Epigenetic Dysregulation Research Articles

The Role of Non-Coding RNAs in Epigenetic Dysregulation in Glioblastoma Development.

Glioblastoma (GBM) is a primary brain tumor arising from glial cells. The tumor is highly aggressive, the reason for which it has become the deadliest brain tumor type with the poorest prognosis. Like other cancers, it compromises molecular alteration on genetic and epigenetic levels. Epigenetics refers to changes in gene expression or cellular phenotype without the occurrence of any genetic mutations or DNA sequence alterations in the driver tumor-related genes. These epigenetic changes are reversible, making them convenient targets in cancer therapy. Therefore, we aim to review critical epigenetic dysregulation processes in glioblastoma. We will highlight the significant affected tumor-related pathways and their outcomes, such as regulation of cell cycle progression, cell growth, apoptosis, angiogenesis, cell invasiveness, immune evasion, or acquirement of drug resistance. Examples of molecular changes induced by epigenetic modifications, such as DNA epigenetic alterations, histone post-translational modifications (PTMs), and non-coding RNA (ncRNA) regulation, are highlighted. As understanding the role of epigenetic regulators and underlying molecular mechanisms in the overall pro-tumorigenic landscape of glioblastoma is essential, this literature study will provide valuable insights for establishing the prognostic or diagnostic value of various non-coding transcripts, including miRNAs.

Super-enhancer driven SOX2 promotes tumor formation by chromatin re-organization in nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is a malignant head and neck cancer with a high incidence in Southern China and Southeast Asia. Patients with remote metastasis and recurrent NPC have poor prognosis. Thus, a better understanding of NPC pathogenesis may identify novel therapies to address the unmet clinical needs. H3K27ac ChIP-seq and HiChIP was applied to understand the enhancer landscapes and the chromosome interactions. Whole genome sequencing was conducted to analyze the relationship between genomic variations and epigenetic dysregulation. CRISPRi and JQ1 treatment were used to evaluate the transcriptional regulation of SOX2 SEs. Colony formation assay, survival analysis and invivo subcutaneous patient-derived xenograft assays were applied to explore the function and clinical relevance of SOX2 in NPC. We globally mapped the enhancer landscapes and generated NPC enhancer connectomes, linking NPC specific enhancers and SEs. We found five overlapped genes, including SOX2, among super-enhancer regulated genes, survival related genes and NPC essential genes. The mRNA expression of SOX2 was repressed when applying CRISPRi targeting different SOX2 SEs or JQ1 treatment. Next, we identified a genetic variation (Chr3:181422197, G>A) in SOX2 SE which is correlated with higher expression of SOX2 and poor survival. In addition, SOX2 was highly expressed in NPC and is correlated with short survival in patients with NPC. Knock-down of SOX2 suppressed tumor growth invitro and invivo. Our study demonstrated the super-enhancer landscape with chromosome interactions and identified super-enhancer driven SOX2 promotes tumorigenesis, suggesting that SOX2 is a potential therapeutic target for patients with NPC. A full list of funding bodies that contributed to this study can be found in the Acknowledgements section.

Maternal Pre-Existing Diabetes: A Non-Inherited Risk Factor for Congenital Cardiopathies.

Congenital heart defects (CHDs) are the most common form of birth defects in humans. They occur in 9 out of 1000 live births and are defined as structural abnormalities of the heart. Understanding CHDs is difficult due to the heterogeneity of the disease and its multifactorial etiology. Advances in genomic sequencing have made it possible to identify the genetic factors involved in CHDs. However, genetic origins have only been found in a minority of CHD cases, suggesting the contribution of non-inherited (environmental) risk factors to the etiology of CHDs. Maternal pregestational diabetes is associated with a three- to five-fold increased risk of congenital cardiopathies, but the underlying molecular mechanisms are incompletely understood. According to current hypotheses, hyperglycemia is the main teratogenic agent in diabetic pregnancies. It is thought to induce cell damage, directly through genetic and epigenetic dysregulations and/or indirectly through production of reactive oxygen species (ROS). The purpose of this review is to summarize key findings on the molecular mechanisms altered in cardiac development during exposure to hyperglycemic conditions in utero. It also presents the various in vivo and in vitro techniques used to experimentally model pregestational diabetes. Finally, new approaches are suggested to broaden our understanding of the subject and develop new prevention strategies.

MODL-12. REGULATION OF OLIGODENDROGLIAL PROGENITOR CELL (OPC) CELL FATE IN DIFFUSE MIDLINE GLIOMA (DMG) BY THE HOMEOBOX TRANSCRIPTION FACTOR DLX2

