Hypomethylating Agents Research Articles

Emerging applications of hypomethylating agents in the treatment of glioblastoma (Review).

DNA hypomethylating agents (HMAs) such as decitabine and 5-azacytidine have established roles in the treatment paradigms for myelodysplastic syndrome and acute myelogenous leukemia, where they are considered to exert their anticancer effects by restoring the expression of tumor suppressor genes. Due to their relatively favorable adverse effect profile and known ability to pass through the blood-brain barrier, applications in the treatment of glioblastoma (GBM) and other central nervous system malignancies are under active investigation. The present review examines the types of HMAs currently available, their known and less-understood antineoplastic mechanisms, and the evidence to date of their preclinical and clinical efficacy in glioblastoma and other solid malignancies. The present review discusses the potential synergies HMAs may have with established and emerging GBM treatments, including temozolomide, immune checkpoint inhibitors and cancer vaccines. Recent successes and setbacks in clinical trials for newly diagnosed and recurrent GBM are summarized in order to highlight opportunities for HMAs to improve therapeutic responses. Challenges for future clinical trials are also assessed.

Downregulation of carnitine acetyltransferase by promoter hypermethylation regulates ultraviolet-induced matrix metalloproteinase-1 expression in human dermal fibroblasts

BackgroundOverexposure to ultraviolet (UV) radiation accelerates skin aging, resulting in wrinkle formation, reduced skin elasticity, and hyperpigmentation. UV irradiation induces increased matrix metalloproteinases (MMPs) that degrade collagen in the extracellular matrix. Skin aging is also accompanied by epigenetic alterations such as promoter methylation by DNA methyltransferases, leading to the activation or suppression of gene expression. Although carnitine acetyltransferase (CRAT) is implicated in aging, the effect of UV on the expression of CRAT and regulatory mechanisms of UV-induced MMP-1 expression remain unknown. ObjectiveWe investigated changes in CRAT expression upon UV irradiation and its effect on MMP-1 expression. MethodsPrimary human dermal fibroblasts were UV irradiated with either control or 5-AZA-dC. CRAT knockdown or overexpression was performed to investigate its effect on MMP-1 expression. The mRNA level was analyzed by quantitative real-time PCR, and protein level by western blotting. ResultsThe expression of CRAT was decreased in UV-irradiated human skin in vivo and in human dermal fibroblasts in vitro. CRAT was downregulated upon UV irradiation by hypermethylation, and treatment with 5-Aza-2′-deoxycytidine, a DNA methyltransferase inhibitor, reversed UV-induced downregulation of CRAT. CRAT knockdown activated the JNK, ERK, and p38 MAPK signaling pathways, which increased MMP-1 expression. Stable overexpression of CRAT alleviated UV-induced MMP-1 induction. ConclusionCRAT downregulation caused by promoter hypermethylation may play an important role in UV-induced skin aging via upregulation of MMP-1 expression.

AML-700 Prolonged Cytopenias After First-Line Treatment With Venetoclax and Hypomethylating Agent in Newly Diagnosed AML/MDS-IB

AML-700 Prolonged Cytopenias After First-Line Treatment With Venetoclax and Hypomethylating Agent in Newly Diagnosed AML/MDS-IB

Inhibition of TOPORS ubiquitin ligase augments the efficacy of DNA hypomethylating agents through DNMT1 stabilization

DNA hypomethylating agents (HMAs) are used for the treatment of myeloid malignancies, although their therapeutic effects have been unsatisfactory. Here we show that CRISPR-Cas9 screening reveals that knockout of topoisomerase 1-binding arginine/serine-rich protein (TOPORS), which encodes a ubiquitin/SUMO E3 ligase, augments the efficacy of HMAs on myeloid leukemic cells with little effect on normal hematopoiesis, suggesting that TOPORS is involved in resistance to HMAs. HMAs are incorporated into the DNA and trap DNA methyltransferase-1 (DNMT1) to form DNA-DNMT1 crosslinks, which undergo SUMOylation, followed by proteasomal degradation. Persistent crosslinking is cytotoxic. The TOPORS RING finger domain, which mediates ubiquitination, is responsible for HMA resistance. In TOPORS knockout cells, DNMT1 is stabilized by HMA treatment due to inefficient ubiquitination, resulting in the accumulation of unresolved SUMOylated DNMT1. This indicates that TOPORS ubiquitinates SUMOylated DNMT1, thereby promoting the resolution of DNA-DNMT1 crosslinks. Consistently, the ubiquitination inhibitor, TAK-243, and the SUMOylation inhibitor, TAK-981, show synergistic effects with HMAs through DNMT1 stabilization. Our study provides a novel HMA-based therapeutic strategy that interferes with the resolution of DNA-DNMT1 crosslinks.

