H3K27me3 Research Articles

Abstract 1423: Regulation of SKP2 expression by methyltransferase-independent function of EZH2 in tumor growth and progression

Abstract Skp2, an F box protein and a substrate-binding component of the SCFSkp2 ubiquitin E3 ligase complex, promotes ubiquitination and degradation of cyclin-dependent kinase inhibitors (CKIs), including p21, p27, and p57. Aberrant Skp2 expression is associated with many human cancers. EZH2 (Enhancer of Zeste Homologue 2) is a core catalytic subunit of the polycomb repressive complex 2 (PRC2) that can regulate gene expression by mediating the methylation of lysine 27 of histone H3 (H3K27me3). Accumulating evidence shows that EZH2 could bind to transcription factors and activate gene expression independently of its methyltransferase activity, leading to the activation of oncogenic signaling pathways and promoting the proliferation of tumor cells. Although the methyltransferase-independent function of EZH2 is important in cell proliferation, its mechanism in cell cycle regulation is still unclear. In this study, we demonstrate that EZH2 positively regulates Skp2 gene expression. We show that suppression of EZH2 expression resulted in a significant decrease of Skp2 protein levels, while the EZH2 enzymatic inhibitors, EPZ6438, had no impact on protein levels of Skp2, which suggests that EZH2 regulates Skp2 expression independently of its methyltransferase activity. Consistently, EZH2 depletion reduced the mRNA levels of Skp2, while EPZ6438 inhibited EZH2 methyltransferase activity without affecting the mRNA levels of Skp2. Importantly, EZH2 expression is positively correlated with the expression of EMT (Epithelial-mesenchymal transition) regulators (YAP1/TAZ, ZEB1, and Snail) and immune checkpoint proteins (FGL1 and Siglec-15) in cervical and pancreatic cancer cells, but reversely correlated with the expression of CKIs (p21, p27, and p57). Taken together, our findings suggest a novel mechanism by which EZH2 regulates tumorigenesis through its methyltransferase-independent function by regulating Skp2 expression. Targeting the methyltransferase-independent function of EZH2 may have therapeutic efficacy for cancer patients. Citation Format: Tania Marlyn Colon, Wei Dai, Byeong Hyeok Choi. Regulation of SKP2 expression by methyltransferase-independent function of EZH2 in tumor growth and progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1423.

Front Cover: Dioxygen Binding Is Controlled by the Protein Environment in Non‐heme FeII and 2‐Oxoglutarate Oxygenases: A Study on Histone Demethylase PHF8 and an Ethylene‐Forming Enzyme (Chem. Eur. J. 24/2023)

The cover shows the oxygen diffusion channel in class 7 histone demethylase (PHF8) and ethylene-forming enzyme (EFE). PHF8 catalyzes the hydroxylation of its H3 K9me2 histone substrate, and EFE catalyzes two competing reactions of ethylene generation and substrate l-Arg hydroxylation. Although both enzymes initially have an off-line 2-oxoglutarate (2OG) conformation, upon oxygen binding, PHF8 forms an in-line superoxo complex, whereas EFE uses the off-line superoxo complex. More information can be found in the Research Article by C. Z. Christov and co-workers. (DOI: 10.1002/chem.202300138).

H3K27me3-H3K4me1 transition at bivalent promoters instructs lineage specification in development