Abstract Diffuse midline glioma, K27-altered (DMG) is a treatment resistant, uniformly fatal cancer that arises predominantly in children. 80% of patients with DMG harbor K27M mutations in canonical histone variants H3.3 and H3.1, leading to global epigenetic dysregulation and an oligodendroglial progenitor cell (OPC) transcriptional profile. DLX2 is a homeobox transcription factor that directly represses OPC cell fate and promotes a GABAergic interneuron cell fate during neurogenesis. Our lab has previously identified DLX2 transcription factor occupancy of genes required for OPC differentiation in vivo (Olig2, Nkx2.2, Myt1, Plp1), and shown that co-expression of DLX2 to target sequences reduced reporter gene expression in vitro. Likewise, increased expression of Olig2, Nkx2.2, and Plp-1 are observed in a Dlx1/Dlx2 double knockout (DKO) mouse model. Transient overexpression of a Dlx2-GFP construct into cells of a genetically engineered mouse model (GEMM) of DMG resulted in increased expression of Gad1/2 isoforms and decreased Olig2 and Nkx2.2, global restoration of H3K27me3, and a reduction in migration, invasion, and colony formation in vitro. Inverse expression of DLX2 and OLIG1/2 was observed in a bulk RNAseq database of 45 human DMG cell lines. We have established a pipeline to evaluate cell fate changes following changes in DLX2 expression levels. OLIG1/2high/DLX2low DMG expressing cell lines underwent stable transfection of a DLX2-mCherry overexpression construct. Successful alteration of DLX2 expression was confirmed by qPCR and immunoblot in knockout and overexpressing human DMG cell lines. Analysis of histone marks (H3K27me3, H3K27ac), K27M, and OPC signature genes will be presented with comparison of altered OPC genes from the DKO mouse as determined by RNA-seq. The manipulation of DLX2 expression in human DMG cells in vitro and in vivo provides a model system for assessing the contribution of a homeobox transcription factor required for neurogenesis to cell fate regulation and effects on tumorigenicity in DMG.

EXTH-28. ISOCITRATE DEHYDROGENASE MUTATIONS PREDICT SENSITIVITY TO HISTONE DEACETYLASE INHIBITION AND ENHANCED HISTONE ACETYLATION DYNAMICS ONLY IN THE CONTEXT OF GLIOMA

Abstract Isocitrate dehydrogenase 1/2 mutations (IDHmut) define a subtype of glioma with profound epigenetic dysregulation through genomic hypermethylation. IDHmut gliomas are incurable and fatal, demonstrating new treatments are needed. These mutations are common in other solid tumors, including chondrosarcoma (CS) and intrahepatic cholangiocarcinoma (ICC). Histone deacetylase (HDAC) enzymes compose a class of epigenetic drug targets utilized clinically to treat cancer, but their potential against IDHmut gliomas has not been extensively studied. We previously showed that IDHmut glioma cultures exhibit pronounced sensitivity to the effects mediated by the FDA-approved HDAC inhibitor (HDACi) panobinostat. Here, we integrate proliferation and cytoxicity data to show that this effect is unique to gliomas. In the context of histone H3 acetylation (H3KAc), panobinostat increased H3KAc, on average, by 7.6-fold and 30.5-fold in IDHwt and IDHmut glioma cultures, respectively (p< 0.001). This difference in HDACi-mediated H3KAc based on IDH status was not significant for CS and ICC. IDHmut gliomas are more sensitive two other FDA-approved HDACis: belinostat and vorinostat. RNA-Seq analysis showed that panobinostat more greatly affected gene expression in IDHmut glioma cells, particularly in the context of gene downregulation. Similarly, H3KAc ChIP-Seq showed a decrease in promoter histone acetylation moreso in IDHmut glioma cultures treated with panobinostat: there is an average loss of 1,236 and 14,759 H3KAc peaks at gene promoters in IDHwt and IDHmut cultures, respectively. This suggests that IDHmut gliomas exhibit enhanced histone acetylation dynamics in response to HDACi. Furthermore, using a PDX glioma model, we show that only mice engrafted with IDHmut glioma exhibit a significant survival increase in response to the HDACi belinostat: median survival was extended by 14 days in IDHmut glioma-bearing mice (p= 0.01), whereas belinostat extended median survival by 4 days in IDHwt glioma-bearing mice (p= 0.11). These results suggest that HDACi may be an effective in treating IDHmut gliomas.

EPCO-08. CONVERGENCE OF NON-CODING SOMATIC MUTATIONS ON CANCER DRIVER PLEXUSES IN PEDIATRIC HIGH-GRADE GLIOMA

Abstract Central nervous system tumors are the most common pediatric malignancies after leukemia, and the most common cause of cancer death among persons ages 0 – 14 [1,2]. Despite its low incidence rate, pediatric high-grade gliomas (pHGGs) account for over 40% of all childhood brain tumor deaths [3, 4]. As such, it remains the focus of many research teams across the globe. The discovery of recurrent mutations in genes encoding histone H3.3 [5] was a breakthrough in the genetic basis of pHGGs and highlighted the relevance of epigenetic reconfiguration for oncogene activation. However, much work is still needed to uncover driver alterations responsible for tumor initiation or progression. pHGGs have frequent epigenetic dysregulation [6,7] and abundant somatic mutations in the noncoding regions of the genome [8], but the functional relevance of the noncoding somatic mutations has not been evaluated systematically. Here, we assess correlations between noncoding somatic mutations in pediatric brain-specific cis-regulatory elements (CREs) and changes in gene expression. We demonstrate the sparsity of recurrent mutations in CREs. Then illustrate the power of graph database in consolidating multi-omics datasets for efficient queries. Our network approach for charting the genomic, epigenetic, and transcriptomic landscape of pHGG allow us to 1) identify clusters of CREs associated with dysregulated gene expression, 2) nominate transcription factors whose binding affinities are recurrently impacted by somatic mutations found in pHGGs, and 3) establish regulatory networks potentially guiding pHGG tumor growth and progression.

