid tumors. However, less than 5 % of colorectal cancer patients respond to immune checkpoint therapy. Thus, it is of great scientific significance to develop "potentiators" for immune checkpoint therapy. In this study, we found that knocking down different DNMT and HDAC isoforms could increase the expression of IFNs in colorectal cancer cells, which can enhance the effectiveness of immune checkpoint therapy. Therefore, the combined inhibition of DNMT and HDAC cloud synergistically enhance the effect of immunotherapy. We found that dual DNMT and HDAC inhibitors C02S could inhibit tumor growth in immunocompetent mice but not in immunocompromised nude mice, which indicates that C02S exerts its antitumor effects through the immune system. Mechanistically, C02S could increase the expression of ERVs, which generated the intracellular levels of dsRNA in tumor cells, and then promotes the expression of IFNs through the RIG-I/MDA5-MAVS signaling pathway. Moreover, C02S increased the immune infiltration of DCs and T cells in microenvironment, and enhanced the efficacy of anti-PD-L1 therapy in MC38 and CT26 mice model. These results confirmed that C02S can activate IFNs through the RIG-I/MDA5-MAVS signaling pathway, remodel the tumor immune microenvironment and enhance the efficacy of immune checkpoint therapy, which provides new evidence and solutions for the development of "potentiator" for colorectal cancer immunotherapy.

Biological function molecular pathways and druggability of DNMT2/TRDMT1.

5-methylcytosine (m5C) is among the most common epigenetic modification in DNA and RNA molecules, and plays an important role in the animal development and disease pathogenesis. Interestingly, unlike other m5C DNA methyltransferases (DNMTs), DNMT2/TRDMT1 has the double-substrate specificity and adopts a DNMT-similar catalytic mechanism to methylate RNA. Moreover, it is widely involved in a variety of physiological regulatory processes, such as the gene expression, precise protein synthesis, immune response, and disease occurrence. Thus, comprehending the epigenetic mechanism and function of DNMT2/TRDMT1 will probably provide new strategies to treat some refractory diseases. Here, we discuss recent studies on the spatiotemporal expression pattern and post-translational modifications of DNMT2/TRDMT1, and summarize the research advances in substrate characteristics, catalytic recognition mechanism, DNMT2/TRDMT1-related genes or proteins, pharmacological application, and inhibitor development. This review will shed light on the pharmacological design by targeting DNMT2/TRDMT1 to treat parasitic, viral and oncologic diseases.

XPO1 inhibition displays anti-leukemia efficacy against DNMT3A-mutant acute myeloid leukemia via downregulating glutathione pathway.

Acute myeloid leukemia (AML) patients with DNA methyltransferase 3A (DNMT3A) mutation display poor prognosis, and targeted therapy is not available currently. Our previous study identified increased expression of Exportin1 (XPO1) in DNMT3AR882H AML patients. Therefore, we further investigated the therapeutic effect of XPO1 inhibition on DNMT3AR882H AML. Three types of DNMT3AR882H AML cell lines were generated, and XPO1 was significantly upregulated in all DNMT3AR882H cells compared with the wild-type (WT) cells. The XPO1 inhibitor selinexor displayed higher potential in the inhibition of proliferation, promotion of apoptosis, and blockage of the cell cycle in DNMT3AR882H cells than WT cells. Selinexor also significantly inhibited the proliferation of subcutaneous tumors in DNMT3AR882H AML model mice. Primary cells with DNMT3A mutations were more sensitive to selinexor in chemotherapy-naive AML patients. RNA sequencing of selinexor treated AML cells revealed that the majority of metabolic pathways were downregulated after selinexor treatment, with the most significant change in the glutathione metabolic pathway. Glutathione inhibitor L-Buthionine-(S, R)-sulfoximine (BSO) significantly enhanced the apoptosis-inducing effect of selinexor in DNMT3AWT/DNMT3AR882H AML cells. In conclusion, our work reveals that selinexor displays anti-leukemia efficacy against DNMT3AR882H AML via downregulating glutathione pathway. Combination of selinexor and BSO provides novel therapeutic strategy for AML treatment.

DNMT1 inhibition by pUG-fold quadruplex RNA.