BackgroundBivalent genes, of which promoters are marked by both H3K4me3 (trimethylation of histone H3 on lysine 4) and H3K27me3 (trimethylation of histone H3 on lysine 27), play critical roles in development and tumorigenesis. Monomethylation on lysine 4 of histone H3 (H3K4me1) is commonly associated with enhancers, but H3K4me1 is also present at promoter regions as an active bimodal or a repressed unimodal pattern. Whether the co-occurrence of H3K4me1 and bivalent marks at promoters plays regulatory role in development is largely unknown.ResultsWe report that in the process of lineage differentiation, bivalent promoters undergo H3K27me3-H3K4me1 transition, the loss of H3K27me3 accompanies by bimodal pattern loss or unimodal pattern enrichment of H3K4me1. More importantly, this transition regulates tissue-specific gene expression to orchestrate the development. Furthermore, knockout of Eed (Embryonic Ectoderm Development) or Suz12 (Suppressor of Zeste 12) in mESCs (mouse embryonic stem cells), the core components of Polycomb repressive complex 2 (PRC2) which catalyzes H3K27 trimethylation, generates an artificial H3K27me3-H3K4me1 transition at partial bivalent promoters, which leads to up-regulation of meso-endoderm related genes and down-regulation of ectoderm related genes, thus could explain the observed neural ectoderm differentiation failure upon retinoic acid (RA) induction. Finally, we find that lysine-specific demethylase 1 (LSD1) interacts with PRC2 and contributes to the H3K27me3-H3K4me1 transition in mESCs.ConclusionsThese findings suggest that H3K27me3-H3K4me1 transition plays a key role in lineage differentiation by regulating the expression of tissue specific genes, and H3K4me1 pattern in bivalent promoters could be modulated by LSD1 via interacting with PRC2.

The promise of metabolic imaging in diffuse midline glioma

Recent insights into histopathological and molecular subgroups of glioma have revolutionized the field of neuro-oncology by refining diagnostic categories. An emblematic example in pediatric neuro-oncology is the newly defined diffuse midline glioma (DMG), H3 K27–altered. DMG represents a rare tumor with a dismal prognosis. The diagnosis of DMG is largely based on clinical presentation and characteristic features on conventional magnetic resonance imaging (MRI), with biopsy limited by its delicate neuroanatomic location. Standard MRI remains limited in its ability to characterize tumor biology. Advanced MRI and positron emission tomography (PET) imaging offer additional value as they enable non-invasive evaluation of molecular and metabolic features of brain tumors. These techniques have been widely used for tumor detection, metabolic characterization and treatment response monitoring of brain tumors. However, their role in the realm of pediatric DMG is nascent. By summarizing DMG metabolic pathways in conjunction with their imaging surrogates, we aim to elucidate the untapped potential of such imaging techniques in this devastating disease.

Loss of MLL3/4 decouples enhancer H3K4 monomethylation, H3K27 acetylation, and gene activation during embryonic stem cell differentiation

BackgroundEnhancers are essential in defining cell fates through the control of cell-type-specific gene expression. Enhancer activation is a multi-step process involving chromatin remodelers and histone modifiers including the monomethylation of H3K4 (H3K4me1) by MLL3 (KMT2C) and MLL4 (KMT2D). MLL3/4 are thought to be critical for enhancer activation and cognate gene expression including through the recruitment of acetyltransferases for H3K27.ResultsHere we test this model by evaluating the impact of MLL3/4 loss on chromatin and transcription during early differentiation of mouse embryonic stem cells. We find that MLL3/4 activity is required at most if not all sites that gain or lose H3K4me1 but is largely dispensable at sites that remain stably methylated during this transition. This requirement extends to H3K27 acetylation (H3K27ac) at most transitional sites. However, many sites gain H3K27ac independent of MLL3/4 or H3K4me1 including enhancers regulating key factors in early differentiation. Furthermore, despite the failure to gain active histone marks at thousands of enhancers, transcriptional activation of nearby genes is largely unaffected, thus uncoupling the regulation of these chromatin events from transcriptional changes during this transition. These data challenge current models of enhancer activation and imply distinct mechanisms between stable and dynamically changing enhancers.ConclusionsCollectively, our study highlights gaps in knowledge about the steps and epistatic relationships of enzymes necessary for enhancer activation and cognate gene transcription.

ETHYLENE INSENSITIVE3/EIN3-LIKE1 modulate FLOWERING LOCUS C expression via histone demethylase interaction.