The Mechanism of Therapy Resistance By Lineage Plasticity in AML and How to Overcome It

Acute myeloid leukemia (AML) is an aggressive blood cancer driven predominantly by mutations in epigenetic regulators. This characteristic allows AML to thrive in a hostile and rapidly changing environment where it needs to adapt to stressors both extrinsic (chemotherapy) and intrinsic (replicative and oxidative stress) to survive. Our study is the first to demonstrate the mechanism of how AML uses epigenetic reprogramming to adjust to these stressors via lineage plasticity. Venetoclax and Azacitidine (Ven/Aza) co-treatment is increasingly the front-line therapy for AML, functioning through BH3 inhibition and DNA hypomethylation. However, most patients will relapse with a highly aggressive refractory AML. Some studies suggest that monocytic character of the AML is responsible for relapse, but more recent evidence demonstrates that Ven/Aza treatment itself can induce a lineage switch to monocytic character. A potent selection mechanism facilitated by epigenetic dysregulation generating plastic clones is likely driving resistance, though the mechanism remains unclear and represents a significant barrier for AML treatment. Using a combination of single cell transcriptomics with surface immunophenotype sequencing in multiple human xenograft and primary patient samples, we discovered that this plasticity is widespread and mediated by epigenetic reprogramming of the tumor in direct response to the therapy. Broadly, we found that AML responds to Ven/Aza by transitioning into a reversible state of cell cycle arrest with transcriptional character similar to hematopoietic stem cells. This transient state protects AML from Azacitidine through replication arrest and protects from Venetoclax by upregulation of anti-apoptotic transcriptional programs. These protections operate simultaneously with reprogramming events driven by a switch from MYC-driven transcriptional programs to a PU.1-driven program. Reprogramming is both facilitated by and dependent on repression of the Chromatin Assembly Factor 1 (CAF1) complex, a chromatin-bound master epigenetic regulator responsible for maintaining lineage fidelity through transcriptional regulation via its role as a nucleosome assembly factor. Repressing CAF1 facilitates reprogramming where the output is an altered cell population that is distinct from the initial tumor both transcriptionally and by ATACseq. AML can repeatedly return to this plastic state, regardless of the number of cycles of Ven/Aza, and introducing other cytotoxic compounds does not remove this plastic stem-like population but instead initiates the iterative process of reprogramming anew until a different resistant population emerges. To find therapeutic vulnerabilities, we generated several PDX models of Ven/Aza resistance so that we could track the stages of acquired resistance in real time. When single cell data from these experiments were projected onto a reference map of normal hematopoiesis, we found that Ven/Aza resistance stems from a plastic population of cells that actively samples the niche by generating short-lived multi-lineage hybrid differentiated products. While the predominating output of this process was monocytic, we found that the induced plastic stem-like population was durably retained even after Ven/Aza withdrawal. To undermine this process, we took advantage of the plastic state to drive differentiation through an external stimulus. We expected this strategy to be particularly effective because CAF1 was repressed (CAF1 repression facilitates differentiation in every metazoan model) and, based on our single cell data, these plastic populations of AML cells express pro-differentiation surface receptors. In particular, Ven/Aza induced the expression of CSF3R (the G-CSF receptor) which when stimulated, drives neutrophil maturation through the JAK/STAT pathway. While G-CSF as a single agent or in sequence with Ven/Aza was unable to achieve maturation, when used concurrently with Ven/Aza (or recapitulated by CAF1 depletion using shRNA) we were able to drive neutrophil differentiation in vitro and fully resolve primary mouse and PDX tumors in vivo, even when using Ven/Aza resistant tumors as starting material. Together, these data demonstrate the possibility that Ven/Aza resistance can be completely subverted by taking advantage of the plastic state induced by the therapy to stimulate differentiation and resolve the disease.

Changes in Methylation and Chromatin Accessibility Underlie Subtype Classification and Disease Evolution in Waldenström's Macroglobulinemia