TOPORS E3 ligase mediates resistance to hypomethylating agent cytotoxicity in acute myeloid leukemia cells

Hypomethylating agents (HMAs) are frontline therapies for Myelodysplastic Neoplasms (MDS) and Acute Myeloid Leukemia (AML). However, acquired resistance and treatment failure are commonplace. To address this, we perform a genome-wide CRISPR-Cas9 screen in a human MDS-derived cell line, MDS-L, and identify TOPORS as a loss-of-function target that synergizes with HMAs, reducing leukemic burden and improving survival in xenograft models. We demonstrate that depletion of TOPORS mediates sensitivity to HMAs by predisposing leukemic blasts to an impaired DNA damage response (DDR) accompanied by an accumulation of SUMOylated DNMT1 in HMA-treated TOPORS-depleted cells. The combination of HMAs with targeting of TOPORS does not impair healthy hematopoiesis. While inhibitors of TOPORS are unavailable, we show that inhibition of protein SUMOylation with TAK-981 partially phenocopies HMA-sensitivity and DDR impairment. Overall, our data suggest that the combination of HMAs with inhibition of SUMOylation or TOPORS is a rational treatment option for High-Risk MDS (HR-MDS) or AML.

Intensive chemotherapy after hypomethylating agent and venetoclax in adult acute myeloid leukemia

AbstractThe combination of a hypomethylating agent (HMA) and venetoclax (VEN) is approved for adults aged >75 years with newly diagnosed acute myeloid leukemia (AML) as well as those ineligible for intensive chemotherapy (IC). HMA/VEN is increasingly substituted for IC in adults with AML aged <75 years, particularly in those with adverse cytogenetic and molecular features. When patients fail to respond or relapse after HMA/VEN, the utility of salvage IC is largely unknown. We performed a retrospective single-institution study and identified 46 patients who received IC after HMA/VEN, including 24 patients who received HMA/VEN as their first treatment for AML. This population had complete remission (CR)/CR with incomplete count recovery (CRi)/morphologic leukemia-free state rate of 37%, CR/CRi rate of 28%, and a median overall survival (mOS) of 7.2 months (95% confidence interval, 5.0-10.3). Patients who relapsed after an initial response to HMA/VEN and subsequently received IC were more likely to achieve a CR/CRi than those refractory to HMA/VEN (50% vs 19%; P = .04), although there was no statistically significant difference in survival (mOS, 8.8 vs 5.4 months; P = .64). Age >65 years predicted poorer survival (mOS, 4.3 vs 10.6 months; P < .001). IC after HMA/VEN should be further studied and chosen with caution.

Identification of DNA methylation-regulated WEE1 with potential implications in prognosis and immunotherapy for low-grade glioma.

WEE1 is a critical kinase in the DNA damage response pathway and has been shown to be effective in treating serous uterine cancer. However, its role in gliomas, specifically low-grade glioma (LGG), remains unclear. The impact of DNA methylation on WEE1 expression and its correlation with the immune landscape in gliomas also need further investigation. This study used data from The Cancer Genome Atlas (TCGA), Chinese Glioma Genome Atlas (CGGA), and Gene Expression Omnibus (GEO) and utilized various bioinformatics tools to analyze gene expression, survival, gene correlation, immune score, immune infiltration, genomic alterations, tumor mutation burden, microsatellite instability, clinical characteristics of glioma patients, WEE1 DNA methylation, prognostic analysis, single-cell gene expression distribution in glioma tissue samples, and immunotherapy response prediction based on WEE1 expression. WEE1 was upregulated in LGG and glioblastoma (GBM), but it had a more significant prognostic impact in LGG compared to other cancers. High WEE1 expression was associated with poorer prognosis in LGG, particularly when combined with wild-type IDH. The WEE1 inhibitor MK-1775 effectively inhibited the proliferation and migration of LGG cell lines, which were more sensitive to WEE1 inhibition. DNA methylation negatively regulated WEE1, and high DNA hypermethylation of WEE1 was associated with better prognosis in LGG than in GBM. Combining WEE1 inhibition and DNA methyltransferase inhibition showed a synergistic effect. Additionally, downregulation of WEE1 had favorable predictive value in immunotherapy response. Co-expression network analysis identified key genes involved in WEE1-mediated regulation of immune landscape, differentiation, and metastasis in LGG. Our study shows that WEE1 is a promising indicator for targeted therapy and prognosis evaluation. Notably, significant differences were observed in the role of WEE1 between LGG and GBM. Further investigation into WEE1 inhibition, either in combination with DNA methyltransferase inhibition or immunotherapy, is warranted in the context of LGG.