Time to flowering (vegetative to reproductive phase) is tightly regulated by endogenous factors and environmental cues to ensure proper and successful reproduction. How endogenous factors coordinate with environmental signals to regulate flowering time in plants is unclear. Transcription factors ETHYLENE INSENSITIVE 3 (EIN3) and its homolog EIN3 LIKE 1 (EIL1) are the core downstream regulators in ethylene signal transduction, and their null mutants exhibit late flowering in Arabidopsis (Arabidopsis thaliana); however, the precise mechanism of floral transition remains unknown. Here, we reveal that FLOWERING LOCUS D (FLD), encoding a histone demethylase acting in the autonomous pathway of floral transition, physically associates with EIN3 and EIL1. Loss of EIN3 and EIL1 upregulated transcriptional expression of the floral repressor FLOWERING LOCUS C (FLC) and its homologs in Arabidopsis, and ethylene-insensitive mutants displayed inhibited flowering in an FLC-dependent manner. We further demonstrated that EIN3 and EIL1 directly bind to FLC loci, modulating their expression by recruiting FLD and thereafter removing di-methylation of lysine 4 on histone H3 (H3K4me2). In plants treated with 1-aminocyclopropane-1-carboxylic acid, decreased expression of FLD resulted in increased enrichment of H3K4me2 at FLC loci and transcriptional activation of FLC, leading to floral repression. Our study reveals the role of EIN3 and EIL1 in FLC-dependent and ethylene-induced floral repression and elucidates how phytohormone signals are transduced into chromatin-based transcriptional regulation.

EZH2 W113C is a gain-of-function mutation in B-cell lymphoma enabling both PRC2 methyltransferase activation and tazemetostat resistance

Polycomb Repressive Complex2 (PRC2) suppresses gene transcription by methylating lysine 27 of histone H3 (H3K27) and plays critical roles in embryonic development. Among the core PRC2 subunits, EZH2 is the catalytic subunit and EED allosterically activates EZH2 upon binding trimethylated H3K27 (H3K27me3). Activating mutations on Y641, A677, and A687 within the enzymatic SET domain of EZH2 have been associated with enhanced H3K27me3 and tumorigenicity of many cancers including B-cell lymphoma and melanoma. To tackle the critical residues outside the EZH2 SET domain, we examined EZH2 mutations in lymphoma from cancer genome databases and identified a novel gain-of-function (GoF) mutation W113C, which increases H3K27me3 in vitro and in vivo and promotes CDKN2A silencing to a similar level as the EZH2 Y641F. Different from other GoF mutations, this mutation is located in the SAL motif at the EZH2 N-terminus, which stabilizes the SET domain and facilitates substrate binding. This may explain how the W113C mutation increases PRC2 activity. Tazemetostat is an FDA-approved EZH2 binding inhibitor for follicular lymphoma treatment. Intriguingly, the W113C mutation leads to tazemetostat-resistance in cell and in vivo in both H3K27 methylation and tumor proliferation. Another class of allosteric PRC2 inhibitor binding EED overcomes the resistance, effectively decreases H3K27me3, and blocks tumor proliferation in cells expressing EZH2 W113C. As this mutation is originally identified from lymphoma samples, our results demonstrated its activating characteristic and the deleterious consequence, provide insights on PRC2 regulation, and support the continued exploration of treatment optimization for lymphoma patients.

High levels of histone H3 K27 acetylation and tri-methylation are associated with shorter survival in oral squamous cell carcinoma patients.

