Histone Mutations Research Articles

DIPG-45. ELONGIN B REGULATES H3K27M CHROMATIN INCORPORATION TO PROMOTE MALIGNANCY IN DIFFUSE MIDLINE GLIOMA

Abstract Chromatin dysregulation is both a cause and consequence of aberrant RNA Polymerase II (Pol2) transcription in cancer. Some of the most striking examples of chromatin disruption in cancer include somatic mutations in genes encoding histone H3 (H3.3/H3.1K27M) observed in ~80% of diffuse midline glioma (DMG). H3K27M mutant histones act in a dominant manner to disrupt chromatin regulation and Pol2-dependent transcription in DMG. How H3K27M impinges on the Pol2 transcriptional machinery and whether aberrant Pol2 transcription can reciprocally induce chromatin dysregulation in DMG remain open questions. We conducted epigenome-focused CRISPR dropout screens and identified multiple regulators of Pol2 elongation, including Elongin B and C (ELOB/ELOC) as DMG genetic dependencies. Follow-up studies confirm that knockout (KO) of ELOB inhibits DMG proliferation in neurosphere culture and abrogates DMG tumorigenesis in orthotopic xenografts. Additional chromatin profiling studies reveal that ELOB binding sites are enriched in H3K27M mutant histones, suggesting that ELOB works together with H3K27M to promote malignant gene expression in DMG. Indeed, reciprocal knockout studies suggest that while ELOB chromatin binding patterns were not altered by H3K27M KO, ELOB KO significantly reduced H3K27M chromatin incorporation at thousands of genomic loci. Further RNAseq and PROseq analyses reveal that ELOB KO alters Pol2 transcription resulting in the up-regulation of genes associated with neuronal and muscle differentiation and the repression of glial stem cell genes. Together, these findings suggest that H3K27M chromatin binding patterns and aberrant gene regulation in DMG are, in part, dictated by ELOB and the Pol2 elongation machinery. Finally, analyses of patient datasets indicate that ELOB complex genes are overexpressed in high grade glioma and DMG and correlate with decreased patient survival. Collectively, these studies suggest that a functional interaction between ELOB and H3K27M mutant histones induces DMG-specific aberrations in chromatin and gene regulation to drive malignancy.

DIPG-46. TARGETING THE METABOLIC ERK5-PFKFB3 AXIS IN PEDIATRIC DIFFUSE MIDLINE GLIOMA

Abstract Diffuse midline glioma is an aggressive brain tumor with a median age at diagnosis of 6.3 years and 5-year survival rate of 2.2%.The defining histone mutation H3K27M precludes docking of a protein responsible for epigenetic modification resulting in erroneously accessible chromatin and subsequent transcription of oncogenic pathways, including the RAS-MERK5-ERK5 signaling cascade. To determine which oncogenic processes are regulated by extracellular signal-regulated kinase 5 (ERK5), gene-set enrichment analysis of patient-derived datasets demonstrated gene networks involved in glycolysis to be enriched with ERK5 expression. In confirmation, loss of ERK5 via shRNA interference reduced cell proliferation and glycolysis in DIPG IV and SF8628 cells. Reintroduction of ERK5 wildtype (WT) into ERK5 knockdown lines rescued cell proliferation and glycolysis, while the addition of ERK5 kinase dead domain (KDD) only partially rescued these survival and metabolic defects. Targeted evaluation of glycolysis enzyme expression via qRT-PCR revealed a direct relationship between ERK5 and proglycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3). Mechanistically, ERK5 activation of PFKFB3 was mediated by activation of transcriptional factor myocyte enhancer factor 2A (MEF2A) as demonstrated by coimmunoprecipitation and luciferase promoter assays. Expression of PFKFB3 was elevated at both the mRNA and protein level in DMG patient-derived samples. Genetic knockdown of PFKFB3 via shRNA interference resulted in reduced proliferation and glycolysis in DIPG IV and SF8628 cells. Similarly, pharmacologic inhibition of PFKFB3 with small molecule inhibitor PFK-158 capitulated these results. This inhibitor demonstrated blood brain barrier penetrance in silico and extended survival of in vivo mouse models. Multitargeted drug therapy against both ERK5 and PFKFB3 produced a synergetic in vitro response with increased sensitivity of these cells to apoptosis compared to single treatment alone. In conclusion, these results support ERK5 regulation of glycolysis through the critical metabolic effector PFKFB3. Multitargeted drug therapy against this axis represents a therapeutic vulnerability in pediatric diffuse midline 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-25. MAPPING TUMOR MICROENVIRONMENT USING EMERGING TECHNOLOGIES FOR PEDIATRIC BRAIN CANCERS