Prolonged Cytopenias After First-Line Treatment With Venetoclax and Hypomethylating Agent in Newly Diagnosed AML/MDS-IB

Prolonged Cytopenias After First-Line Treatment With Venetoclax and Hypomethylating Agent in Newly Diagnosed AML/MDS-IB

MDS-772 Time Toxicity for Patients Receiving Oral Versus Parenteral Hypomethylating Agents for Myelodysplastic Syndromes/Neoplasms

MDS-772 Time Toxicity for Patients Receiving Oral Versus Parenteral Hypomethylating Agents for Myelodysplastic Syndromes/Neoplasms

Time Toxicity for Patients Receiving Oral Versus Parenteral Hypomethylating Agents for Myelodysplastic Syndromes/Neoplasms

Time Toxicity for Patients Receiving Oral Versus Parenteral Hypomethylating Agents for Myelodysplastic Syndromes/Neoplasms

Identification of epigenetic modifiers essential for growth and survival of AML1/ETO-positive leukemia.

Aberrant gene expression patterns in acute myeloid leukemia (AML) with balanced chromosomal translocations are often associated with dysregulation of epigenetic modifiers. The AML1/ETO (RUNX1/MTG8) fusion protein, caused by the translocation (8;21)(q22;q22), leads to the epigenetic repression of its target genes. We aimed in this work to identify critical epigenetic modifiers, on which AML1/ETO-positive AML cells depend on for proliferation and survival using shRNA library screens and global transcriptomics approaches. Using shRNA library screens, we identified 41 commonly depleted genes in two AML1/ETO-positive cell lines Kasumi-1 and SKNO-1. We validated, genetically and pharmacologically, DNMT1 and ATR using several AML1/ETO-positive and negative cell lines. We also demonstrated in vivo differentiation of myeloblasts after treatment with the DNMT1 inhibitor decitabine in a patient with an AML1/ETO-positive AML. Bioinformatic analysis of global transcriptomics after AML1/ETO induction in 9/14/18-U937 cells identified 973 differentially expressed genes (DEGs). Three genes (PARP2, PRKCD, and SMARCA4) were both downregulated after AML1/ETO induction, and identified in shRNA screens. In conclusion, using unbiased shRNA library screens and global transcriptomics, we have identified several driver epigenetic regulators for proliferation in AML1/ETO-positive AML. DNMT1 and ATR were validated and are susceptible to pharmacological inhibition by small molecules showing promising preclinical and clinical efficacy.

Epigenetics and Control of Tumor Angiogenesis in Melanoma: An Update with Therapeutic Implications.

Angiogenesis, the formation of new blood vessels from pre-existing ones, is a crucial process in the progression and metastasis of melanoma. Recent research has highlighted the significant role of epigenetic modifications in regulating angiogenesis. This review comprehensively examines the current understanding of how epigenetic mechanisms, including DNA methylation, histone modifications, and non-coding RNAs, influence angiogenic pathways in melanoma. DNA methylation, a key epigenetic modification, can silence angiogenesis inhibitors such as thrombospondin-1 and TIMP3 while promoting pro-angiogenic factors like vascular endothelial growth factor (VEGF). Histone modifications, including methylation and acetylation, also play a pivotal role in regulating the expression of angiogenesis-related genes. For instance, the acetylation of histones H3 and H4 is associated with the upregulation of pro-angiogenic genes, whereas histone methylation patterns can either enhance or repress angiogenic signals, depending on the specific histone mark and context. Non-coding RNAs, particularly microRNAs (miRNAs) further modulate angiogenesis. miRNAs, such as miR-210, have been identified as key regulators, with miR-9 promoting angiogenesis by targeting E-cadherin and enhancing the expression of VEGF. This review also discusses the therapeutic potential of targeting epigenetic modifications to inhibit angiogenesis in melanoma. Epigenetic drugs, such as DNA methyltransferase inhibitors (e.g., 5-azacytidine) and histone deacetylase inhibitors (e.g., Vorinostat), have shown promise in preclinical models by reactivating angiogenesis inhibitors and downregulating pro-angiogenic factors. Moreover, the modulation of miRNAs and lncRNAs presents a novel approach for anti-angiogenic therapy.

