H3 Mutations Research Articles

Pediatric glioma histone H3.3 K27M/G34R mutations drive abnormalities in PML nuclear bodies

BackgroundPoint mutations in histone variant H3.3 (H3.3K27M, H3.3G34R) and the H3.3-specific ATRX/DAXX chaperone complex are frequent events in pediatric gliomas. These H3.3 point mutations affect many chromatin modifications but the exact oncogenic mechanisms are currently unclear. Histone H3.3 is known to localize to nuclear compartments known as promyelocytic leukemia (PML) nuclear bodies, which are frequently mutated and confirmed as oncogenic drivers in acute promyelocytic leukemia.ResultsWe find that the pediatric glioma-associated H3.3 point mutations disrupt the formation of PML nuclear bodies and this prevents differentiation down glial lineages. Similar to leukemias driven by PML mutations, H3.3-mutated glioma cells are sensitive to drugs that target PML bodies. We also find that point mutations in IDH1/2—which are common events in adult gliomas and myeloid leukemias—also disrupt the formation of PML bodies.ConclusionsWe identify PML as a contributor to oncogenesis in a subset of gliomas and show that targeting PML bodies is effective in treating these H3.3-mutated pediatric gliomas.

Serology and genomic analysis of para-Bombay individuals in a hospital in Hunan Province

To investigate the serological and genetic characteristics of para-Bombay patients in a hospital in Hunan Province. A retrospective analysis was conducted on the blood type results of 175 439 hospitalized patients born in Hunan Province from the Third Xiangya Hospital, Central South University from 2016 to 2021. Phenotypes of ABO blood group was analyzed by blood group serology, and molecular biological methods were used to analyze the genotype, including ABO genotyping by polymerase chain reaction-sequence specific primers (PCR-SSP) and fucosyltransferase 1 (FUT1) and fucosyltransferase 2 (FUT2) gene sequencing. The results showed that 3 cases of Ah and 1 case of Bh were detected. FUT1 sequencing showed that there were 2 cases of h3h3, 1 case of h1h1 and 1 case of h302h1, of which h302 (c.302C>T) was the first discovered mutation. FUT2 sequencing revealed that 4 cases were all Se357Se357. The pedigree study showed that the inheritance of para-Bombay blood group was consistent with autosomal dominant inheritance. In conclusion, the FUT1 gene mutations leading to para-Bombay blood group mainly include h3, h1 and h302, of which h3 mutation is the most common.

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.

TMIC-04. IMMUNE PROFILING OF PEDIATRIC ONCOHISTONE GLIOMAS REVEALS DIVERSE MYELOID POPULATIONS AND TUMOR-PROMOTING BEHAVIORS

Abstract Pediatric high-grade gliomas pHGG are lethal and frequently bear missense mutations in histone H3, which drive tumorigenesis by altering the epigenome and cell fate/differentiation. While previous studies showed intrinsic contingencies associated with tumor development, limited information exists on the tumor microenvironment (TME) and immune cells for these tumors. To define the immune landscape of pediatric tumors at single-cell resolution, we profiled and compared 69 pediatric gliomas samples using the Chromium 10X technologies, H3.3 K27M (N=19) and G34R (N=16) mutant tumors, low-grade gliomas (N=11) and ependymomas (N=23). Additionally, to enable the spatial resolution of immune lineages, we performed Imaging Mass Cytometry (IMC) on H3-mutant samples (H3.3 K27M (N=7) and G34R (N=5)). We demonstrate that pediatric histone H3-mutant gliomas are highly infiltrated by myeloid cells, more specifically resident microglia, bone-marrow derived macrophages (BMDM) and monocytes, and are devoid of lymphoid infiltration. Different myeloid subsets were found to interact with H3-mutant cancer cells. Classical BMDM showed strong interactions with H3-mutant cancer cells while monocytes subsets showed tendency to interact only with G34R cells. We have generated a novel H3.3K27M glioma immunocompetent murine model, establishing a highly-penetrant and reliable tool. H3.3K27M murine tumors recapitulated the TME from human tumors, showing great myeloid infiltration. Using in vivo orthotopic serial engraftments, we observed that secondaries transplanted tumors had a significantly worse survival, possessing a less diverse immune infiltration compared to initial engraftments and a dominant presence of myeloid cells. Interestingly, in vivo myeloid depletion combined with PD-1 blockade extended overall mice survival. Our findings provide a valuable characterization of the biology of these tumors, reinforcing the several roles of myeloid cells in the context of pediatric brain tumours, providing a framework for understanding the H3.3K27M and G34R tumors, including other pediatric gliomas, and designing future immunotherapies.