Background: We previously identified three subtypes of Waldenstrom's Macroglobulinemia (WM): B-cell like (BCL), Plasma cell like (PCL), and an intermediate group enriched for early/smoldering WM from which the other two subtypes evolved (Hunter et al, ASH 2022). Diffusion pseudo-time (DPT) analysis suggested all WM samples could be placed on a shared evolutionary path independent of subtype, and when samples were separated into Early and Late DPT values, it mirrored disease progression. The role of epigenetic dysregulation that underlies subtype classification and disease evolution remains poorly understood in WM. Methods: We therefore performed chromatin availability analysis using ATACSeq and bisulfite sequencing of WM patients. ATAC-Seq libraries were constructed from 37 CD19+ BM cells from treatment-naive WM patients along with 6 paired CD19+CD27- and CD19+CD27+ selected healthy donor (HD) peripheral blood (PB) controls and 3 HD CD138+ plasma cell (PC) samples. Enhanced reduced representational bisulfite sequencing (ERRBS) was also performed on 65 MYD88 mutated and 13 MYD88 wild type (WT) WM patients using CD19-selected BM lymphoplasmacytic cells. For 11 WM MYD88 mutated patients, CD138+ cells were also selected. In addition ERRBS was also performed on paired CD19+ and CD138+ selected samples from 5 IgM multiple myeloma (MM) patients, 6 paired CD19+CD27- and CD19+CD27+ selected healthy donor (HD) peripheral blood cells; 8 HD CD138+ selected PC; as well as MYD88 mutated BCWM.1, BCWM.2, MWCL-1, RPCI-WM1, OCI-Ly3, TMD-8, HBL-1 and SU-DHL2 cells; MYD88 wild-type OCI-Ly7, OCI-Ly19, Ramos B-cells. To infer 5-hydroxymethylcytosine (5HMC) levels, oxidative bisulfite sequencing was also performed for 73 samples including the HD, MM, WT and WM CD19/138 pairs. All ATAC-Seq and 37 of the WM ERRBS samples were part of our multi-omic study that included 253 treatment naïve patients who underwent whole exome and RNASeq sequencing. Results: A UMAP projection of the CpG ERRBS data clustered WM patients into two distinct groups ( Figure 1). One group clustered directly with HD Memory B-cells (MB) and was greatly enriched for CXCR4 mutated WM patients. The other WM group clustered with IgM MM patients and was largely CXCR4 WT. The findings are consistent with the BCL and PCL WM subtypes, which were confirmed by a perfect assignment correlation with our larger multi-omic data set wherein these subtypes were defined. Notably, BCWM.1, BCWM.2 and MWCL-1 WM cell lines clustered with the WM samples. The 5HMC data displayed no clear clustering of the patient samples but clearly separated all HD samples from those of WM and IgM MM samples, consistent with a role for 5HMC dysregulation in oncogenesis. Differential methylation analysis showed differences by MYD88 and CXCR4 mutation status, deletions in Chr6q, familial history for WM, WM subtype, and stratification by early and late DPT values. Notably, the only comparison that did not yield any differences was WM CD19+ to WM CD138+, suggesting that this may not be a meaningful epigenetic distinction in WM. Similar comparisons were performed on the ATAC peaks. Differential chromatin accessibility was noted near many of the corresponding differentially expressed genes. ATAC peak coverage significantly correlated with the expression of top genes. This was particularly true for WM subtype and DPT comparisons ( Table 1). DNA sequences from differentially accessible peaks between BCL and PCL subtypes were also evaluated for relative TF motif enrichment in open vs. closed chromatin revealing PAX5, FUBP1, and ZNF121 enrichment in BCL subtypes using PCL subtype peaks as a background control. TFs REL, SP1, SPIB, BC11A and JUN were enriched PCL subtype compared with BCL subtype. Correlation between DPT associated genes revealed two clear groups, one associated with stem cell and pre-B-cell genes (Group 1) and the other with inflammatory, myeloid, and T-cell genes (Group 2). Motif analysis of differential peaks correlating with genes in each group demonstrated comparative enrichment for MYC, MXI1, and E2F2 in group 1 while RUNX1, NFKB1, and ATF1 were in enriched in group 2. Conclusions: This is the first independent validation of our previously reported multi-omic driven WM subtype classification. The studies underscore that epigenetic differences underlie the biology of WM subclassification and support a strong role for epigenetic changes driving WM evolution.

Methyl-CpG Binding Domain Protein MBD2 Is a Targetable Vulnerability in TET2 Mutant Myeloid Neoplasia