Determination of global DNA methylation level by methylation-sensitive comet assay in patients with urinary bladder cancer.

DNA methylation is an important mechanism in the regulation of gene expression and maintenance of genomic integrity. Aberrant DNA methylation is an early event in carcinogenesis. DNA methyltransferase inhibitors are used to restore aberrant DNA methylation and inhibit tumor growth. Evaluation of DNA methylation level is important for an effective anti-cancer therapy. In the present study, the determination of global DNA methylation levels in patients with urinary bladder cancer was proposed. The methylation-sensitive comet assay determined the global DNA methylation level at the level of single cells. McrBC enzyme, a methylation-sensitive restriction endonuclease was used for enzymatic digestion to generate additional breaks at methylated sites. % DNA methylation level was significantly higher in patients with bladder cancer compared to the control group. The clinical performance of % DNA methylation analysis by methylation-sensitive comet assay was evaluated by ROC curve. Using the cut-off value of 6,5% DNA methylation, 92% sensitivity, and 42% specificity were obtained. In conclusion, global DNA methylation measured by methylation-sensitive comet assay may be a promising non-invasive biomarker that reduces interventional tests required in the diagnosis and follow-up of urinary bladder cancer.

Prospects for breast cancer immunotherapy using microRNAs and transposable elements as objects.

One of the directions in treatment of chemoresistant breast cancer (BC) may include new methods of activating the immune response against tumor cells. Clinically used checkpoint inhibition using antibodies to PD-1 and PD-L1 works in some patients, but the lack of biomarkers means number of respondents is low. The possibility of combining this method with chemotherapy is limited by an increased risk of toxic liver damage, development of immune-related pneumonitis, and thyroid dysfunction. This article includes introduction into the clinic of new methods of immunotherapy for BC, among which epigenetic activation of retroelements, double-stranded transcripts of which stimulate the interferon response against the tumor, is promising. For this purpose, inhibitors of DNA methyltransferase*, histone deacetylase* and histone methyltransferase* are used (* subtitles in the main text). Their antitumor effect is also mediated by removal of repressive epigenetic marks from tumor suppressor genes. However, numerous studies have proven the role of retroelements in the carcinogenesis of various malignant neoplasms, including BC. Moreover, endogenous retroviruses HERV-K and LINE1 retrotransposons are planned to be used as diagnostic biomarkers for BC. Therefore, a rational approach to using viral mimicry in antitumor therapy of BC may be the simultaneous suppression of specific retrotransposons (drivers for carcinogenesis) using reverse transcriptase inhibitors and silencing of specific transposons involved in carcinogenesis using complementary microRNAs. To determine possible pathways of influence in this direction, 35 specific transposon-derived microRNAs* changes in BC were identified, which can become guides for targeted therapy of BC.

Hypomethylating Agents are Effective in Treatment for Relapsed Myelofibrosis After Allogeneic Hematopoietic Cell Transplantation

Myelofibrosis (MF) is a myeloproliferative neoplasm with a relapse rate of 10% to 30% after allogeneic transplantation (alloHCT). Current recommendations to treat relapse include withdrawal of immunosuppression, donor lymphocyte infusion, and potentially a second alloHCT. Hypomethylating agents (HMAs) have shown efficacy as salvage therapy by inducing an immune response and improving donor chimerism for myeloid neoplasm post-HCT. Data is limited on use of HMAs for MF post-alloHCT relapse. To determine the benefit of using HMAs for MF patients relapsing after alloHCT, we retrospectively analyzed 12 patients with MF post-alloHCT relapse who received HMA to determine response via restoration of donor chimerism and clearance of molecular mutation. The median age was 61 years (range 41-72) with 92% classified as intermediate-2/high-risk by the Dynamic International Prognostic Scoring System (DIPSS) and 83% as high/very high risk by the MIPSS70+ (Molecular International Prognostic Scoring System). The median time to relapse post-alloHCT was 282.5 days (range 96-2388) with median donor chimerism 57.82% (range 2.48-84.0) prior to starting an HMA. After two cycles of HMA, 58% experienced restoration of donor chimerism. Molecular clearance of pre-HCT driver mutations occurred in 50% of patients at the most recent follow-up. New chronic graft-vs.-host disease (cGVHD) occurred in 50% of patients, with most being mild to moderate that resolved after treatment. HMA was safe and effective in a high-risk population after post-alloHCT relapse and is an option for patients in the future.

