Hypomethylating Agents Research Articles

Acute myeloid leukemia management and research in 2025.

The first 5 decades of research in acute myeloid leukemia (AML) were dominated by the cytarabine plus anthracyclines backbone, with advances in strategies including allogeneic hematopoietic stem cell transplantation, high-dose cytarabine, supportive care measures, and targeted therapies for the subset of patients with acute promyelocytic leukemia. Since 2017, a turning point in AML research, 12 agents have received regulatory approval for AML in the United States: venetoclax (BCL2 inhibitor); gemtuzumab ozogamicin (CD33 antibody-drug conjugate); midostaurin, gilteritinib, and quizartinib (fms-like tyrosine kinase 3 inhibitors); ivosidenib, olutasidenib, and enasidenib (isocitrate dehydrogenase 1 and 2 inhibitors); oral azacitidine (a partially absorbable formulation); CPX351 (liposomal encapsulation of cytarabine:daunorubicin at a molar ratio of 5:1); glasdegib (hedgehog inhibitor); and recently revumenib (menin inhibitor; approved November 2024). Oral decitabine-cedazuridine, which is approved as a bioequivalent alternative to parenteral hypomethylating agents in myelodysplastic syndrome, can be used for the same purpose in AML. Menin inhibitors, CD123 antibody-drug conjugates, and other antibodies targeting CD123, CD33, and other surface markers are showing promising results. Herein, the authors review the frontline and later line therapies in AML and discuss important research directions.

Efficacy and safety of hypomethylating agents in the treatment of AML/MDS patients relapsed post allogenetic hematopoietic stem cell transplantation

IntroductionAcute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) constitute myeloid malignancies, and allogeneic hematopoietic stem cell transplantation (allo-HSCT) is considered as a potentially optimal approach for achieving a long term cure. However, post-allo-HSCT relapse remains a leading cause of mortality and therapeutic failure.MethodsTo evaluate the efficacy and safety of combining hypomethylating agents (HMAs) with Bcl-2 inhibitors in the treatment of AML/MDS relapse following allo-HSCT, we retrospectively collected data from 42 patients who experienced relapse between April 2012 and March 2022 at Peking University First Hospital. Among these patients, 21 underwent intensive chemotherapy (IC) alone, while the other 21 received treatment with HMAs after IC treatment, either alone or in combination with the Bcl-2 inhibitor venetoclax (VEN).ResultsThe median overall survival (OS) was 9 ± 2.153 months, and the one-year OS rate was 41.5%. The overall response rate (ORR) in the chemotherapy group and the IC+HMAs ± VEN group was 52.38% (11/21) and 76.19% (16/21), respectively, with no significant difference found (P=0.107). Kaplan-Meier analysis revealed a significant difference in OS between the chemotherapy group and the IC+HMAs ± VEN group in our retrospective cohort study (P=0.041, χ2= 4.016). Additionally, a significant difference in overall survival (OS) rates was observed between the two groups for patients categorized as intermediate/high risk (P=0.008). The secondary relapse rate was 45.45% (5/11) in the IC cohort and 25% (4/16) in the IC+HMAs ± VEN group, respectively, with no significant difference identified between the two cohorts (P=0.268). Furthermore, upon assessing the risk of graft-versus-host disease (GvHD), infection, and agranulocytosis, no notable differences were observed with the use of HMAs, suggesting that HMAs did not increase the risk. In the IC+HMAs ± VEN group, 7 patients received VEN in addition to HMAs, and no significant statistical difference was found in OS when comparing patients who received HMAs alone and those who received HMA+VEN (P=0.183), also, a statistically significant difference in OS was noted between the two groups whenaccounting for competing risks (P=0.028).ConclusionsThis retrospective study highlights the efficacy of IC+HMAs ± VEN in treating AML/MDS patients experiencing relapse post allo-HSCT, improving survival rates, especially for those classified as intermediate/high risk, with favorable tolerability.

Semi-mechanistic population PK/PD model to aid clinical understanding of myelodysplastic syndromes following treatment with Venetoclax and Azacitidine.