EPCO-30. UNCOVERING MECHANISMS OF RESISTANCE IN PEDIATRIC HIGH-GRADE GLIOMA (PHGG)

Abstract Pediatric high-grade gliomas (pHGGs) are currently the leading cause of cancer-related deaths in children, remaining incurable in most cases. A molecular subgroup of pHGGs harbor G34R mutations in the histone H3.3, although the mechanisms of the H3.3-G34R/V are not well established. In most of the patients, the tumors resist radiation and relapse after standard of care (SOC). We aim to study the molecular mechanisms mediating resistance in these tumors. Knowing why certain tumors resist treatment allows us to implement targeted therapies. We performed single cell RNA sequencing on 3 non-treated (NT) glioma bearing mice, 3 mice resistant to treatment (R), and 3 mice susceptible to treatment (S). We isolated the tumor tissue (including tumor microenvironment) and processed the cells for single cell RNA sequencing. Using R, the single-cell RNA sequencing data was converted to Seurat datasets for analysis and subsequently combined and clustered using UMAP. We used the ScType and SingleR software tools to annotate cell types. After cell annotation, Gene Set Enrichment Analysis (GSEA) was performed between R and S tumors for each cell cluster as well as NT vs Treated. GSEA showed that susceptible tumor cells showed increased immune responses when compared to resistant cells. Additionally, resistant cells showed increased neuronal phenotypes as well as suppression of the ERK 1/2 cascade pathway in comparison to susceptible cells. There were also numerous differences in immune cell subpopulations, which may help uncover mechanisms of resistance. For example, certain immune cells show signs of increased epigenetic modifications in resistant vs. susceptible glioma cells. Our results indicate that the resistance of pHGG to treatment is associated with the establishment of immunosuppressive signals from tumor cells, and the suppression of tumor growth mediated by downregulation of the MAPK/ERK cascade.

BIOM-46. STANDARDIZATION OF LIQUID BIOPSY FOR PEDIATRIC DIFFUSE MIDLINE GLIOMA USING DDPCR

Abstract BACKGROUND Diffuse midline glioma (DMG) is a highly morbid pediatric brain tumor. Up to 80% of DMGs harbor mutations in histone H3-encoding genes, associated with poor prognosis. We previously showed the feasibility of detecting H3 mutations in circulating tumor DNA (ctDNA) in the liquid biome of children diagnosed with DMG. However, detection of low levels of ctDNA is highly dependent on platform sensitivity and sample type. To address this, we optimized ctDNA detection sensitivity and specificity across two commonly used digital droplet PCR (ddPCR) platforms (RainDance and BioRad), and validated methods for detecting H3F3A c.83A >T (H3.3K27M) mutations in DMG CSF, plasma, and primary tumor specimens across three different institutions. METHODS DNA was extracted from H3.3K27M mutant and H3 wildtype (H3WT) specimens, including H3.3K27M tumor tissue (n = 4), CSF (n = 6), plasma (n = 4), and human primary pediatric glioma cells (H3.3K27M, n = 2; H3WT, n = 1). ctDNA detection was enhanced via PCR pre-amplification and use of distinct custom primers and fluorescent LNA probes for c.83 A >T H3F3A mutation detection. Mutation allelic frequency (MAF) was determined and validated through parallel analysis of matched H3.3K27M tissue specimens (n = 3). RESULTS We determined technical nuances between ddPCR instruments, and optimized sample preparation and sequencing protocols for H3.3K27M mutation detection and quantification. We observed 100% sensitivity and specificity for mutation detection in matched DMG tissue and CSF across assays, platforms and institutions. CONCLUSION ctDNA is reliably and reproducibly detected in the liquid biome using ddPCR, representing a clinically feasible, reproducible, and minimally invasive approach for DMG diagnosis, molecular subtyping and therapeutic monitoring.