Acetylation and trimethylation of histone H3 lysine 27 (H3K27ac and H3K27me3) generally activate and repress transcription, respectively. Concurrent activation of H3K27ac and H3K27me3 has been reported to correlate with poor prognosis in hepatocellular carcinoma. A high level of H3K27me3 has been shown to be associated with advanced oral squamous cell carcinoma (OSCC) tumour stage, but prognostic impact of H3K27ac level alone/or in combination with H3K27me3 in OSCC patients had not yet been reported. In this study, immunohistochemistry using specific antibodies against H3K27ac and H3K27me3 was performed on a series of 72 OSCC samples to investigate the association between H3K27ac and H3K27me3 levels and OSCC patient's survival. For each mark, the proportion of labelled cells (percentage) and the intensity of the labelling were measured, and a score of percentage x intensity was calculated. A high percentage of H3K27me3 positive cells was significantly associated with survival in univariate and multivariate analyses (logrank p-value < 0.05). Patients with high total scores of H3K27ac and H3K27me3 labelling also showed significantly shorter survival probabilities (logrank p-value < 0.05) compared to patients with low total scores of labelling for these histone posttranslational modifications (PTMs). Our findings suggest that the detection of both H3K27ac and H3k27me3 could help evaluating prognosis in OSCC patients.

Chromatin Remodeling Complex SWR1 Regulates Root Development by Affecting the Accumulation of Reactive Oxygen Species (ROS)

Reactive oxygen species (ROS), a type of oxygen monoelectronic reduction product, play integral roles in root growth and development. The epigenetic mechanism plays a critical role in gene transcription and expression; however, its regulation of ROS metabolism in root development is still limited. We found that the chromatin remodeling complex SWR1 regulates root length and lateral root formation in Arabidopsis. Our transcriptome results and gene ontology (GO) enrichment analysis showed that the oxidoreductase activity-related genes significantly changed in mutants for the Arabidopsis SWR1 complex components, such as arp6 and pie1, and histone variant H2A.Z triple mutant hta8 hta9 hta11. The three encoding genes in Arabidopsis are the three H2A.Z variants hta8, hta9, and hta11. Histochemical assays revealed that the SWR1 complex affects ROS accumulation in roots. Furthermore, chromatin immunoprecipitation quantitative real-time PCR (ChIP-qPCR) analysis showed that the reduced H2A.Z deposition in oxidoreductase activity-related genes caused ROS to accumulate in arp6, pie1, and hta8 hta9 hta11. H2A.Z deposition-deficient mutants decreased after the trimethylation of lysine 4 on histone H3 (H3K4me3) modifications and RNA polymerase II (Pol II) enrichment, and increased after the trimethylation of lysine 27 on histone H3 (H3K27me3) modifications, which may account for the expression change in oxidoreductase activity-related genes. In summary, our results revealed that the chromatin complex SWR1 regulates ROS accumulation in root development, highlighting the critical role of epigenetic mechanisms.

Lysine 27 of histone H3.3 is a fine modulator of developmental gene expression and stands as an epigenetic checkpoint for lignin biosynthesis in Arabidopsis.

Chromatin is a dynamic platform within which gene expression is controlled by epigenetic modifications, notably targeting amino acid residues of histone H3. Among them is lysine 27 of H3 (H3K27), the trimethylation of which by the Polycomb Repressive Complex 2 (PRC2) is instrumental in regulating spatiotemporal patterns of key developmental genes. H3K27 is also subjected to acetylation and is found at sites of active transcription. Most information on the function of histone residues and their associated modifications in plants was obtained from studies of loss-of-function mutants for the complexes that modify them. To decrypt the genuine function of H3K27, we expressed a non-modifiable variant of H3 at residue K27 (H3.3K27A ) in Arabidopsis, and developed a multi-scale approach combining in-depth phenotypical and cytological analyses, with transcriptomics and metabolomics. We uncovered that the H3.3K27A variant causes severe developmental defects, part of them are reminiscent of PRC2 mutants, part of them are new. They include early flowering, increased callus formation and short stems with thicker xylem cell layer. This latest phenotype correlates with mis-regulation of phenylpropanoid biosynthesis. Overall, our results reveal novel roles of H3K27 in plant cell fates and metabolic pathways, and highlight an epigenetic control point for elongation and lignin composition of the stem.