Myelodysplastic syndromes (MDS) represent a group of bone marrow disorders involving cytopenias, hypercellular bone marrow, and dysplastic hematopoietic progenitors. MDS remains a challenge to treat due to the complex interplay between disease-induced and treatment-related cytopenias. Venetoclax, a selective BCL-2 inhibitor, in combination with azacitidine, a hypomethylating agent, is currently being investigated in patients with previously untreated higher-risk MDS. We present an integrated semi-mechanistic pharmacokinetic/pharmacodynamic (PK/PD) model developed using preliminary clinical data from an ongoing Phase 1b study evaluating the safety and efficacy of venetoclax in combination with azacitidine in treatment-naïve patients with higher-risk MDS. Longitudinal data from 57 patients were used to develop the model, which accounted for venetoclax PK and azacitidine treatment to describe time dynamics of bone marrow blasts, neutrophils, red blood cells, and platelets. The proliferation and maturation of progenitor cells in the bone marrow to peripheral cells is described via three parallel connected transit models including feedback terms. The model also accounted for bone marrow crowding and its impact on hematopoiesis. Model validation demonstrated adequate goodness-of-fit, visual and numerical predictive checks. Model predicted complete remission (CR) rates and marrow complete remission (mCR) rates closely matched observed rates in the clinical study, and simulated efficacy (recovery of blast count, CR, and mCR rates) and safety (neutropenia and thrombocytopenia) endpoints aligned with expected outcomes from various dosing regimens. Importantly, the semi-mechanistic model may aid understanding and discriminating between disease-driven and drug-induced cytopenias.

DNA hypermethylation of tumor suppressor genes among oral squamous cell carcinoma patients: a prominent diagnostic biomarker.

Oral Squamous Cell Carcinoma is a globally revealing form of oral malignancy. Epigenetics, which studies genetic modifications in gene expression without altering the sequence of DNA, is crucial for understanding OSCC. Key epigenetic modifications such as histone modifications, DNA methylation, and microRNA regulation play significant roles in Oral carcinoma. Aberrant methylation of DNA of tumor suppressor genes which leads to their inactivation, promoting cancer development, and specific methylation patterns are emerging as biomarkers for early OSCC detection.Current treatments like surgery, radiotherapy, and chemotherapy often fall short, prompting research into epigenetic therapies. Agents like DNMT and HDAC inhibitors demonstrate the potential for reversing aberrant epigenetic patterns, perhaps reactivating silenced TSGs, and suppressing oncogenes. Despite early promise, the development of effective combination medicines and the identification of reliable biomarkers continue to present challenges.In OSCC, resistance to therapy is also influenced by epigenetic processes. Aberrant DNA methylation and changes in histone modifications impact genes involved in medication metabolism and the survival of cells. Enhancing treatment efficacy and overcoming medication resistance may be possible by recognizing and focusing on these processes. This review explores the interplay between epigenetic changes and OSCC, their role in the disease's initiation and progression, and their impact on diagnosis and treatment. It also discusses the potential of epigenetic drugs (epi-drugs) to improve diagnostic precision and treatment outcomes.

Treatment of high risk myelodysplastic syndrome.

The myelodysplastic syndrome (MDS) is considered to be a heterogeneous myeloid malignancy with a common origin in the hematopoietic stem cell compartment, generally divided into lower and higher risk. While treatment goal for lower risk MDS (LR-MDS) is to decrease transfusion burden and transformation into acute leukemia major aim for high risk MDS is to prolong survival and ultimately cure. While novel agents such as luspatercept or imetelstat have recently been approved as new treatment options for LR-MDS, hypomethylating agents (HMA) remain currently the only approved non-transplant option for HR-MDS and is the standard of care for non-transplant-eligible patients. Combinations with other drugs as first-line treatment has to date not proven more efficacious than monotherapy in HR-MDS, and outcome after HMA failure is poor. The only potential cure and standard of care for eligible patients is allogeneic stem cell transplantation (HSCT) and even if the number of transplanted - especially older - MDS patients increased over time due to a better management and donor availability the majority of MDS patients will not be eligible for this curative approach. Current challenges encompass to decrease the relapse risk, the main cause of HSCT failure. This review will summarize current knowledge of options of transplant- and non-transplant treatment approaches for these patients and demonstrate the unmet clinical need for more effective therapies.