IDENTIFYING ROLE OF YB1 AND NPM1 AS PART OF HISTONE 3 MUTANT PROTEIN INTERACTOME, IN H3 MUTATED PHGG

Abstract AIMS Paediatric high-grade gliomas (pHGG) are the leading cause of childhood cancer deaths, accounting for over 40% of deaths in the UK. pHGG tumours harbour histone H3 mutations, defining 50% of pHGG cases. Studies show prominent mutations as being H3.3K27M and H3.3G34R/V. Our previous research has identified unique and common interacting binding partners of H3-G34R and H3-G34V mutant proteins. Among these we have focused on YB-1 and Nucleophosmin (NPM1). YB-1 is multifunctional transcription factor and a well-known oncogenic protein. NPM1 is a molecular chaperone and frequently over- expressed in cancer as well. Our aim is to analyse the role of YB-1 and NPM1 in H3 mutated pHGG, in order to identify targeted therapeutic option for these diffcult to treat tumours. METHOD Further data analyses of mass spectrometric proteomic data were performed. Subsequently, H3 mutated glioma cell lines, and control H3 wild type pHGG cells were cultured and then subjected to proteomic analyses. A comparative immunofluorescence and microscopy, and western blot analyses were performed to investigate the role of YB-1 and NPM-1. RESULTS Results verified the proteomic data and further showed altered protein expression of YB-1 and altered localization of protein NPM1 in H3 mutated pHGG. CONCLUSIONS Results of the study support that YB-1 and NPM1 are important interacting proteins in H3G34-mutated pHGGs and can be explored as therapeutic targets in H3 mutated pHGG.

Pediatric-Type Diffuse High-Grade Gliomas

Pediatric-type diffuse high-grade glioma is a new tumor class defined in the 5th edition of the WHO Classification of Tumors of the Central Nervous System(WHO2021). The class includes the following four tumor types: diffuse midline glioma, H3 K27-altered; diffuse hemispheric glioma, H3 G34-mutant; diffuse pediatric-type high-grade glioma, H3-wildtype and IDH-wildtype; and infant-type hemispheric glioma. Genetic detection of histone H3 mutations as well as detection of fusion genes and even methylation classifiers may be necessary to diagnose these tumors. Therefore, understanding their clinical and radiographical features is vital. Urgent establishment of new treatments is necessary for diffuse midline glioma, H3 K27-altered, as it is associated with very poor prognosis and is generally resistant to temozolomide. In this chapter, we focus on the clinical, radiographical, pathological, and molecular features of pediatric-type diffuse high-grade gliomas and introduce promising new treatments for diffuse midline glioma, H3 K27-altered.

Correlation between Multiparametric MR Imaging and Molecular Genetics in Pontine Pediatric High-Grade Glioma.

Molecular profiling is a crucial feature in the "integrated diagnosis" of CNS tumors. We aimed to determine whether radiomics could distinguish molecular types of pontine pediatric high-grade gliomas that have similar/overlapping phenotypes on conventional anatomic MR images. Baseline MR images from children with pontine pediatric high-grade gliomas were analyzed. Retrospective imaging studies included standard precontrast and postcontrast sequences and DTI. Imaging analyses included median, mean, mode, skewness, and kurtosis of the ADC histogram of the tumor volume based on T2 FLAIR and enhancement at baseline. Histone H3 mutations were identified through immunohistochemistry and/or Sanger or next-generation DNA sequencing. The log-rank test identified imaging factors prognostic of survival from the time of diagnosis. Wilcoxon rank-sum and Fisher exact tests compared imaging predictors among groups. Eighty-three patients had pretreatment MR imaging and evaluable tissue sampling. The median age was 6 years (range, 0.7-17 years); 50 tumors had a K27M mutation in H3-3A, and 11, in H3C2/3. Seven tumors had histone H3 K27 alteration, but the specific gene was unknown. Fifteen were H3 wild-type. Overall survival was significantly higher in H3C2/3- compared with H3-3A-mutant tumors (P = .003) and in wild-type tumors compared with any histone mutation (P = .001). Lower overall survival was observed in patients with enhancing tumors (P = .02) compared with those without enhancement. H3C2/3-mutant tumors showed higher mean, median, and mode ADC_total values (P < .001) and ADC_enhancement (P < .004), with lower ADC_total skewness and kurtosis (P < .003) relative to H3-3A-mutant tumors. ADC histogram parameters are correlated with histone H3 mutation status in pontine pediatric high-grade glioma.