Aberrant DNA methylation is one of the earliest hallmarks of cancer. DNMT1 is responsible for methylating newly replicated DNA, but the precise regulation of DNMT1 to ensure faithful DNA methylation remains poorly understood. A link between RNA and chromatin-associated proteins has recently emerged, and several studies have shown that DNMT1 can be regulated by a variety of RNAs. In this study, we have confirmed that human DNMT1 indeed interacts with multiple RNAs, including its own nuclear mRNA. Unexpectedly, we found that DNMT1 exhibits a strong and specific affinity for GU-rich RNAs that form a pUG-fold, a noncanonical G-quadruplex. We find that pUG-fold-capable RNAs inhibit DNMT1 activity by inhibiting binding of hemimethylated DNA, and we additionally provide evidence for multiple RNA binding modes with DNMT1. Together, our data indicate that a human chromatin-associated protein binds to and is regulated by pUG-fold RNA.

Inhibition of DNA methyltransferase aberrations reinstates antioxidant aging suppressors and ameliorates renal aging.

DNA methylation alterations play mechanistic roles in aging; however, the epigenetic regulators/mediators causally involved in renal aging remain elusive. Here, we report that natural and D-galactose (D-gal)-induced aging kidneys display marked suppression of antiaging factor NRF2 (nuclear factor erythroid-derived 2-like 2) and KLOTHO, accompanied by upregulations of DNA methyltransferase (DNMT) 1/3a/3b and NRF2/KLOTHO gene promoter hypermethylations. Administration of a DNMT inhibitor SGI-1072 effectively hypomethylated the promoters, derepressed NRF2/KLOTHO, and mitigated the structural and functional alterations of renal aging in D-gal mice. Moreover, oleuropein (OLP), an olive-derived polyphenol, also displayed similar epigenetic modulation and antiaging effects. OLP inhibited the epigenetic NRF2/KLOTHO suppressions in a gain of DNMT-sensitive manner in cultured renal cells, demonstrating a strong DNA-demethylating capacity. In NRF2 knockout and KLOTHO knockdown D-gal mice, OLP exhibited reduced antiaging effects with KLOTHO displaying a prominent gene effect and effect size; consistently in KLOTHO knockdown mice, the antiaging effects of SGI-1027 were largely abrogated. Therefore, the KLOTHO recovery is critical for the antiaging effects of DNA demethylation. Collectively, our data indicate that aberrant DNMT1/3a/3b elevations and the resultant suppression of antiaging factors contribute significantly to epigenetic renal aging, which might be targeted for epigenetic intervention by synthetic or natural DNA-demethylating agents.

Methylation Status of the miR-141-3p Promoter Regulates miR-141-3p Expression, Inflammasome Formation, and the Invasiveness of HTR-8/SVneo Cells.

MicroRNA-141 (miR-141-3p) is upregulated in preeclampsia. This study investigated the effect of methylation of the miR-141-3p promoter on cell viability, invasion capability, and inflammasomes in vitro. The expression of miR-141-3p and methylation status of the miR-141-3p promoter were examined by RT-qPCR and pyrosequencing in villus tissues of women with spontaneous delivery (VTsd), villus tissues of women with preeclampsia (VTpe), and also in HTR-8/SVneo cells treated with a miR-141-3p inhibitor and 20 µmol/L 5-aza-2'-deoxycytidine (5-Aza), a DNA methyltransferase inhibitor. Cell viability and invasion were evaluated by CCK-8 and transwell assays. In addition, the levels of CXCL12, CXCR4, CXCR2, MMPs, NLRP3, and ASC expression were assessed by western blotting, and IL-1ß and IL-18 concentrations were assayed by ELISA. miR-141-3p expression was upregulated, and the levels of miR-141-3p promoter methylation and CXCL12, CXCR4, and CXCR2 expression were decreased in VTpe relative to VTsd. In HTR-8/SVneo cells, hypomethylation caused by 5-Aza treatment increased miR-141-3p expression, while DNA methyltransferase 3 (DNMT3) transfection decreased miR-141-3p expression. miRNA-141-3p induced NLRP3, IL-1ß, and IL-18 production, decreased CXCR4, MMP, and MMP2 production, and suppressed cell growth and invasion. Furthermore, we observed that NLRP3 plays an important mediatory role in the effects of miR-141-3p described above. Decreased methylation of the miR-141-3p promoter increases miR-141-3p expression, which in turn increases NLRP3 expression, resulting in higher IL-1ß and IL-18 levels and lower levels of MMP2/9 and CXCR4. We conclude that modification of the miR-141-3p promoter might be a curial mediator in preeclampsia.