Trial eligibility, treatment patterns and outcome for venetoclax-based therapy in AML: a prospective cohort study.

Venetoclax (Ven) plus hypomethylating agents are considered standard-of-care for patients with acute myeloid leukemia (AML) judged ineligible for intensive chemotherapy (IC). Real-world studies complement clinical trials, since patterns of patient selection, treatment-exposure and post-remission management may vary. This prospective observational multi-center study included 209 newly diagnosed IC-ineligible patients with a median age 75 years (interquartile range, 71-81 years). A high proportion of patients had secondary AML (53.7%), adverse-risk disease (35.3%), and complex karyotype (15.5%). At a median follow-up of 22.5 months (range 0.1-43), median overall-survival (mOS) was 11.7 months (95% Confidence Interval [CI] 9.9,15.4). Composite complete remission (CRc) was achieved in 65.2% (CR 44.4%; CRi 20.8%). Of responding patients, 21.1% underwent stem-cell transplantation. When stratified based on VIALE-A original eligibility criteria, mOS was 17.8 months for patients meeting eligibility criteria and 10.7 months for patients who did not (p=0.027). AML ontogeny (p=0.024), reduced kidney function (p=0.001), Charlson co-morbidity index (CCI; p=0.0017), ELN-risk (p=0.01) and body-mass index (p=0.0298) were significantly associated with OS. Multivariant Cox-regression analysis confirmed independent association of OS with AML ontogeny (p=0.012), CCI (p=0.033) and ELN-risk (p=0.019). Patients enrolled in the latter half of the study period demonstrated improved OS compared to those enrolled earlier (p=0.026). This prospective observational study highlights outcomes of patient subgroups, including those excluded from registration trials. (clinicaltrials.gov: #NCT03987958).

DNMT aberration-incurred GPX4 suppression prompts osteoblast ferroptosis and osteoporosis

Osteoporosis (OP) is a common and fracture-prone skeletal disease characterized by deteriorated trabecular microstructure and pathologically involving various forms of regulated bone cell death. However, the exact role, cellular nature and regulatory mechanisms of ferroptosis in OP are not fully understood. Here, we reported that OP femurs from ovariectomized (Ovx) mice exhibited pronounced iron deposition, ferroptosis, and transcriptional suppression of a key anti-ferroptotic factor GPX4 (glutathione peroxidase 4). GPX4 suppression was accompanied by hypermethylation of the Gpx4 promoter and an increase in DNA methyltransferases DNMT1/3a/3b and was transcriptionally promoted by repressive KLF5 and the transcriptional corepressors NCoR and SnoN. Conversely, DNMT inhibition with SGI-1027 reversed promoter hypermethylation, GPX4 suppression and ferroptotic osteoporosis. In cultured primary bone cells, ferric ammonium citrate (FAC) mimicking iron loading similarly induced GPX4 suppression and ferroptosis in osteoblasts but not in osteoclasts, which were rescued by siRNA-mediated individual knockdown of DNMT 1/3a/3b. Intriguingly, SGI-1027 alleviated the ferroptotic changes caused by FAC, but not by a GPX4 inactivator RSL3. More importantly, we generated a strain of osteoblast-specific Gpx4 haplo-deficient mice Gpx4Ob+/− that developed spontaneous and more severe ferroptotic OP alterations after Ovx operation, and showed that GPX4 inactivation by RSL3 or semi-knockout in osteoblasts largely abolished the anti-ferroptotic and osteoprotective effects of SGI-1027. Taken together, our data suggest that GPX4 epigenetic suppression caused by DNMT aberration and the resulting osteoblastic ferroptosis contribute significantly to OP pathogenesis, and that the strategies preserving GPX4 by DNMT intervention are potentially effective to treat OP and related bone disorders.