DIPG-35. IDENTIFYING DRIVER-SPECIFIC VULNERABILITIES IN PAEDIATRIC HIGH GRADE GLIOMA SUBTYPES

Abstract Paediatric high-grade gliomas (pHGGs) are incurable malignant brain tumours and a leading cause of cancer-related death in children. The majority of pHGGs carry lysine-to-methionine (K27M) or glycine-to-arginine/valine (G34R/V) mutations in histone variants H3.1 or H3.3. Moreover, histone mutations associate with different anatomical locations and co-segregating mutations defining distinct tumour subtypes within pHGG. However whether these co-occuring mutations can act as drivers to modify tumour phenotypes and drug sensitivities is currently unknown. In order to functionally evaluate the role of partner alterations and to identify new therapeutic targets, we developed in vivo tumour models of pHGG subtypes using in utero electroporation (IUE) in combination with piggyBac transposon and CRISPR technology. We also established ex vivo glioma stem cell (GSCs) lines from mouse models with different co-segregating mutations, able to engraft in syngeneic immune-competent mice. Then, we performed transcriptome analysis and drug screening identifying selective pharmacological vulnerabilities. Our approach represents a preclinical platform to evaluate subtype-specific precision therapies identifying new pathways involved in brain tumour initiation, progression and maintenance.

DIPG-21. LINKING DETERMINATION OF CELL FATE IN THE DEVELOPING CENTRAL NERVOUS SYSTEM TO DIFFUSE MIDLINE GLIOMA

Abstract Diffuse midline glioma, K27-altered (DMG) is a treatment resistant, uniformly fatal cancer that arises predominantly in children. Next-generation sequencing has revealed that the majority of patients with DMG harbor K27M mutations in canonical histone variants H3.1 and H3.3, leading to global epigenetic dysregulation and an oligodendrocyte precursor 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. We identified DLX2 occupancy of early (Olig2, Nkx2.2) and late (Myt1, Plp1) genes required in OPC differentiation in vivo. Co-expression of DLX2 to target sequences reduced reporter gene expression in vitro. The Dlx1/Dlx2 double knockout (DKO) mouse showed increases in Olig2, Nkx2.2, and Plp-1 expression in vivo. Transient over-expression of a Dlx2-GFP construct into murine DMG cells resulted in significant increased expression of Gad1/2 isoforms and decreased Olig2 and Nkx2.2, global restoration of H3K27me3, as well as a reduction in migration, invasion, and colony formation in vitro. An RNAseq database containing 45 human DMG cell lines showed an inverse correlation between DLX2 expression and OLIG1/2. Cell fate changes in DLX2high/OLIG1/2low cell lines were evaluated by knockout of DLX2 using CRISPR/Cas9. Concurrently, OLIG1/2high/DLX2low expressing cell lines underwent stable transfection of an 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 H3 marks (K27me3, K27ac), K27M, and OPC signature genes will be presented along with comparison of altered OPC genes from the Dlx1/Dlx2 DKO mouse as determined by RNAseq. The manipulation of DLX2 expression in human DMG cells both 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.