Clinical outcomes of hypomethylating agents and venetoclax in newly diagnosed unfit and relapsed/refractory paediatric, adolescent and young adult acute myeloid leukaemia patients.

Venetoclax (VEN) combined with hypomethylating agents (HMA) decitabine or azacitidine is used for adult acute myeloid leukaemia (AML), but its application in paediatric, adolescent and young adult (AYA) AML lacks prospective studies. We performed a retrospective chart review of paediatric and AYA AML patients treated with HMA + VEN at Cincinnati Children's Hospital Medical Centre. Twenty-seven patients received 30 HMA + VEN treatment courses for relapsed/refractory (R/R, n = 21) or newly diagnosed (n = 9) AML due to ineligibility for intensive chemotherapy. The R/R cohort had high-risk cytomolecular genetic alterations and prior extensive treatments, with 50% (n = 9) of relapse patients (n = 18) having undergone haematopoietic stem cell transplantation (HSCT). Venetoclax treatment using the 400 mg adult exposure-equivelant dosing (AED) had a median duration of 21 days (range 7-30 days). Grade 3-4 toxicities included neutropenia (90%), anaemia (64%), thrombocytopenia (64%) and febrile neutropenia (44%). The overall complete remission (CR)/CR with incomplete blood count recovery (CRi) rate was 73% (77% minimal residual disease [MRD] negativity <0.1%), with 60% undergoing HSCT. Among newly diagnosed patients (n = 9), 89% achieved CR/CRi (78% MRD negativity) and 78% proceeded to HSCT. The R/R cohort (n = 21) showed a 67% CR/CRi rate (71% MRD negativity), with 52% undergoing HSCT. These findings support the safety and efficacy of HMA + VEN in paediatric/AYA AML, indicating it as a viable option for patients unfit for intensive chemotherapy. Further studies are necessary to determine optimal venetoclax dosing, chemotherapy combinations and pharmacokinetics in this population.

Epigenetic Targeting of Heparan Sulfate 3-O- and 6-O-Sulfation in Breast Cancer Cells: Prospects for Attenuating Prothrombotic Tumor Cell Activities.

The deregulation of cell surface heparan sulfate proteoglycans (HSPGs) is a main issue of cancer cells for increasing their malignancy. In these terms, the sulfation pattern of HS, created by an orchestrated activity of enzymes balancing a site-specific sulfation, is of key importance. These enzymes are often deregulated by epigenetic processes in cancer, e.g., being silenced by DNA hypermethylation. Here, we address this issue in human breast cancer cell lines aiming to target epigenetic processes to reactivate HS sulfation, shifting HS into an antithrombotic phenotype for which 3-O-sulfation is particularly important. Treatment of MCF-7 and MDA-MB-231 cells with nontoxic concentrations of 5-azacytidine (azacytidine) and 5-fluoro-2'-deoxycytidine (FdCyd) as DNMT inhibitors or vorinostat for targeting HDAC increased HS3-O-sulfation remarkably, as confirmed by fluorescence microscopy, by upregulating HS3-O-sulfotransferases, detected by quantitative real-time polymerase chain reaction and Western blot. Flow cytometry and microscopic approaches confirm that upon inhibitor treatment, increased HS3-O-sulfation improves cell binding to antithrombin, leading to an antithrombotic activity. Nevertheless, only azacytidine- and vorinostat-treated cells display anticoagulative properties, represented by attenuated thrombin formation, a lower activation of human platelet aggregation, or ATP release. In contrast, FdCyd additionally upregulated tissue factor expression in both cell lines, overshadowing the anticoagulant effects of HS, leading to an overall prothrombotic phenotype. Our data provide evidence for the first time that targeting epigenetic processes in HS sulfation is a valuable means to foster anticoagulative cell properties for decreasing malignancy and metastatic potency. These data warrant further investigations to fine-tune epigenetic targeting and to search for potential biomarkers attributed to these activities.