Background & Significance. Epigenetic dysregulation leading to acquired disorganization of CpG methylation (mCpG) is a hallmark of cancer in general and myeloid neoplasia (MN) in particular. TET dioxygenases (TET1, TET2 and TET3) progressively oxidize 5-methyl cytosine (mC) to facilitate CpG demethylation, a process critical to efficient transcription, lineage differentiation, and proliferation. Loss of function TET2 mutation ( TET2 MT), the predominant TET in HSPCs, isone of the most common genetic lesions in MN and often occurs early in MDS/AML as a founder lesion. It is frequently observed in clonal hematopoiesis of indeterminate potential (CHIP) 1,2, a prodromal condition linked with increased risk of MN, coronary heart disease 3, and pulmonary arterial hypertension 4. TET2 MT results in abnormal accumulation of mC in the genome resulting in loss of lineage plasticity due to transcriptional repression of differentiation-promoting genes and tumor suppressor genes (TSGs), contributing to a volatile pre-neoplastic state. Indeed, the accumulation of mC at otherwise unmethylated promoter- and enhancer-associated CpGs is a hallmark cancer 2,5. The epigenetic reader mCpG binding domain protein 2 (MBD2) specifically recognizes mCpG, thereby modulating regional gene transcription 6. This modulation occurs through the recruitment of co-repressor complexes which deliver enzymatic machinery to reorganize chromatin into compact, inaccessible, and stably silenced regions. This tightly regulated process, required for normal development and cell lineage differentiation, is hijacked by malignant cells to maintain a pro-oncogenic transcriptional program. Therefore, MBD2 inhibition may restore the normal hematopoiesis in TET2 MT HSPCs and may represent a selective therapeutic target. Methods. Genetic and epigenetic in vitro and in vivo model systems of clonal evolution including competitive repopulation assays and CRISPR-engineered cells coupled with NGS and biochemical analysis were used to establish the functional relevance of MBD2 in the clonal evolution of TET2 MT HSPCs. In addition, gene knockout or knockdown (ko/kd) were used to establish MBD2 as a novel therapeutic vulnerability in TET2 MT MN. Results. Here we report that loss of TET activity, either by TET2 MT or by neomorphic IDH1/2 MT, selectively enriches promoter/enhancer mCpG contributing to malignant evolution of TET2 MT HSPCs that creates sensitivity to MBD2 inhibition. TET2 MT creates regional accumulation of mC that recruits the MBD2-NuRd complex, resulting in silencing of TSGs as demonstrated via MBD2 chromatin immunoprecipitation assay. MBD2 ko/kd in TET2 MT cells (SIG-M5, OCIAML-5) or isogenic CRISPR-engineered leukemic cells (THP1, K562) resulted in the termination of the malignant program as reflected in a complete loss of colony forming ability in vitro. Orthotropic transplant of SIG-M5 cells (10 5 cells/mice) created leukemia in SGM3-NSGS mice with a maximum survival of 45 days, however, MBD2 ko in transplanted cells resulted in 100% disease-free survival of these mice. To further test if Mbd2 loss is sufficient to restore normal hematopoiesis and if it can abrogate the proliferative advantage of Tet2 -/- HSPCs, we developed a Tet2 -/-;Mbd2 -/- double ko (DKO) murine model by crossing Tet2 -/-with Mbd2 -/- mice. The DKO mice developed normally with restored hematopoiesis within the myeloid lineage compared to Tet2 -/-. Most importantly, the proliferative advantage of Tet2 -/-HSPCs was ablated, as reflected in the mutant fraction of total WBC and the restoration of CD11b + myeloid cells following Mbd2 loss in competitive bone marrow repopulation assays ( Fig. A).This restoration was mainly driven by the transcriptional rescue of differentiation-promoting genes which were silenced by promoter methylation in Tet2 -/- HSPCs ( Fig. B). Discussion & Conclusion. Here, for the first time, we demonstrate that MBD2 is required for maintaining the undifferentiated state of TET2 MT HSPCs with myeloid skewing. We provide several lines of evidence in human cells as well as in murine models that MBD2 is a targetable vulnerability in TET2 MT-associated leukemia. Our current findings provide a rationale for development of novel and potent non-cytotoxic therapeutic agents targeting MBD2 and their application in a biomarker-driven fashion for a large proportion of leukemias.

SRCAP mutations drive clonal hematopoiesis through epigenetic and DNA repair dysregulation.

Somatic mutations accumulate in all cells with age and can confer a selective advantage, leading to clonal expansion over time. In hematopoietic cells, mutations in a subset of genes regulating DNA repair or epigenetics frequently lead to clonal hematopoiesis (CH). Here, we describe the context and mechanisms that lead to enrichment of hematopoietic stem cells (HSCs) with mutations in SRCAP, which encodes a chromatin remodeler that also influences DNA repair. We show that SRCAP mutations confer a selective advantage in human cells and in mice upon treatment with the anthracycline-class chemotherapeutic doxorubicin and bone marrow transplantation. Furthermore, Srcap mutations lead to a lymphoid-biased expansion, driven by loss of SRCAP-regulated H2A.Z deposition and increased DNA repair. Altogether, we demonstrate that SRCAP operates at the intersection of multiple pathways in stem and progenitor cells, offering a new perspective on the functional impact of genetic variants that promote stem cell competition in the hematopoietic system.

Developmental, cytogenetic and epigenetic consequences of removing complex proteins and adding melatonin during in vitro maturation of bovine oocytes.

In vitro maturation (IVM) of germinal vesicle intact oocytes prior to in vitro fertilization (IVF) is practiced widely in animals. In human assisted reproduction it is generally reserved for fertility preservation or where ovarian stimulation is contraindicated. Standard practice incorporates complex proteins (CP), in the form of serum and/or albumin, into IVM media to mimic the ovarian follicle environment. However, the undefined nature of CP, together with batch variation and ethical concerns regarding their origin, necessitate the development of more defined formulations. A known component of follicular fluid, melatonin, has multifaceted roles including that of a metabolic regulator and antioxidant. In certain circumstances it can enhance oocyte maturation. At this stage in development, the germinal-vesicle intact oocyte is prone to aneuploidy and epigenetic dysregulation. To determine the developmental, cytogenetic and epigenetic consequences of removing CP and including melatonin during bovine IVM. The study comprised a 2 x 2 factorial arrangement comparing (i) the inclusion or exclusion of CP, and (ii) the addition (100 nM) or omission of melatonin, during IVM. Cumulus-oocyte complexes (COCs) were retrieved from stimulated cycles. Following IVM and IVF, putative zygotes were cultured to Day 8 in standard media. RNAseq was performed on isolated cumulus cells, cytogenetic analyses (SNP-based algorithms) on isolated trophectoderm cells, and DNA methylation analysis (reduced representation bisulfite sequencing) on isolated cells of the inner-cell mass. Removal of CP during IVM led to modest reductions in blastocyst development, whilst added melatonin was beneficial in the presence but detrimental in the absence of CP. The composition of IVM media did not affect the nature or incidence of chromosomal abnormalities but cumulus-cell transcript expression indicated altered metabolism (primarily lipid) in COCs. These effects preceded the establishment of distinct metabolic and epigenetic signatures several days later in expanded and hatching blastocysts. These findings highlight the importance of lipid, particularly sterol, metabolism by the COC during IVM. They lay the foundation for future studies that seek to develop chemically defined systems of IVM for the generation of transferrable embryos that are both cytogenetically and epigenetically normal.