DIPG-17. TREATMENT OF DIFFUSE INTRINSIC PONTINE GLIOMA USING ALLELE-SPECIFIC SIRNA TARGETING H3.3 K27M MUTANT HISTONE

Abstract In recent years CNS tumors have surpassed leukemia and lymphoma as the deadliest forms of pediatric cancer, yet many of these tumors remain resistant to treatment. Understanding of the molecular pathology of a particularly fatal pediatric glioma, diffuse intrinsic pontine glioma (DIPG), has improved dramatically over the past decade. However, this knowledge has not yet yielded new targeted therapeutics or advances in treatment. The majority of DIPG tumors (~70-80%) harbor a K27M mutation in the histone H3.3 gene resulting in significant epigenetic changes which drive tumorigenesis. We are utilizing cutting edge siRNA chemistries to develop a therapeutic specifically targeting this oncohistone H3.3 K27M. It has been shown by others that ex vivo disruption of the H3.3 K27M mutant significantly delays tumor development in orthotopic xenograft models and could be a promising target for treatment of disease. New siRNA chemistries allow broad distribution and silencing throughout the brain with durable silencing for months. Utilizing this technology, we have engineered siRNA compounds that can discriminate between wild-type (WT) and K27M mutant H3.3 alleles. Our first-generation compounds achieved &amp;gt;95% silencing of K27M mutant with only 40% silencing of WT H3.3. We have developed second-generation compounds to improve the discrimination between the two alleles. These compounds allow us to selectively knockdown the mutant driver of DIPG tumorigenesis and we hypothesize that silencing of this oncohistone will revert the epigenetic landscape of DIPG to a more wild-type form detrimentally impacting tumor growth and progression. Together the in vivo potency of the new siRNA chemistries combined with the precision allele-specific targeting of mutant K27M oncohistone may be a promising path towards development of an effective and safe therapy for DIPG.

Screen time: an unbiased search for histone mutations that affect quiescence and chronological aging.

Quiescence, reversible cell cycle arrest, is essential for survival during nutrient limitations and the execution of precise developmental patterns. In yeast, entry into quiescence is associated with a loss of histone acetylation as the chromatin becomes tightly condensed. In this issue, Small and Osley performed an unbiased screen of mutations in histone H3 and H4 amino acids in budding yeast and identified histone residues that are critical for quiescence and chronological lifespan. The results indicate that multiple histone amino acids, likely affecting nucleosome structure and a wide range of chromatin-associated processes, can promote or inhibit quiescence entry. Many of the same histone amino acids are also critical regulators of chronological lifespan.

Oncohistones and disrupted development in pediatric-type diffuse high-grade glioma.

Recurrent, clonal somatic mutations in histone H3 are molecular hallmarks that distinguish the genetic mechanisms underlying pediatric and adult high-grade glioma (HGG), define biological subgroups of diffuse glioma, and highlight connections between cancer, development, and epigenetics. These oncogenic mutations in histones, now termed "oncohistones", were discovered through genome-wide sequencing of pediatric diffuse high-grade glioma. Up to 80% of diffuse midline glioma (DMG), including diffuse intrinsic pontine glioma (DIPG) and diffuse glioma arising in other midline structures including thalamus or spinal cord, contain histone H3 lysine 27 to methionine (K27M) mutations or, rarely, other alterations that result in a depletion of H3K27me3 similar to that induced by H3 K27M. This subgroup of glioma is now defined as diffuse midline glioma, H3K27-altered. In contrast, histone H3 Gly34Arg/Val (G34R/V) mutations are found in approximately 30% of diffuse glioma arising in the cerebral hemispheres of older adolescents and young adults, now classified as diffuse hemispheric glioma, H3G34-mutant. Here, we review how oncohistones modulate the epigenome and discuss the mutational landscape and invasive properties of histone mutant HGGs of childhood. The distinct mechanisms through which oncohistones and other mutations rewrite the epigenetic landscape provide novel insights into development and tumorigenesis and may present unique vulnerabilities for pHGGs. Lessons learned from these rare incurable brain tumors of childhood may have broader implications for cancer, as additional high- and low-frequency oncohistone mutations have been identified in other tumor types.