Asperosaponin VI inhibition of DNMT alleviates GPX4 suppression-mediated osteoblast ferroptosis and diabetic osteoporosis.

Diabetic osteoporosis (DOP) is an insidious complication of diabetes with limited therapeutic options. DOP is pathologically associated with various types of regulated cell death, but the precise role of ferroptosis in the process remains poorly understood. Asperosaponin VI (AVI), known for its clinical efficacy in treating bone fractures and osteoporosis, may exert its osteoprotective effects through mechanisms involving ferroptosis, however this has not been established. This study aimed to investigate the role of AVI in modulating ferroptosis in a mouse model of DOP and to explore the underlying mechanisms. We assessed OP alterations in femurs of DOP-conditioned mice and primary bone cells. We generated a strain of osteoblast-specific Gpx4-deficient mice. A combination of micro-CT, immunohistochemistry, immunofluorescence, methylation-specific PCR (MSP), bisulfite sequencing PCR (BSP), western blotting (WB), and AVI pull-down assays were employed to elucidate the mechanism and therapeutic target of AVI in DOP. Our findings revealed that femurs from DOP-conditioned mice exhibited significant ferroptosis and suppression of the core anti-ferroptosis factor GPX4, mainly due to hypermethylation of the Gpx4 promoter mediated by DNA methyltransferases DNMT1and DNMT3a. Notably, treatment with AVI effectively reversed the hypermethylation, restored GPX4 expression, and reduced ferroptotic pathologies associated with DOP by inhibiting DNMT1/3a. In primary osteoblasts, AVI alleviated GPX4 suppression and reduced ferroptosis in DOP-conditioned primary osteoblasts through a mechanism dependent on DNMT inhibition and GPX4 restoration. Importantly, the anti-ferroptotic and osteoprotective effects of AVI were abolished in osteoblastic Gpx4 haplo-deficient mice (Gpx4Ob-/+) or when GPX4 was pharmacologically inactivated with RSL3. Our study identifies a pivotal epigenetic ferroptotic pathway that contributes significantly to DOP and uncovers a crucial pharmacological property of AVI that is potentially effective in treating patients with DOP and related osteoporotic disorders.

DNA hypomethylation promotes UHRF1-and SUV39H1/H2-dependent crosstalk between H3K18ub and H3K9me3 to reinforce heterochromatin states.

Mono-ubiquitination of lysine 18 on histone H3 (H3K18ub), catalyzed by UHRF1, is a DNMT1 docking site that facilitates replication-coupled DNA methylation maintenance. Its functions beyond this are unknown. Here, we genomically map simultaneous increases in UHRF1-dependent H3K18ub and SUV39H1/H2-dependent H3K9me3 following DNMT1 inhibition. Mechanistically, transient accumulation of hemi-methylated DNA at CpG islands facilitates UHRF1 recruitment and E3 ligase activity toward H3K18. Notably, H3K18ub enhances SUV39H1/H2 methyltransferase activity and, in colon cancer cells, nucleates new H3K9me3 domains at CpG island promoters of DNA methylation-silenced tumor suppressor genes (TSGs). Disrupting UHRF1 enzyme activity prevents H3K9me3 accumulation while promoting PRC2-dependent H3K27me3 as a tertiary layer of gene repression in these regions. By contrast, disrupting H3K18ub-dependent SUV39H1/H2 activity enhances the transcriptional activating and antiproliferative effects of DNMT1 inhibition. Collectively, these findings reveal roles for UHRF1 and H3K18ub in regulating a hierarchy of repressive histone methylation signaling and rationalize a combination strategy for epigenetic cancer therapy.

SAM-DNMT3A, a strategy for induction of genome-wide DNA methylation, identifies DNA methylation as a vulnerability in ER-positive breast cancers.