The role of histone methyltransferases in neurocognitive disorders associated with brain size abnormalities.

Brain size is controlled by several factors during neuronal development, including neural progenitor proliferation, neuronal arborization, gliogenesis, cell death, and synaptogenesis. Multiple neurodevelopmental disorders have co-morbid brain size abnormalities, such as microcephaly and macrocephaly. Mutations in histone methyltransferases that modify histone H3 on Lysine 36 and Lysine 4 (H3K36 and H3K4) have been identified in neurodevelopmental disorders involving both microcephaly and macrocephaly. H3K36 and H3K4 methylation are both associated with transcriptional activation and are proposed to sterically hinder the repressive activity of the Polycomb Repressor Complex 2 (PRC2). During neuronal development, tri-methylation of H3K27 (H3K27me3) by PRC2 leads to genome wide transcriptional repression of genes that regulate cell fate transitions and neuronal arborization. Here we provide a review of neurodevelopmental processes and disorders associated with H3K36 and H3K4 histone methyltransferases, with emphasis on processes that contribute to brain size abnormalities. Additionally, we discuss how the counteracting activities of H3K36 and H3K4 modifying enzymes vs. PRC2 could contribute to brain size abnormalities which is an underexplored mechanism in relation to brain size control.

PGC7 Regulates Genome-Wide DNA Methylation by Regulating ERK-Mediated Subcellular Localization of DNMT1.

DNA methylation is an epigenetic modification that plays a vital role in a variety of biological processes, including the regulation of gene expression, cell differentiation, early embryonic development, genomic imprinting, and X chromosome inactivation. PGC7 is a maternal factor that maintains DNA methylation during early embryonic development. One mechanism of action has been identified by analyzing the interactions between PGC7 and UHRF1, H3K9 me2, or TET2/TET3, which reveals how PGC7 regulates DNA methylation in oocytes or fertilized embryos. However, the mechanism by which PGC7 regulates the post-translational modification of methylation-related enzymes remains to be elucidated. This study focused on F9 cells (embryonic cancer cells), which display high levels of PGC7 expression. We found that both knockdown of Pgc7 and inhibition of ERK activity resulted in increased genome-wide DNA methylation levels. Mechanistic experiments confirmed that inhibition of ERK activity led to the accumulation of DNMT1 in the nucleus, ERK phosphorylated DNMT1 at ser717, and DNMT1 Ser717-Ala mutation promoted the nuclear localization of DNMT1. Moreover, knockdown of Pgc7 also caused downregulation of ERK phosphorylation and promoted the accumulation of DNMT1 in the nucleus. In conclusion, we reveal a new mechanism by which PGC7 regulates genome-wide DNA methylation via phosphorylation of DNMT1 at ser717 by ERK. These findings may provide new insights into treatments for DNA methylation-related diseases.

WDR5 represents a therapeutically exploitable target for cancer stem cells in glioblastoma.

Glioblastomas (GBMs) are heterogeneous, treatment-resistant tumors driven by populations of cancer stem cells (CSCs). However, few molecular mechanisms critical for CSC population maintenance have been exploited for therapeutic development. We developed a spatially resolved loss-of-function screen in GBM patient-derived organoids to identify essential epigenetic regulators in the SOX2-enriched, therapy-resistant niche and identified WDR5 as indispensable for this population. WDR5 is a component of the WRAD complex, which promotes SET1 family-mediated Lys4 methylation of histone H3 (H3K4me), associated with positive regulation of transcription. In GBM CSCs, WDR5 inhibitors blocked WRAD complex assembly and reduced H3K4 trimethylation and expression of genes involved in CSC-relevant oncogenic pathways. H3K4me3 peaks lost with WDR5 inhibitor treatment occurred disproportionally on POU transcription factor motifs, including the POU5F1(OCT4)::SOX2 motif. Use of a SOX2/OCT4 reporter demonstrated that WDR5 inhibitor treatment diminished cells with high reporter activity. Furthermore, WDR5 inhibitor treatment and WDR5 knockdown altered the stem cell state, disrupting CSC in vitro growth and self-renewal, as well as in vivo tumor growth. These findings highlight the role of WDR5 and the WRAD complex in maintaining the CSC state and provide a rationale for therapeutic development of WDR5 inhibitors for GBM and other advanced cancers.