Abstract Diffuse midline glioma (DMG) is a fatal childhood brain cancer with a survival rate of less than one year from diagnosis. Pharmacological approaches as well as immunotherapy have failed to make a clinical impact. Incomplete understanding of the tumor microenvironment (TME) and tumor associated antigens have contributed to the observed poor prognosis. We hypothesize that characterization of the DMG TME will identify biology-informed targeted treatments. We thus obtained postmortem specimens from 70 patients diagnosed with brain cancers, including 50 DMGs, 20 other types and 10 non-CNS-cancer patients. Up to four anatomical brain locations were selected including the primary tumor, metastatic and adjacent healthy sites. Formalin-fixed-paraffin-embedded (FFPE) specimens were processed for constructing a tissue microarray (TMA). The TMA was stained for a number of markers (H&E, H3K27M, H3K27me3, KI67) and was scored by a neuropathologist. We then used a multiplexed immunofluorescence (MxIF) technology, Cell DIVE™, to iteratively probe 33 biomarkers on a single tissue slide, focusing on immune cell type profiling and activation, and histone mutation status. Analysis of biomarker density and spatial relationships are underway. The main expressed immune markers across all patients were CD163, CD68, and CD8. CD8, a cytotoxic T-cell biomarker, was highly detected in pons, cerebellum, thalamus and the frontal lobe (tumor and healthy) of DMG patients and varied according to clinical intervention, including ONC201 treatment. Furthermore, in comparison to other tumors, DMGs exhibited a higher expression of CD3, T- cell marker, and CD4, a T-helper cell biomarker. Analysis of Iba1, a microglial marker, confirmed a higher difference in microglial activation in primary tumor compared to metastatic and adjacent healthy tissue. In contrast, CD68 was significantly increased in metastatic sites. Analysis is still ongoing. We report establishment of the most comprehensive TMA for pediatric brain tumors, which provides insights into TME comparing critically important clinical variables.

MDB-16. TARGETING EPIGENETIC DYSREGULATION IN PAEDIATRIC BRAIN TUMOURS

Abstract Medulloblastoma (MB) is the most common paediatric malignant brain tumour and is classified into four distinct molecular subgroups (WNT, SHH, G3 and G4), each of them further divided into subtypes with different prognosis and responses to therapy. Deregulation of chromatin modifier genes play an essential role in MB, particularly in the G4 subgroup. A BMI1High;CHD7Low molecular signature identifies patients with poor survival. We have shown that BMI1High;CHD7Low sustains MB growth through regulation of MAPK/ERK signalling and via epigenetic regulation of inositol metabolism in both G4 MB cells and patients. These tumours display over-activation of MAPK/ERK and AKT/mTOR pathways leading to energetic rewiring characterised by enhanced glycolytic capacity and reduced mitochondrial function. We demonstrate that inositol administration counteracts this metabolic alteration and significantly extends survival in an in vivo pre-clinical model. Additionally, we identify a synergistic vulnerability of G4 MB to a combination treatment with BMI1 and MAPK/ERK inhibitors that overcomes the acquired resistance induced by single drug therapies. Importantly, we have now analysed recurrent G4 MB and found that the BMI1High;CHD7Low signature is maintained at recurrence and it continues to predict the pharmacological vulnerabilities we previously described. Finally, we extended the analysis to other paediatric brain tumours, including histone mutant gliomas and ependymomas, and identified molecularly defined subgroups that exhibit differential response to the compounds predicted to be effective by the BMI1High;CHD7Low signature, hence expanding the spectrum of tumour potentially amenable to these novel pharmacological approaches.

Chromatin structure related to oncogenesis.