DNA methylation is an epigenetic mark that plays a critical role in regulating gene expression. DNA methyltransferase (DNMT) inhibitors, inhibit global DNA methylation and have been a key tool in studies of DNA methylation. A major bottleneck is the lack of tools to induce global DNA methylation. Here, we engineered a CRISPR based approach, that we initially designed, to enable site-specific DNA methylation. Using the synergistic activation mediator (SAM) system, we unexpectedly find that regardless of the targeted sequence any sgRNA induces global genome-wide DNA methylation. We term this method SAM-DNMT3A and show that induction of global DNA methylation is a unique vulnerability in ER-positive breast cancer suggesting a therapeutic approach. Our findings highlight the need of caution when using CRISPR based approaches for inducing DNA methylation and demonstrate a method for global induction of DNA methylation.

DNA Hypomethylation Is One of the Epigenetic Mechanisms Involved in Salt-Stress Priming in Soybean Seedlings.

Salt-stress priming enhances the tolerance of plants against subsequent exposure to a similar stress. Priming-induced transcriptomic reprogramming is mediated by multiple epigenetic mechanisms, the best known of which is histone modifications. However, not much is known about other epigenetic responses. In this study, salt-stress priming resulted in global DNA hypomethylation in the leaves of soybean seedlings. The DNA methyltransferase activities in primed seedlings were reduced, contributing to the overall DNA hypomethylation. Genes associated with the hypomethylated DNA regions in primed seedlings also showed a higher mean level of the active histone mark, histone 3 lysine 4 trimethylation (H3K4me3), and a lower mean level of the repressive histone mark, H3K4me2. Transcriptomic analyses supported that DNA hypomethylation played a role in fine-tuning the chromatin status in primed seedlings to potentiate gene expressions. Motif and transcriptional network analyses revealed that DNA hypomethylation may facilitate the responses mediated by key transcription factors in the abscisic acid (ABA)-dependent pathway. A pre-treatment using a DNA methyltransferase inhibitor, 5-azacytidine, could enhance salt tolerance in non-primed soybean seedlings, similar to the priming effect, suggesting the role of DNA hypomethylation in salt-stress priming. Overall, this research furthers our understanding of the epigenetic mechanisms involved in salt-stress priming in soybean.

Mutation- and MRD-informed treatments for transplant-ineligible patients.

The ongoing development of molecularly targeted therapies in addition to the new standard of care combination of azacitidine and venetoclax (AZA-VEN) has transformed the prognostic outlook for older, transplant-ineligible patients with acute myeloid leukemia (AML). While conventional treatments, such as standard anthracycline and cytarabine- based chemotherapy or hypomethylating agent (HMA) monotherapy, are associated with a generally poor prognosis in this patient population, the use of these novel regimens can result in long-lasting, durable remissions in select patient subgroups. Furthermore, the simultaneous discovery of resistance mechanisms to targeted therapies and AZA-VEN has enabled the identification of patient subgroups with inferior outcomes, leading to the development, of new risk-stratification models and clinical investigations incorporating targeted therapies using an HMA-VEN-based platform. Treatments inclusive of IDH1, IDH2, FLT3, and menin inhibitors combined with HMA-VEN have additionally demonstrated safety and high rates of efficacy in early-phase clinical trials, suggesting these regimens may further improve outcomes within select subgroups of patients with AML in the near future. Additional studies defining the prognostic role of measurable residual disease following VEN-based treatment have further advanced prognostication capabilities and increased the ability for close disease monitoring and early targeted intervention prior to morphologic relapse. This review summarizes these recent developments and their impact on the treatment and survival of transplant-ineligible patients living with AML.