Targeting SWI/SNF ATPases in H3.3K27M diffuse intrinsic pontine gliomas

Diffuse midline gliomas (DMGs) including diffuse intrinsic pontine gliomas (DIPGs) bearing lysine-to-methionine mutations in histone H3 at lysine 27 (H3K27M) are lethal childhood brain cancers. These tumors harbor a global reduction in the transcriptional repressive mark H3K27me3 accompanied by an increase in the transcriptional activation mark H3K27ac. We postulated that H3K27M mutations, in addition to altering H3K27 modifications, reprogram the master chromatin remodeling switch/sucrose nonfermentable (SWI/SNF) complex. The SWI/SNF complex can exist in two main forms termed BAF and PBAF that play central roles in neurodevelopment and cancer. Moreover, BAF antagonizes PRC2, the main enzyme catalyzing H3K27me3. We demonstrate that H3K27M gliomas show increased protein levels of the SWI/SNF complex ATPase subunits SMARCA4 and SMARCA2, and the PBAF component PBRM1. Additionally, knockdown of mutant H3K27M lowered SMARCA4 protein levels. The proteolysis targeting chimera (PROTAC) AU-15330 that simultaneously targets SMARCA4, SMARCA2, and PBRM1 for degradation exhibits cytotoxicity in H3.3K27M but not H3 wild-type cells. AU-15330 lowered chromatin accessibility measured by ATAC-Seq at nonpromoter regions and reduced global H3K27ac levels. Integrated analysis of gene expression, proteomics, and chromatin accessibility in AU-15330-treated cells demonstrated reduction in the levels of FOXO1, a key member of the forkhead family of transcription factors. Moreover, genetic or pharmacologic targeting of FOXO1 resulted in cell death in H3K27M cells. Overall, our results suggest that H3K27M up-regulates SMARCA4 levels and combined targeting of SWI/SNF ATPases in H3.3K27M can serve as a potent therapeutic strategy for these deadly childhood brain tumors.

Common molecular features of H3K27M DMGs and PFA ependymomas map to hindbrain developmental pathways

Globally decreased histone 3, lysine 27 tri-methylation (H3K27me3) is a hallmark of H3K27-altered diffuse midline gliomas (DMGs) and group-A posterior fossa ependymomas (PFAs). H3K27-altered DMGs are largely characterized by lysine-to-methionine mutations in histone 3 at position 27 (H3K27M). Most PFAs overexpress EZH inhibitory protein (EZHIP), which possesses a region of similarity to the mutant H3K27M. Both H3K27M and EZHIP inhibit the function of the polycomb repressive complex 2 (PRC2) responsible for H3K27me3 deposition. These tumors often arise in neighboring regions of the brainstem and posterior fossa. In rare cases PFAs harbor H3K27M mutations, and DMGs overexpress EZHIP. These findings together raise the possibility that certain cell populations in the developing hindbrain/posterior fossa are especially sensitive to modulation of H3K27me3 states. We identified shared molecular features by comparing genomic, bulk transcriptomic, chromatin-based profiles, and single-cell RNA-sequencing (scRNA-seq) data from the two tumor classes. Our approach demonstrated that 1q gain, a key biomarker in PFAs, is prognostic in H3.1K27M, but not H3.3K27M gliomas. Conversely, Activin A Receptor Type 1 (ACVR1), which is associated with mutations in H3.1K27M gliomas, is overexpressed in a subset of PFAs with poor outcome. Despite diffuse H3K27me3 reduction, previous work shows that both tumors maintain genomic H3K27me3 deposition at select sites. We demonstrate heterogeneity in shared patterns of residual H3K27me3 for both tumors that largely segregated with inferred anatomic tumor origins and progenitor populations of tumor cells. In contrast, analysis of genes linked to H3K27 acetylation (H3K27ac)-marked enhancers showed higher expression in astrocytic-like tumor cells. Finally, common H3K27me3-marked genes mapped closely to expression patterns in the human developing hindbrain. Overall, our data demonstrate developmentally relevant molecular similarities between PFAs and H3K27M DMGs and support the overall hypothesis that deregulated mechanisms of hindbrain development are central to the biology of both tumors.

Developing H3K27M mutant selective radiosensitization strategies in diffuse intrinsic pontine glioma

Diffuse intrinsic pontine glioma (DIPG) is a rare but highly lethal pediatric and adolescent tumor located in the pons of the brainstem. DIPGs harbor unique and specific pathological and molecular alterations, such as the hallmark lysine 27-to-methionine (H3K27M) mutation in histone H3, which lead to global changes in the epigenetic landscape and drive tumorigenesis. While fractionated radiotherapy, the current standard of care, improves symptoms and delays tumor progression, DIPGs inevitably recur, and despite extensive efforts chemotherapy-driven radiosensitization strategies have failed to improve survival. Advances in our understanding of the role of epigenetics in the cellular response to radiation-induced DNA damage, however, offer new opportunities to develop combinational therapeutic strategies selective for DIPGs expressing H3K27M. In this review, we provide an overview of preclinical studies that explore potential radiosensitization strategies targeting the unique epigenetic landscape of H3K27M mutant DIPG. We further discuss opportunities to selectively radiosensitize DIPG through strategic inhibition of the radiation-induced DNA damage response. Finally, we discuss the potential for using radiation to induce anti-tumor immune responses that may be potentiated in DIPG by radiosensitizing-therapeutic strategies.