Genome-wide identification and characterization of polycomb repressive complex 2 core components in upland cotton (Gossypium hirsutum L.)

BackgroundThe evolutionarily conserved Polycomb Repressive Complex 2 (PRC2) plays a vital role in epigenetic gene repression by depositing tri-methylation on lysine residue K27 of histone H3 (H3K27me3) at the target loci, thus participating in diverse biological processes. However, few reports about PRC2 are available in plant species with large and complicated genomes, like cotton.ResultsHere, we performed a genome-wide identification and comprehensive analysis of cotton PRC2 core components, especially in upland cotton (Gossypium hirsutum). Firstly, a total of 8 and 16 PRC2 core components were identified in diploid and tetraploid cotton species, respectively. These components were classified into four groups, E(z), Su(z)12, ESC and p55, and the members in the same group displayed good collinearity, similar gene structure and domain organization. Next, we cloned G. hirsutum PRC2 (GhPRC2) core components, and found that most of GhPRC2 proteins were localized in the nucleus, and interacted with each other to form multi-subunit complexes. Moreover, we analyzed the expression profile of GhPRC2 genes. The transcriptome data and quantitative real-time PCR (qRT-PCR) assays indicated that GhPRC2 genes were ubiquitously but differentially expressed in various tissues, with high expression levels in reproductive organs like petals, stamens and pistils. And the expressions of several GhPRC2 genes, especially E(z) group genes, were responsive to various abiotic and biotic stresses, including drought, salinity, extreme temperature, and Verticillium dahliae (Vd) infection.ConclusionWe identified PRC2 core components in upland cotton, and systematically investigated their classifications, phylogenetic and synteny relationships, gene structures, domain organizations, subcellular localizations, protein interactions, tissue-specific and stresses-responsive expression patterns. Our results will provide insights into the evolution and composition of cotton PRC2, and lay the foundation for further investigation of their biological functions and regulatory mechanisms.

The clinicopathological features and prognosis of multifocal high-grade gliomas in adults withH3F3Amutation

To explore the clinicopathological features and prognosis of multifocal high-grade gliomas (M-HGGs) with H3F3A mutation in adults. Four adult patients with H3F3A-mutant M-HGGs who were treated at our institution from August 2020 to December 2021 were reviewed, including clinical, pathological and radiologic data. A series of 16 adult patients with M-HGGs without H3F3A mutation was used as a comparative group. Progression-free survival (PFS) and overall survival (OS) were compared between the groups using the Kaplan-Meier method. All patients were IDH wild-type and TERT wild-type, and P53 was overexpressed. A patient with the H3 G34R mutation and 1 of 3 patients with the H3 K27M mutation had MGMT promoter methylation. The lesions with the H3 G34R mutation were located in the cerebral hemisphere; the lesions with H3 K27 alterations were mainly in the midline structure, and the cerebral hemisphere could also be involved. One patient underwent subtotal resection (STR), and 3 patients underwent biopsy. All patients received radiotherapy, and the median PFS and OS were 9.5 months and 14.5 months, respectively. The clinical outcomes were similar to those of non-H3F3A-mutated M-HGGs patients (median PFS and OS were 7.0 months and 18.0 months, respectively). We describe the clinicopathological features and outcomes of 4 adult M-HGGs patients with H3F3A mutation, and found this mutation doesn't appear to have a negative outcome with the administration of current therapies.