Combining azole antifungals with venetoclax plus azacitidine in patients with newly diagnosed acute myeloid leukemia

ABSTRACT The combination of venetoclax (VEN) with hypomethylating agents (HMAs) improves survival in patients with acute myeloid leukemia (AML) and may cause neutropenia requiring combined antifungal therapy or prophylaxis. The inhibition of cytochrome P450 activity by azole antifungal agents leads to elevated blood concentrations of VEN. This study aimed to evaluate the efficacy and safety of venetoclax plus azacitidine (AZA) with azoles in newly diagnosed AML patients. The primary endpoints included complete remission (CR), complete remission with incomplete blood cell recovery (CRi), composite CR (CRc, CR + CRi), blood cell recovery time and incidence of infections. The CRc was 50.0% in the azole group and 56% in the nonazole group (p > 0.05). In the azole group, the median recovery times for patients with ANC >500 cells/mm3 and ANC >1,000 cells/mm3 were 19 and 25 days, respectively. For the nonazole group, the corresponding times were 16 and 19 days (p < 0.05). In the azole group, the median durations for patients with a PLT >50,000/mm3 and >100,000/mm3 were 18 and 20 days, respectively. For the nonazole group, the corresponding times were 16 and 19 days (p > 0.05). The incidences of fungal and bacterial infections were not significantly different (30.8% vs 26.1% and 50.0% vs 56.0%) (p > 0.05). The cost-effectiveness ratio of the azole group is lower. There was no significant difference between VEN + AZA with or without azole in terms of efficacy, infection, or partial hematological toxicity. However, the combination of azoles may prolong the neutrophil recovery time. Azole combination could reduce the amount of venetoclax and improve health economics.

Targeting heterochromatin eliminates chronic myelomonocytic leukemia malignant stem cells through reactivation of retroelements and immune pathways

Chronic myelomonocytic leukemia (CMML) is a severe myeloid malignancy affecting the elderly, for which therapeutic options are limited. DNA hypomethylating agents (HMAs) provide transient responses, failing to eradicate the malignant clone. Hematopoietic stem cell (HSC) aging involves heterochromatin reorganization, evidenced by alterations in histone marks H3K9me2 and H3K9me3. These repressive marks together with DNA methylation are essential for suppressing transposable elements (TEs). In solid cancers, the antitumor efficacy of HMAs involves the derepression of TEs, mimicking a state of viral infection. In this study, we demonstrate a significant disorganization of heterochromatin in CMML HSCs and progenitors (HSPCs) characterized by an increase in the repressive mark H3K9me2, mainly at the level of TEs, and a repression of immune and age-associated transcripts. Combining HMAs with G9A/GLP H3K9me2 methyltransferase inhibitors reactivates these pathways, selectively targeting mutated cells while preserving wild-type HSCs, thus offering new therapeutic avenues for this severe myeloid malignancy.

Activation of Cryptic Secondary Metabolite Biosynthesis in Tobacco BY-2 Suspension Cells by Epigenetic Modifiers.

Cultured plant cells often biosynthesize secondary metabolites to a lesser extent relative to the mother plants. This phenomenon is associated with epigenetic alterations of the biosynthetic gene(s). Here we investigated the effectiveness of epigenetic modifiers, such as inhibitors of histone deacetylase (HDAC) and DNA methyltransferase (DNMT), to activate cryptic secondary metabolite biosynthesis in tobacco (Nicotiana tabacum) BY-2 cells. The BY-2 suspension cells cultured with an HDAC inhibitor, suberoyl bis-hydroxamic acid, exhibited strong biosynthesis of four compounds that were originally present at trace concentrations. The induced compounds were identified as caffeoylputrescine (1), 4-O-β-D-glucopyranosylferulic acid (2), 5-O-caffeoylquinic acid (3), and feruloylputrescine (4). Biosynthetic activation of compounds 1-4 was reproduced by two other HDAC inhibitors. Treatment of the cells with a DNMT inhibitor (zebularine) also activated the biosynthesis of compounds 1-4, but had a limited effectiveness relative to the HDAC inhibitors, indicating that histone acetylation levels are involved more than DNA methylation levels in the epigenetic regulation of the biosynthesis of compounds 1-4 in the BY-2 cells. Following our previous demonstration using cultured cells of a monocotyledonous plant, this study demonstrates the utility of epigenetic modifiers to activate cryptic secondary metabolite biosynthesis in cultured cells of a dicotyledonous plant.