Gut Microbiota Regulates Epigenetic Remodelling in the Amygdala: A Role in Repeated Mild Traumatic Brain Injury (rMTBI)-Induced Anxiety.

Gut microbiota serves in the development and maintenance of phenotype. However, the underlying mechanisms are still in its infancy. The current study shows epigenetic remodelling in the brain as a causal mechanism in the gut microbiota-brain axis. Like in trauma patients, gut dysbiosis and anxiety were comorbid in adult male Wistar rats subjected to repeated mild traumatic brain injuries (rMTBI). rMTBI caused epigenetic dysregulation of brain-derived neurotrophic factor (Bdnf) expression in the amygdala, owing to the formation of transcriptional co-repressor complex due to dynamic interaction between histone deacetylase and DNA methylation modification at the Bdnf gene promoter. The probiosis after faecal microbiota transplantation (FMT) from healthy naïve rats or by administration of single strain probiotic (SSP), Lactobacillus rhamnosus GG (LGG), recuperated rMTBI-induced anxiety. Concurrently, LGG infusion or naïve FMT also dislodged rMTBI-induced co-repressor complex resulting in the normalization of Bdnf expression and neuronal plasticity as measured by Golgi-Cox staining. Furthermore, sodium butyrate, a short-chain fatty acid, produced neurobehavioural effects similar to naïve FMT or LGG administration. Interestingly, the gut microbiota from rMTBI-exposed rats per se was able to provoke anxiety in naïve rats in parallel with BDNF deficits. Therefore, gut microbiota seems to be causally linked with the chromatin remodelling necessary for neuroadaptations via neuronal plasticity which drives experience-dependent behavioural manifestations.

Ketamine Produces Antidepressant Effects by Inhibiting Histone Deacetylases and Upregulating Hippocampal Brain-Derived Neurotrophic Factor Levels in a Diisopropyl Fluorophosphate-Based Rat Model of Gulf War Illness.

Approximately one-third of Gulf War veterans suffer from Gulf War Illness (GWI), which encompasses mood disorders and depressive symptoms. Deployment-related exposure to organophosphate compounds has been associated with GWI development. Epigenetic modifications have been reported in GWI veterans. We previously showed that epigenetic histone dysregulations were associated with decreased brain-derived neurotrophic factor (BDNF) expression in a GWI rat model. GWI has no effective therapies. Ketamine (KET) has recently been approved by the Food and Drug Administration for therapy-resistant depression. Interestingly, BDNF upregulation underlies KET's antidepressant effect in GWI-related depression. Here, we investigated whether KET's effect on histone mechanisms signals BDNF upregulations in GWI. Male Sprague-Dawley rats were injected once daily with diisopropyl fluorophosphate (DFP; 0.5 mg/kg, s.c., 5 days). At 6 months following DFP exposure, KET (10 mg/kg, i.p.) was injected, and brains were dissected 24 hours later. Western blotting was used for protein expression, and epigenetic studies used chromatin immunoprecipitation methods. Dil staining was conducted for assessing dendritic spines. Our results indicated that an antidepressant dose of KET inhibited the upregulation of histone deacetylase (HDAC) enzymes in DFP rats. Furthermore, KET restored acetylated histone occupancy at the Bdnf promoter IV and induced BDNF protein expression in DFP rats. Finally, KET treatment also increased the spine density and altered the spine diversity with increased T-type and decreased S-type spines in DFP rats. Given these findings, we propose that KET's actions involve the inhibition of HDAC expression, upregulation of BDNF, and dendritic modifications that together ameliorates the pathologic synaptic plasticity and exerts an antidepressant effect in DFP rats. SIGNIFICANCE STATEMENT: This study offers evidence supporting the involvement of epigenetic histone pathways in the antidepressant effects of ketamine (KET) in a rat model of Gulf War Illness (GWI)-like depression. This effect is achieved through the modulation of histone acetylation at the Bdnf promoter, resulting in elevated brain-derived neurotrophic factor expression and subsequent dendritic remodeling in the hippocampus. These findings underscore the rationale for considering KET as a potential candidate for clinical trials aimed at managing GWI-related depression.