Histone lysine-specific demethylase 1 regulates the proliferation of hemocytes in the oyster Crassostrea gigas.

Lysine-specific demethylase 1 (LSD1) is an essential epigenetic regulator of hematopoietic differentiation, which can specifically mono-methylate H3K4 (H3K4me1) and di-methylate H3K4 (H3K4me2) as a transcriptional corepressor. Previous reports have been suggested that it participated in hematopoiesis and embryonic development process. Here, a conserved LSD1 (CgLSD1) with a SWIRM domain and an amino oxidase (AO) domain was identified from the Pacific oyster Crassostrea gigas. We conducted a comprehensive analysis by various means to verify the function of CgLSD1 in hematopoietic process, including quantitative real-time PCR (qRT-PCR) analysis, western blot analysis, immunofluorescence assay, RNA interference (RNAi) and flow cytometry. The qRT-PCR analysis revealed that the transcripts of CgLSD1 were widely expressed in oyster tissues with the highest level in the mantle. And the transcripts of CgLSD1 were ubiquitously expressed during larval development with the highest expression level at the early D-veliger larvae stage. In hemocytes after Vibrio splendidus stimulation, the transcripts of CgLSD1 were significantly downregulated at 3, 6, 24, and 48 h with the lowest level at 3 h compared to that in the Seawater group (SW group). Immunocytochemical analysis showed that CgLSD1 was mainly distributed in the nucleus of hemocytes. After the CgLSD1 was knocked down by RNAi, the H3K4me1 and H3K4me2 methylation level significantly increased in hemocyte protein. Besides, the percentage of hemocytes with EdU-positive signals in the total circulating hemocytes significantly increased after V. splendidus stimulation. After RNAi of CgLSD1, the expression of potential granulocyte markers CgSOX11 and CgAATase as well as oyster cytokine-like factor CgAstakine were increased significantly in mRNA level, while the transcripts of potential agranulocyte marker CgCD9 was decreased significantly after V. splendidus stimulation. The above results demonstrated that CgLSD1 was a conserved member of lysine demethylate enzymes that regulate hemocyte proliferation during the hematopoietic process.

Transient Methionine Deprivation Triggers Histone Modification and Potentiates Differentiation of Induced Pluripotent Stem Cells.

Human induced pluripotent stem cells (iPSCs) require high levels of methionine (Met). Met deprivation results in a rapid decrease in intracellular S-adenosyl-methionine (SAM), poising human iPSCs for differentiation and leading to the apoptosis of undifferentiated cells. Met deprivation triggers rapid metabolic changes, including SAM, followed by reversible epigenetic modifications. Here, we show that short-term Met deprivation impairs the pluripotency network through epigenetic modification in a 3D suspension culture. The trimethylation of lysine 4 on histone H3 (H3K4me3) was drastically affected compared with other histone modifications. Short-term Met deprivation specifically affects the transcription start site (TSS) region of genes, such as those involved in the transforming growth factor β pathway and cholesterol biosynthetic process, besides key pluripotent genes such as NANOG and POU5F1. The expression levels of these genes decreased, correlating with the loss of H3K4me3 marks. Upon differentiation, Met deprivation triggers the upregulation of various lineage-specific genes, including key definitive endoderm genes, such as GATA6. Upon differentiation, loss of H3K27me3 occurs in many endodermal genes, switching from a bivalent to a monovalent (H3K4me3) state. In conclusion, Met metabolism maintains the pluripotent network with histone marks, and their loss potentiates differentiation.