Somatic co‐alteration signatures are prognostic in high‐grade TP53‐mutated myeloid neoplasms

SummaryTo assess the relevance of co‐occurring somatic mutations in TP53‐mutated myeloid neoplasms with ≥10% blasts, we pooled 325 individuals from 10 centres. We focused on comparing three published somatic co‐alteration signatures comprising (1) nine MDS‐related genes (‘ICC‐MDSR’), (2) ICC‐MDSR + additional secondary mutations‐related genes (‘Tazi signature’) and (3) EPI6 (comprising six genes). Outcomes examined were 24‐month overall survival (OS24) and front‐line complete response (CR1). The median age was 69 years with 77% receiving front‐line hypomethylating agents (HMA). All three signatures ICC‐MDSR (p = 0.009), Tazi signature (p = 0.001) and EPI6 (p = 0.025) predicted inferior CR1. In the low‐intensity (HMA) subgroup, only Tazi signature (p = 0.026) predicted inferior CR1. In OS24 analysis of the HMA‐treated subgroup (N = 200), only Tazi signature was adverse (hazard ratio, HR = 1.6 [1.1–2.2]; p = 0.011). However, a forward stepwise multivariable age‐adjusted Cox model including all three signatures picked EPI6 as the sole significant adverse predictor in the entire cohort (p = 0.0001) as well as within the HMA‐treated subgroup (p = 0.0071). These data confirm the value of testing co‐occurring somatic alterations even within a high‐grade TP53‐mutated myeloid neoplasm cohort.

The Impact of Alcohol-Induced Epigenetic Modifications in the Treatment of Alcohol use Disorders.

Alcohol use disorders are responsible for 5.9% of all death annually and 5.1% of the global disease burden. It has been suggested that alcohol abuse can modify gene expression through epigenetic processes, namely DNA and histone methylation, histone acetylation, and microRNA expression. The alcohol influence on epigenetic mechanisms leads to molecular adaptation of a wide number of brain circuits, including the hypothalamus-hypophysis-adrenal axis, the prefrontal cortex, the mesolimbic-dopamine pathways and the endogenous opioid pathways. Epigenetic regulation represents an important level of alcohol-induced molecular adaptation in the brain. It has been demonstrated that acute and chronic alcohol exposure can induce opposite modifications in epigenetic mechanisms: acute alcohol exposure increases histone acetylation, decreases histone methylation and inhibits DNA methyltransferase activity, while chronic alcohol exposure induces hypermethylation of DNA. Some studies investigated the chromatin status during the withdrawal period and the craving period and showed that craving was associated with low methylation status, while the withdrawal period was associated with elevated activity of histone deacetylase and decreased histone acetylation. Given the effects exerted by ethanol consumption on epigenetic mechanisms, chromatin structure modifiers, such as histone deacetylase inhibitors and DNA methyltransferase inhibitors, might represent a new potential strategy to treat alcohol use disorder. Further investigations on molecular modifications induced by ethanol might be helpful to develop new therapies for alcoholism and drug addiction targeting epigenetic processes.

EPCO-08. EPIGENETIC THERAPY POTENTIATES TRANSPOSABLE ELEMENT TRANSCRIPTION TO CREATE TUMOR-ENRICHED ANTIGENS IN GLIOBLASTOMA