SPOP mutation induces DNA methylation via stabilizing GLP/G9a.

Mutations in SPOP E3 ligase gene are reportedly associated with genome-wide DNA hypermethylation in prostate cancer (PCa) although the underlying mechanisms remain elusive. Here, we demonstrate that SPOP binds and promotes polyubiquitination and degradation of histone methyltransferase and DNMT interactor GLP. SPOP mutation induces stabilization of GLP and its partner protein G9a and aberrant upregulation of global DNA hypermethylation in cultured PCa cells and primary PCa specimens. Genome-wide DNA methylome analysis shows that a subset of tumor suppressor genes (TSGs) including FOXO3, GATA5, and NDRG1, are hypermethylated and downregulated in SPOP-mutated PCa cells. DNA methylation inhibitor 5-azacytidine effectively reverses expression of the TSGs examined, inhibits SPOP-mutated PCa cell growth in vitro and in mice, and enhances docetaxel anti-cancer efficacy. Our findings reveal the GLP/G9a-DNMT module as a mediator of DNA hypermethylation in SPOP-mutated PCa. They suggest that SPOP mutation could be a biomarker for effective treatment of PCa with DNA methylation inhibitor alone or in combination with taxane chemotherapeutics.

TCF/LEF regulation of the topologically associated domain ADI promotes mESCs to exit the pluripotent ground state.

Mouse embryonic stem cells (mESCs) can be maintained in vitro in defined N2B27 medium supplemented with two chemical inhibitors for GSK3 and MEK (2i) and the cytokine leukemia inhibitory factor (LIF), which act synergistically to promote self-renewal and pluripotency. Here, we find that genetic deletion of the four genes encoding the TCF/LEF transcription factors confers mESCs with the ability to self-renew in N2B27 medium alone. TCF/LEF quadruple knockout (qKO) mESCs display dysregulation of several genes, including Aire, Dnmt3l, and IcosL, located adjacent to each other within a topologically associated domain (TAD). Aire, Dnmt3l, and IcosL appear to be regulated by TCF/LEF in a ß-catenin independent manner. Moreover, downregulation of Aire and Dnmt3l in wild-type mESCs mimics the loss of TCF/LEF and increases mESC survival in the absence of 2iL. Hence, this study identifies TCF/LEF effectors that mediate exit from the pluripotent state.

Combined effects of cigarette smoking, DNA methyltransferase 3B genetic polymorphism, and DNA damage on lung cancer.

BACKGROUND: Smoking increases DNA methylation and DNA damage, and DNA damage acts as a vital cause of tumor development. The DNA methyltransferase 3B (DNMT3B) enhances promoter activity and methylation of tumor suppressor genes. Tea polyphenols may inhibit DNMT activity. We designed a case-control study to evaluate the combined effects of smoking, green tea consumption, DNMT3B - 149 polymorphism, and DNA damage on lung cancer occurrence. METHODS: Questionnaires were administered to obtain demographic characteristics, life styles, and family histories of lung cancer from 190 primary lung cancer cases and 380 healthy controls. Genotypes and cellular DNA damage were determined by polymerase chain reaction and comet assay, respectively. RESULTS: The mean DNA tail moment for lung cancer cases was significantly higher than that for healthy controls. Compared to nonsmokers carrying the DNMT3B - 149 CT genotype, smokers carrying the TT genotype had a greater lung cancer risk (odds ratio [OR]: 2.83, 95% confidence interval [CI]: 1.62-4.93). DNA damage levels were divided by the tertile of the healthy controls' values. Compared to nonsmokers with low DNA damage, smokers with moderate DNA damage (OR: 2.37, 95% CI: 1.54-3.63) and smokers with high DNA damage (OR: 3.97, 95% CI: 2.63-5.98) had elevated lung cancer risks. Interaction between smoking and DNA damage significantly affected lung cancer risk. CONCLUSIONS: Our study suggested that the DNMT3B - 149 TT genotype, which has higher promoter activity, can increase the lung cancer risk elicited by smoking, and DNA damage may further promote smoking related lung cancer development.