Epigenetic and Metabolic Changes in Diffuse Intrinsic Pontine Glioma.

Diffuse midline glioma (DMG), hitherto known as diffuse intrinsic pontine glioma (DIPG), is a rare and aggressive form of brain cancer that primarily affects children. Although the exact cause of DMG/DIPG is not known, a large proportion of DMG/DIPG tumors harbor mutations in the gene encoding the histone H3 protein, specifically the H3K27M mutation. This mutation decreases the level of H3K27me3, a histone modification that plays a vital role in regulating gene expression through epigenetic regulation. The mutation also alters the function of polycomb repressive complex 2 (PRC2), thereby preventing the repression of genes associated with cancer development. The decrease in H3K27me3 caused by the histone H3 mutation is accompanied by an increase in the level of H3K27ac, a post-translational modification related to active transcription. Dysregulation of histone modification markedly affects gene expression, contributing to cancer development and progression by promoting uncontrolled cell proliferation, tumor growth, and metabolism. DMG/DIPG alters the metabolism of methionine and the tricarboxylic acid cycle, as well as glucose and glutamine uptake. The role of epigenetic and metabolic changes in the development of DMG/DIPG has been studied extensively, and understanding these changes is critical to developing therapies targeting these pathways. Studies are currently underway to identify new therapeutic targets for DMG/DIPG, which may lead to the development of effective treatments for this devastating disease.

High temperature increases centromere-mediated genome elimination frequency and enhances haploid induction in Arabidopsis

Double haploid production is the most effective way to create true-breeding lines in a single generation. In Arabidopsis, haploid induction via mutation of the centromere-specific histone H3 (cenH3) has been shown when the mutant is outcrossed to the wild-type, and the wild-type genome remains in the haploid progeny. However, factors that affect haploid induction are still poorly understood. Here, we report that a mutant of the cenH3 assembly factor Kinetochore Null2 (KNL2) can be used as a haploid inducer when pollinated by the wild-type. We discovered that short-term temperature stress of the knl2 mutant increased the efficiency of haploid induction 10-fold. We also demonstrated that a point mutation in the CENPC-k motif of KNL2 is sufficient to generate haploid-inducing lines, suggesting that haploid-inducing lines in crops can be identified in a naturally occurring or chemically induced mutant population, avoiding the generic modification (GM) approach at any stage. Furthermore, a cenh3-4 mutant functioned as a haploid inducer in response to short-term heat stress, even though it did not induce haploids under standard conditions. Thus, we identified KNL2 as a new target gene for the generation of haploid-inducer lines and showed that exposure of centromeric protein mutants to high temperature strongly increases their haploid induction efficiency.

Epigenetic programming of pediatric high-grade glioma: Pushing beyond proof of concept to clinical benefit.

Pediatric high-grade gliomas (pHGG) are a molecularly diverse group of malignancies, each incredibly aggressive and in dire need of treatment advancements. Genomic analysis has revolutionized our understanding of these tumors, identifying biologically relevant subgroups with differing canonical mutational profiles that vary based on tumor location and age. In particular, the discovery of recurrent histone H3 mutations (H3K27M in diffuse midline glioma, H3G34R/V in hemispheric pediatric high-grade gliomas) as unique "oncohistone" drivers revealed epigenetic dysregulation as a hallmark of pediatric high-grade gliomas oncogenesis. While reversing this signature through epigenetic programming has proven effective in several pre-clinical survival models, early results from pediatric high-grade gliomas clinical trials suggest that epigenetic modifier monotherapy will likely not provide long-term disease control. In this review we summarize the genetic, epigenetic, and cellular heterogeneity of pediatric high-grade gliomas, and highlight potential paths forward for epigenetic programming in this devastating disease.