Abstract Epigenetic treatment has been studied as a means to augment anti-tumor immune cell activity by increasing the expression of antiviral response genes. Previous studies, including our own, have revealed an additional consequence of epigenetic treatment: reactivation of transposable elements (TEs). Normally, most TEs are epigenetically repressed in healthy somatic tissues. However, epigenetic treatment can reactivate TEs as previously unannotated promoters, particularly in rapidly proliferating cells. These reactivated TEs could generate unique proteins that may act as novel antigens for immunotherapy. To study epigenetic treatment as a novel method to augment immunotherapy in cancers with a low number of somatic mutation-derived antigens, we chose glioblastoma as a model. Specifically, we investigated if drug-induced TE expression increases the antigen repertoire upon combinatorial treatment with DNMT inhibitor (Decitabine) and HDAC inhibitor (Panobinostat), agents currently being tested for clinical use in cancers, including CNS tumors. Using patient-derived primary glioblastoma stem cell (GSC) lines and control cell lines (astrocytes and fibroblasts), we first profiled treatment-induced changes in the epigenetic landscape using WGBS-seq and ATAC-seq. Then, using nanoCAGE-seq, both short-read and long-read RNA-seq, we identified treatment-induced, TE-derived transcripts that are predominantly expressed in glioblastoma cells along with upregulation of antiviral gene programs. Finally, using LC-MS/MS, we validated that tumor-enriched reactivated TEs, which mostly originate from the LTR class, encode HLA-I presented antigens in primary GSCs. Notably, epigenetic therapy also reactivated many TEs in proliferating control cell lines, and thus we provide evidence that targeted approaches like CRISPRa might reduce the side effects of activating nonspecific TE loci. Overall, our findings suggest that the therapeutic utilization of epigenetic treatment-induced, TE-derived antigens holds great promise but also provides a cautionary tale regarding the careful selection of tumor-enriched antigen targets.

TMOD-32. MURINE MODELLING OF THE STING PATHWAY IN GLIOMAS

Abstract The stimulator of interferon genes (STING) pathway is a critical component of innate antitumor immunity through the recognition of pathogenic cytosolic DNA and stimulating the production of type 1 interferons and pro-inflammatory cytokines. We have previously shown that STING signaling is silenced in the neoplastic compartment of the GBM tumor microenvironment, but remains intact in vascular and immune cells. In human patient derived cell lines, such silencing appears to be mediated by hypermethylation of a region of the STING promoter and can be reversed by treatment with both the DNA methyltransferase (DNMT) inhibitor Decitabine and DNMT1-selective inhibitor GSK-3685032. These results suggest that neoplastic cell STING promoter methylation represents a therapeutic vulnerability that can be exploited by epigenetic therapies to inflame the glioma tumor microenvironment. Murine models are critical tools for exploring the translational implications of STING silencing in vivo. Distinct mouse models have been developed to optimally represent specific aspects of human glioma biology. Here we characterize the STING pathway in commonly used syngeneic murine glioma models, including CT2A, GL261, SB28, and SMA560. We show that the expression of STING pathway components varies significantly across these lines. Importantly, unlike in humans, murine STING (mSTING) expression is not suppressed by promoter methylation and does not respond to decitabine treatment. Additionally, the mSTING protein is expressed in neoplastic cells in vivo. These results demonstrate a fundamental difference in epigenetic regulation of STING between human and murine glioma cells, and suggest that murine modeling of STING epigenetics requires the use of patient derived cell lines implanted in immunodeficient mice. Meanwhile, immune competent syngeneic models are appropriate for exploring the tumor immune microenvironment but would require non-epigenetic approaches (e.g. genetic knock-outs/knock-ins) for modulating STING expression. Humanized mice represent a promising emerging approach for faithfully recapitulating both epigenetic control and immune environment of human gliomas.

Enhancing antitumor activity of herceptin in HER2-positive breast cancer cells: a novel DNMT-1 inhibitor approach

HER2 antagonists remain the cornerstone of therapy for patients with HER2-positive breast cancer. This study introduces a novel small-molecule inhibitor of DNA methyltransferase 1 (DNMT-1), referred to as DI-1, designed to synergize with HER2 antagonists in treating HER2-positive breast cancer cells. Clinical data reveal a negative correlation between DNMT-1 expression and PTEN levels, and a positive correlation with the methylation rates of PTEN's promoter. In experiments with SKBR3 and BT474 cells, DI-1 effectively reduced the methylation of PTEN's promoter region, thereby upregulating PTEN expression. This upregulation, in turn, enhanced the cells' sensitivity to HER2 antagonists, indicating that DI-1’s mechanism involves inhibiting DNMT-1’s recruitment to PTEN's promoter region. Consequently, by increasing PTEN expression, DI-1 amplifies the sensitivity of HER2-positive breast cancer cells to treatment, suggesting its potential as a promising therapeutic strategy in this context.