MPC-16 CHARACTERISTICS AND PROGNOSTIC FACTORS OF H3K27M-MUTANT DIFFUSE MIDLINE GLIOMA

Abstract H3 K27M-mutant diffuse midline gliomas (DMG) is a still challenging disease with no effective medical therapies. We analyzed the characteristics and prognostic factors of 64 patients with midline gliomas including 18 patients with H3 K27M-mutant DMG treated in Kobe University Hospital from 2006 to 2021. The median age at diagnosis in midline gliomas was 50.1 years (range: 14-82 y). All tumors were located in thalamus n=25, corpus callosum n=8, brainstem n=8, spinal cord n=6, cerebellum n=15, and pineal region n=2. Isocitrate dehydrogenase 1 (IDH1) and H3 K27 mutation was recognized in 6 and 18 patients, respectively, which was mutually exclusive. In all midline gliomas, the identification of an H3 K27M mutation was significantly a poor prognosis factor (median 19.8 months vs 27.9 months, p=0.042). Compared to midline gliomas without histone H3 and IDH1 mutations (IDH1/H3 wild type), however, H3 K27M-mutant DMG had no worse overall survival (median 19.8 months vs 15.9 months, p=0.51). H3 K27M-mutant DMG is considered as a distinct WHO Grade 4 regardless of histological features, but the biological properties of H3 K27M mutation is not yet defined in IDH1 wild-type midline gliomas.

Ependymomas

Ependymal neoplasms are a heterogenous group of neoplasms arising from the progenitors of the cells lining the ventricular system and the spinal central canal. During the last few years, significant novel data concerning oncogenesis, molecular characteristics and clinical correlations of these tumours have been collected, with a strong relevance for their pathological classification. The recently published 5th edition of WHO Classification of Central Nervous System Tumours integrates this novel knowledge and represents a substantial update compared to the previous edition. Concerning supratentorial ependymomas, the previous RELA fusion-positive ependymoma has been renamed into ZFTA fusion-positive and the novel YAP1 fusion-positive ependymoma subtype has been added. Posterior fossa ependymomas should now be allocated either to the Type A or Type B subtypes based on molecular profiling or using the H3 K27me3 immunohistochemical surrogate. Regarding spinal ependymomas, a novel subtype has been added based on a distinctive molecular trait, presence of MYCN amplification, and on the unfavourable outcome. Finally, myxopapillary ependymoma is now classified as a grade 2 tumour in accordance with its overall prognosis which mirrors that of conventional spinal ependymomas. The aim of this review is to present these changes and summarize the current diagnostic framework of ependymal tumours, according to the most recent updates.

Epigenetic drug library screening reveals targeting DOT1L abrogates NAD+ synthesis by reprogramming H3K79 methylation in uveal melanoma

Uveal melanoma (UM) is the most frequent and life-threatening ocular malignancy in adults. Aberrant histone methylation contributes to the abnormal transcriptome during oncogenesis. However, a comprehensive understanding of histone methylation patterns and their therapeutic potential in UM remains enigmatic. Herein, using a systematic epi-drug screening and a high-throughput transcriptome profiling of histone methylation modifiers, we observed that disruptor of telomeric silencing-1-like (DOT1L), a methyltransferase of histone H3 lysine 79 (H3K79), was activated in UM, especially in the high-risk group. Concordantly, a systematic epi-drug library screening revealed that DOT1L inhibitors exhibited salient tumor-selective inhibitory effects on UM cells, both in vitro and in vivo. Combining Cleavage Under Targets and Tagmentation (CUT&Tag), RNA sequencing (RNA-seq), and bioinformatics analysis, we identified that DOT1L facilitated H3K79 methylation of nicotinate phosphoribosyltransferase (NAPRT) and epigenetically activated its expression. Importantly, NAPRT served as an oncogenic accelerator by enhancing nicotinamide adenine dinucleotide (NAD+) synthesis. Therapeutically, DOT1L inhibition epigenetically silenced NAPRT expression through the diminishment of dimethylation of H3K79 (H3K79me2) in the NAPRT promoter, thereby inhibiting the malignant behaviors of UM. Conclusively, our findings delineated an integrated picture of the histone methylation landscape in UM and unveiled a novel DOT1L/NAPRT oncogenic mechanism that bridges transcriptional addiction and metabolic reprogramming.