Contribution of Regulatory T Cell Methylation Modifications to the Pathogenesis of Allergic Airway Diseases.

Regulatory T (Treg) cells are a subtype of CD4+ T cells that play a significant role in the protection from autoimmunity and the maintenance of immune tolerance via immune regulation. Epigenetic modifications of Treg cells (i.e., cytosine methylation at the promoter region of the transcription factor, Forkhead Box P3) have been found to be closely associated with allergic diseases, including allergic rhinitis, asthma, and food allergies. In this study, we highlighted the recent evidence on the contribution of epigenetic modifications in Treg cells to the pathogenesis of allergic diseases. Moreover, we also discussed directions for future clinical treatment approaches, with a particular emphasis on Treg cell-targeted therapies for allergic disorders.

Recent Clinical Update of Acute Myeloid Leukemia: Focus on Epigenetic Therapies.

Acute myeloid leukemia (AML) is a complicated disease characterized by genetic heterogeneity and simultaneous alterations in multiple genes. For decades, its only curative method has been intensive induction chemotherapy with or without allogeneic hematopoietic stem cell transplantation, and this approach cannot be applied to elderly patients, who make up more than 50% of AML patients. Recent advances in genomics facilitated the elucidation of various mutations related to AML, and the most frequent mutations were discovered in epigenetic regulators. Alterations to epigenetic modifications that are essential for normal cell biology, including DNA methylation and histone acetylation, have been identified. As epigenetic dysregulation is an important carcinogenic mechanism and some epigenetic changes are reversible, these epigenetic alterations have become targets for novel drug development against AML. This review summarizes the recent advances in epigenetic therapies for AML and discusses future research directions.

Epigenetic modulation of immune synaptic-cytoskeletal networks potentiates γδ T cell-mediated cytotoxicity in lung cancer.

γδ T cells are a distinct subgroup of T cells that bridge the innate and adaptive immune system and can attack cancer cells in an MHC-unrestricted manner. Trials of adoptive γδ T cell transfer in solid tumors have had limited success. Here, we show that DNA methyltransferase inhibitors (DNMTis) upregulate surface molecules on cancer cells related to γδ T cell activation using quantitative surface proteomics. DNMTi treatment of human lung cancer potentiates tumor lysis by ex vivo-expanded Vδ1-enriched γδ T cells. Mechanistically, DNMTi enhances immune synapse formation and mediates cytoskeletal reorganization via coordinated alterations of DNA methylation and chromatin accessibility. Genetic depletion of adhesion molecules or pharmacological inhibition of actin polymerization abolishes the potentiating effect of DNMTi. Clinically, the DNMTi-associated cytoskeleton signature stratifies lung cancer patients prognostically. These results support a combinatorial strategy of DNMTis and γδ T cell-based immunotherapy in lung cancer management.

Targeting Ovarian Cancer Stem Cells by Dual Inhibition of HOTAIR and DNA Methylation.

Ovarian cancer is a chemoresponsive tumor with very high initial response rates to standard therapy consisting of platinum/paclitaxel. However, most women eventually develop recurrence, which rapidly evolves into chemoresistant disease. Persistence of ovarian cancer stem cells (OCSCs) at the end of therapy has been shown to contribute to resistant tumors. In this study, we demonstrate that the long noncoding RNA HOTAIR is overexpressed in HGSOC cell lines. Furthermore, HOTAIR expression was upregulated in OCSCs compared with non-CSC, ectopic overexpression of HOTAIR enriched the ALDH+ cell population and HOTAIR overexpression increased spheroid formation and colony-forming ability. Targeting HOTAIR using peptide nucleic acid-PNA3, which acts by disrupting the interaction between HOTAIR and EZH2, in combination with a DNMT inhibitor inhibited OCSC spheroid formation and decreased the percentage of ALDH+ cells. Disrupting HOTAIR-EZH2 with PNA3 in combination with the DNMTi on the ability of OCSCs to initiate tumors in vivo as xenografts was examined. HGSOC OVCAR3 cells were treated with PNA3 in vitro and then implanted in nude mice. Tumor growth, initiation, and stem cell frequency were inhibited. Collectively, these results demonstrate that blocking HOTAIR-EZH2 interaction combined with inhibiting DNA methylation is a potential approach to eradicate OCSCs and block disease recurrence.