Epigenetic PPARγ preservation attenuates temporomandibular joint osteoarthritis

ObjectivePrevious studies have demonstrated that PPARγ deficiency is associated with osteoarthritis in the knee joint. However, whether epigenetic PPARγ dysregulation has any effect on temporomandibular joint osteoarthritis (TMJOA) is unknown. This study aims to determine the role and mechanism of epigenetic PPARγ dysregulation in TMJOA. MethodsPartial TMJ discectomy was performed to induce TMJOA in rat. Primary condylar chondrocytes were isolated, and TNF-α-induced inflammatory condition was created in vitro. The expressions of PPARγ and DNA methyltransferase were investigated in vivo and in vitro. The association of PPARγ and DNA methylation was further studied by treating chondrocytes with DNA demethylation agent 5-Aza-2′-deoxycytidine (5Aza) and transfecting with siRNA of DNA methyltransferase (DNMT)1 and DNMT3a, and the methylation level of PPARγ promoter was evaluated by Bisulfite-sequencing PCR. The chondroprotective effects of 5Aza were explored in vitro and in vivo. ResultsPPARγ suppression and upregulated DNMT1/DNMT3a expression exist in TMJOA cartilage in vivo and primary condylar chondrocytes under TNF-α-induced inflammatory conditions in vitro. DNMT1 and DNMT3a elevation contributes to PPARγ-promoter hypermethylation in TMJ chondrocytes under TNF-α-induced inflammation conditions. DNA demethylation intervention by 5Aza protects chondrocytes from inflammation response in vitro. Mechanistically, 5Aza reversed the hypermethylation of the PPARγ promoter and subsequently resulted in PPARγ restoration and decreased expression of cartilage-catabolic factors in chondrocytes. Rat TMJOA model revealed that 5Aza, by reversing PPARγ suppression, effectively attenuated cartilage degeneration and stabilized cartilage homeostasis by balancing anabolic factor and catabolic factor expression. ConclusionEpigenetic PPARγ suppression may play a causal role in TMJOA pathogenesis, which can be alleviated by DNA demethylation with 5Aza treatment. This study provides new insights into the pathogenic mechanism and therapeutic strategy of TMJOA.

Integrative analysis reveals early epigenetic alterations in high-grade serous ovarian carcinomas.

High-grade serous ovarian carcinoma (HGSOC) is the most lethal gynecological malignancy. To date, the profiles of gene mutations and copy number alterations in HGSOC have been well characterized. However, the patterns of epigenetic alterations and transcription factor dysregulation in HGSOC have not yet been fully elucidated. In this study, we performed integrative omics analyses of a series of stepwise HGSOC model cells originating from human fallopian tube secretory epithelial cells (HFTSECs) to investigate early epigenetic alterations in HGSOC tumorigenesis. Assay for transposase-accessible chromatin using sequencing (ATAC-seq), chromatin immunoprecipitation sequencing (ChIP-seq), and RNA sequencing (RNA-seq) methods were used to analyze HGSOC samples. Additionally, protein expression changes in target genes were confirmed using normal HFTSECs, serous tubal intraepithelial carcinomas (STICs), and HGSOC tissues. Transcription factor motif analysis revealed that the DNA-binding activity of the AP-1 complex and GATA family proteins was dysregulated during early tumorigenesis. The protein expression levels of JUN and FOSL2 were increased, and those of GATA6 and DAB2 were decreased in STIC lesions, which were associated with epithelial-mesenchymal transition (EMT) and proteasome downregulation. The genomic region around the FRA16D site, containing a cadherin cluster region, was epigenetically suppressed by oncogenic signaling. Proteasome inhibition caused the upregulation of chemokine genes, which may facilitate immune evasion during HGSOC tumorigenesis. Importantly, MEK inhibitor treatment reversed these oncogenic alterations, indicating its clinical effectiveness in a subgroup of patients with HGSOC. This result suggests that MEK inhibitor therapy may be an effective treatment option for chemotherapy-resistant HGSOC.

Deciphering the Mysterious Relationship between the Cross-Pathogenetic Mechanisms of Neurodegenerative and Oncological Diseases.

The relationship between oncological pathologies and neurodegenerative disorders is extremely complex and is a topic of concern among a growing number of researchers around the world. In recent years, convincing scientific evidence has accumulated that indicates the contribution of a number of etiological factors and pathophysiological processes to the pathogenesis of these two fundamentally different diseases, thus demonstrating an intriguing relationship between oncology and neurodegeneration. In this review, we establish the general links between three intersecting aspects of oncological pathologies and neurodegenerative disorders, i.e., oxidative stress, epigenetic dysregulation, and metabolic dysfunction, examining each process in detail to establish an unusual epidemiological relationship. We also focus on reviewing the current trends in the research and the clinical application of the most promising chemical structures and therapeutic platforms that have a modulating effect on the above processes. Thus, our comprehensive analysis of the set of molecular determinants that have obvious cross-functional pathways in the pathogenesis of oncological and neurodegenerative diseases can help in the creation of advanced diagnostic tools and in the development of innovative pharmacological strategies.

Epigenetic targeting to enhance acute myeloid leukemia-directed immunotherapy.

AML is a malignant disease of hematopoietic progenitor cells with unsatisfactory treatment outcome, especially in patients that are ineligible for intensive chemotherapy. Immunotherapy, comprising checkpoint inhibition, T-cell engaging antibody constructs, and cellular therapies, has dramatically improved the outcome of patients with solid tumors and lymphatic neoplasms. In AML, these approaches have been far less successful. Discussed reasons are the relatively low mutational burden of AML blasts and the difficulty in defining AML-specific antigens not expressed on hematopoietic progenitor cells. On the other hand, epigenetic dysregulation is an essential driver of leukemogenesis, and non-selective hypomethylating agents (HMAs) are the current backbone of non-intensive treatment. The first clinical trials that evaluated whether HMAs may improve immune checkpoint inhibitors' efficacy showed modest efficacy except for the anti-CD47 antibody that was substantially more efficient against AML when combined with azacitidine. Combining bispecific antibodies or cellular treatments with HMAs is subject to ongoing clinical investigation, and efficacy data are awaited shortly. More selective second-generation inhibitors targeting specific chromatin regulators have demonstrated promising preclinical activity against AML and are currently evaluated in clinical trials. These drugs that commonly cause leukemia cell differentiation potentially sensitize AML to immune-based treatments by co-regulating immune checkpoints, providing a pro-inflammatory environment, and inducing (neo)-antigen expression. Combining selective targeted epigenetic drugs with (cellular) immunotherapy is, therefore, a promising approach to avoid unintended effects and augment efficacy. Future studies will provide detailed information on how these compounds influence specific immune functions that may enable translation into clinical assessment.