Real-World Treatment Patterns and Clinical Outcomes in Unfit AML Patients: Results of Current Retrospective Study in Romania

Aim: The incidence of acute myeloid leukemia (AML) increases along with the aging population and its prognosis worsens with increasing age. Limited data is available on the real-world survival, clinical outcomes, and treatment patterns in Romanian AML patients unfit for intensive chemotherapy. Given the rapidly changing AML treatment landscape, this retrospective chart review aimed to understand historical AML treatment pathways, their associated outcomes, and the economic impact. Material and methods. Adult patients across Romania with primary or secondary AML, ineligible for intensive induction chemotherapy, for whom the first-line systemic therapy (FLST) or best supportive care (BSC) was initiated between January 2015 and December 2018 were evaluated. The outcomes assessed included overall survival (OS), progression-free survival (PFS), response rates and healthcare resource utilization (HRU). Results. Of the 98 patients included, 85.7% received FLST and 14.3% received BSC as first-line therapy. The median OS was 10.0 months in those who received a hypomethylating agent as FLST and 3.4 months in the BSC group. Median PFS was 8.0, 6.4, and 3.4 for hypomethylating agents and low-dose cytarabine as FLST, and BSC, respectively. During the first line therapy the best response was stable disease and was achieved in 20.2% of the FLST patients and 14.3% of the BSC patients. HRU was generally numerically higher in patients with FLST as compared to the ones who received BSC as first-line therapy. Conclusion. The survival and outcomes of unfit AML patients in Romania remain poor highlighting the unmet need for effective treatment options in this population.

Wnt-5a–Receptor Tyrosine Kinase-Like Orphan Receptor 2 Signaling Provokes Metastatic Colonization and Angiogenesis in Renal Cell Carcinoma, and Prunetin Supresses the Axis Activation

Wnt-5a is a protein that is encoded by the WNT5A gene and is a ligand for the ROR2 receptor. However, its biological impact on clear cell renal cell carcinoma (ccRCC) remains unclear. In this study, the prognostic significance of concurrent WNT5A and ROR2 expression levels was observed to predict unfavorable overall survival and disease-specific survival. High Wnt-5a expression was detected in a ccRCC cell line panel but not in HK-2 cells, a normal proximal tubular cell line. Inhibition of DNA methyltransferase by 5-azaC in 786-O and Caki-2 cells resulted in Wnt-5a upregulation, indicating potential epigenetic modification. Furthermore, the results revealed the repression of cell movement in vitro and metastatic colonization in vivo upon WNT5A and ROR2 knockdown. The suppressions of angiogenesis in vivo and tubular-like structure formation in endothelial cells in vitro were also observed after silencing WNT5A and ROR2 expression. In addition, alteration in the downstream gene signature of the Wnt-5a-ROR2 signaling was discovered to be similar to that in MTA1-CTNNB1 axis. Moreover, prunetin treatment was found to reverse the gene signature derived from Wnt-5a-ROR2 signaling activation and to abolish ccRCC cell migration and proliferation. Overall, this study demonstrates the clinical and functional significance of the Wnt-5a-ROR2 axis and identify prunetin as a potential precision medicine for ccRCC patients harboring aberrant Wnt-5a-ROR2 signaling pathways.

Inhibition of DNMT1 attenuates experimental food allergy

BackgroundThe treatment of food allergy (FA) needs improvement. The treatment of immune disorders can be improved by regulating epigenetic marks, which is a promising method. The objective of this research is to alleviate experimental FA by employing an inhibitor of DNA methyltransferase-1 (DNMT1). MethodsOvalbumin was used as the specific antigen to establish a mouse model of FA. Intestinal IL-35+ regulatory B cells (Breg cells) were isolated from FA mice, and characterized using immunological approaches. ResultsFA mice had a lower frequency of IL-35+ Breg cells, which was inversely correlated with their FA response. The quantity of IL-35 was lower in intestinal Breg cells from FA mice. Hypermethylation status was detected in the Il35 promoter, which was accompanied with high levels of H3K9me3. Enforced expression of DNMT1 hindered the promoter activity of the IL35 gene. Administration of an inhibitor of DNMT1 (RG108) restored the immune regulatory capacity of FA intestinal Bregs, and effectively suppressed the expression of DNMT1, and attenuated experimental FA. ConclusionsThe elevated quantity of DNMT1 in intestinal Breg cells compromises the expression of IL-35 and affects the immune regulatory functions, which facilitates the development of FA. The immune regulatory functions of intestinal Breg cells are restored and experimental FA is attenuated by inhibiting DNMT1.