A novel class of selective non-nucleoside inhibitors of human DNA methyltransferase 3A.

Screening of a small chemical library (Medicines for Malaria Venture Pathogen Box) identified two structurally related pyrazolone (inhibitor 1) and pyridazine (inhibitor 2) DNMT3A inhibitors with low micromolar inhibition constants. The uncompetitive and mixed type inhibition patterns with DNA and AdoMet suggest these molecules act through an allosteric mechanism, and thus are unlikely to bind to the enzyme's active site. Unlike the clinically used mechanism based DNMT inhibitors such as decitabine or azacitidine that act via the enzyme active site, the inhibitors described here could lead to the development of more selective drugs. Both inhibitors show promising selectivity for DNMT3A in comparison to DNMT1 and bacterial DNA cytosine methyltransferases. With further study, this could form the basis of preferential targeting of de novo DNA methylation over maintenance DNA methylation.

Targeting aberrant DNA methylation in mesenchymal stromal cells as a treatment for myeloma bone disease.

Multiple myeloma (MM) progression and myeloma-associated bone disease (MBD) are highly dependent on bone marrow mesenchymal stromal cells (MSCs). MM-MSCs exhibit abnormal transcriptomes, suggesting the involvement of epigenetic mechanisms governing their tumor-promoting functions and prolonged osteoblast suppression. Here, we identify widespread DNA methylation alterations of bone marrow-isolated MSCs from distinct MM stages, particularly in Homeobox genes involved in osteogenic differentiation that associate with their aberrant expression. Moreover, these DNA methylation changes are recapitulated in vitro by exposing MSCs from healthy individuals to MM cells. Pharmacological targeting of DNMTs and G9a with dual inhibitor CM-272 reverts the expression of hypermethylated osteogenic regulators and promotes osteoblast differentiation of myeloma MSCs. Most importantly, CM-272 treatment prevents tumor-associated bone loss and reduces tumor burden in a murine myeloma model. Our results demonstrate that epigenetic aberrancies mediate the impairment of bone formation in MM, and its targeting by CM-272 is able to reverse MBD.

Epigenetic Blockade of Hippocampal SOD2 Via DNMT3b-Mediated DNA Methylation: Implications in Mild Traumatic Brain Injury-Induced Persistent Oxidative Damage.

The recurrent events of mild trauma exacerbate the vulnerability for post-traumatic stress disorder; however, the underlying molecular mechanisms are scarcely known. The repeated mild traumatic brain injury (rMTBI) perturbs redox homeostasis which is primarily managed by superoxide dismutase 2 (SOD2). The current study investigates the role of DNA methylation in SOD2 gene regulation and its involvement in rMTBI-induced persistent neuropathology inflicted by weight drop injury paradigm. The oxidative damage, neurodegenerative indicators, and SOD2 function and its regulation in the hippocampus were analyzed after 48 h and 30 days of rMTBI. The temporal and episodic increase in ROS levels (oxidative stress) heightened 8-hydroxyguanosine levels indicating oxidative damage after rMTBI that was concomitant with decline in SOD2 function. In parallel, occupancy of DNMT3b at SOD2 promoter was higher post 30 days of the first episode of rMTBI causing hypermethylation at SOD2 promoter. This epigenetic silencing of SOD2 promoter was sustained after the second episode of rMTBI causing permanent blockade in SOD2 response. The resultant oxidative stress further culminated into the increasing number of degenerating neurons. The treatment with 5-azacytidine, a pan DNMT inhibitor, normalized DNA methylation levels and revived SOD2 function after the second episode of rMTBI. The release of blockade in SOD2 expression by DNMT inhibition also normalized the post-traumatic oxidative consequences and relieved the neurodegeneration and deficits in learning and memory as measured by novel object recognition test. In conclusion, DNMT3b-mediated DNA methylation plays a critical role in SOD2 gene regulation in the hippocampus, and the perturbations therein post rMTBI are detrimental to redox homeostasis manifesting into neurological consequences.