Deoxyribonucleic acid methylation driven aberrations in pancreatic cancer-related pathways.

Pancreatic cancer (PanCa) presents a catastrophic disease with poor overall survival at advanced stages, with immediate requirement of new and effective treatment options. Besides genetic mutations, epigenetic dysregulation of signaling pathway-associated enriched genes are considered as novel therapeutic target. Mechanisms beneath the deoxyribonucleic acid methylation and its utility in developing of epi-drugs in PanCa are under trails. Combinations of epigenetic medicines with conventional cytotoxic treatments or targeted therapy are promising options to improving the dismal response and survival rate of PanCa patients. Recent studies have identified potentially valid pathways that support the prediction that future PanCa clinical trials will include vigorous testing of epigenomic therapies. Epigenetics thus promises to generate a significant amount of new knowledge of biological and medical importance. Our review could identify various components of epigenetic mechanisms known to be involved in the initiation and development of pancreatic ductal adenocarcinoma and related precancerous lesions, and novel pharmacological strategies that target these components could potentially lead to breakthroughs. We aim to highlight the possibilities that exist and the potential therapeutic interventions.

Regulation of alternative splicing: Functional interplay with epigenetic modifications and its implication to cancer.

Eukaryotic gene expression is intricately regulated at multiple levels. The protein-coding genes are first transcribed as pre-mRNAs in the nucleus and undergo a series of RNA processing steps before being transported into the cytoplasm for translation. During RNA processing, most human genes (>95%) undergo alternative splicing to generate multiple mRNA isoforms from a single gene, which effectively diversifies the genome complexity. Since the splicing of most genes occurs co-transcriptionally, the regulation layers of gene expression often show functional interactions with each other. In this review, we provide a brief overview of alternative splicing regulation in three different layers (controlled by the splicing machinery, transcription process, and chromatin structure), emphasizing the regulatory roles of epigenetic modifications and the crosstalk between these layers. Specifically, we categorize the major effects of the epigenetic modifications on alternative splicing into three different types: by affecting transcription rate, splicing factor recruitment, or the expression/activity of splicing factor. The dysregulation of epigenetics and splicing are extremely common in cancer, we also discuss the potential mechanisms of how epigenetic changes can lead to splicing dysregulation and their functional consequences. We aim to provide insights into the complicated regulation of different gene expression layers, which will shed light on the novel approaches to modulate disease-related splicing dysregulation. This article is categorized under: RNA Processing > 3' End Processing RNA Processing > Splicing Mechanisms RNA Processing > Splicing Regulation/Alternative Splicing RNA in Disease and Development > RNA in Disease.

P03.08.A HIGH-DIMENSIONAL CHARACTERISATION OF THE CELLULAR AND MOLECULAR CHANGES IN THE HUMAN BRAIN AFTER RADIOTHERAPY

Abstract BACKGROUND In the UK ~40,000 intrinsic and metastatic brain tumours are diagnosed each year. Although targeted radiotherapy (RT) is integral to increasing survival of cancer patients, it has significant side-effects, the cellular and molecular mechanisms of which are not fully understood. During RT treatment epigenetic changes occur in the neoplastic tissue, but few studies have assessed these in non-neoplastic brain tissue and results are highly variable. MATERIAL AND METHODS To explore the effects of RT on patient-specific methylation, we retrospectively identified 19 samples, mainly metastatic tumours, that had undergone stereotactic-RT followed by resection, and contained peri-lesional brain tissue, as well as 14 controls. Peri-lesional brain was micro-dissected, followed by DNA methylation profiling and RNA-sequencing. RESULTS Clustering of DNA methylation data showed distinct patterns between the two groups, whilst RNA-seq revealed 1,887 differentially expressed genes. Pathway analysis with GSEA identified differentially represented pathways, including: innate immune system, trans-synaptic signalling, olfactory signalling, regulation of supramolecular fibre organisation, and pattern specification. Computational deconvolution of cellular composition showed increased activated dendritic cells and B-cells, and reduced smooth muscle cells and several sub-sets of astrocytes in the RT samples. CONCLUSION We are currently further investigating these findings using spatial transcriptomics and proteomics to produce the first high-dimensional characterisation of the irradiated human brain. This will also provide the first direct evidence of epigenetic dysregulation after targeted-RT, and assess novel epigenetically-regulated genes, which will be validated with single cell tissue-based imaging techniques. A range of epigenetic-based drugs are undergoing clinical trials and we aim to identify targets for radiomitigation in brain tumour patients.