Chromatin is the fundamental structure of genomic DNA in eukaryotic cells. The nucleosome, the primary unit of chromatin, consists of DNA and histone proteins, and is important for the maintenance of genomic DNA. Histone mutations are present in many types of cancers, suggesting that chromatin and/or nucleosome structures could be closely related to cancer development. Histone modifications and histone variants are also involved in regulating chromatin and nucleosome structures. Chromatin structures are dynamically changed by nucleosome binding proteins. In this review article, we discuss the current progress toward understanding the relationship between chromatin structure and cancer development.

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.

A single N-terminal amino acid determines the distinct roles of histones H3 and H3.3 in the Drosophila male germline stem cell lineage.

Adult stem cells undergo asymmetric cell divisions to produce 2 daughter cells with distinct cell fates: one capable of self-renewal and the other committed for differentiation. Misregulation of this delicate balance can lead to cancer and tissue degeneration. During asymmetric division of Drosophila male germline stem cells (GSCs), preexisting (old) and newly synthesized histone H3 are differentially segregated, whereas old and new histone variant H3.3 are more equally inherited. However, what underlies these distinct inheritance patterns remains unknown. Here, we report that the N-terminal tails of H3 and H3.3 are critical for their inheritance patterns, as well as GSC maintenance and proper differentiation. H3 and H3.3 differ at the 31st position in their N-termini with Alanine for H3 and Serine for H3.3. By swapping these 2 amino acids, we generated 2 mutant histones (i.e., H3A31S and H3.3S31A). Upon expressing them in the early-stage germline, we identified opposing phenotypes: overpopulation of early-stage germ cells in the H3A31S-expressing testes and significant germ cell loss in testes expressing the H3.3S31A. Asymmetric H3 inheritance is disrupted in the H3A31S-expressing GSCs, due to misincorporation of old histones between sister chromatids during DNA replication. Furthermore, H3.3S31A mutation accelerates old histone turnover in the GSCs. Finally, using a modified Chromatin Immunocleavage assay on early-stage germ cells, we found that H3A31S has enhanced occupancy at promoters and transcription starting sites compared with H3, while H3.3S31A is more enriched at transcriptionally silent intergenic regions compared to H3.3. Overall, these results suggest that the 31st amino acids for both H3 and H3.3 are critical for their proper genomic occupancy and function. Together, our findings indicate a critical role for the different amino acid composition of the N-terminal tails between H3 and H3.3 in an endogenous stem cell lineage and provide insights into the importance of proper histone inheritance in specifying cell fates and regulating cellular differentiation.

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 epigenetic aberrations of sarcoma in CRISPR era.

Sarcomas are rare malignancies that exhibit diverse biological, genetic, morphological, and clinical characteristics. Genetic alterations, such as gene fusions, mutations in transcriptional machinery components, histones, and DNA methylation regulatory molecules, play an essential role in sarcomagenesis. These mutations induce and/or cooperate with specific epigenetic aberrations required for the growth and maintenance of sarcomas. Appropriate mouse models have been developed to clarify the significance of genetic and epigenetic interactions in sarcomas. Studies using the mouse models for human sarcomas have demonstrated major advances in our understanding the developmental processes as well as tumor microenvironment of sarcomas. Recent technological progresses in epigenome editing will not only improve the studies using animal models but also provide a direct clue for epigenetic therapies. In this manuscript, we review important epigenetic aberrations in sarcomas and their representative mouse models, current methods of epigenetic editing using CRISPR/dCas9 systems, and potential applications in sarcoma studies and therapeutics.

Abstract 3484: Oncohistone H2B-E76K alters gene expression & cellular phenotype in a bronchial epithelial cell line

Abstract The nucleosome is the basic unit of chromatin, comprising 147 bp of DNA wrapped around an octamer of core histone proteins H2A, H2B, H3 and H4. Compact chromatin acts as a barrier to gene expression and is a key property governing DNA replication and gene expression. Analyzing over 46,000 in cancer patient samples in cBioPortal, we characterized mutations located in canonical histone genes and found several recurrent missense mutations. Foremost among the identified mutations was a glutamate to lysine missense mutation at amino acid 76 of histone H2B (H2B-E76K) that disrupts histone H2B-H4 interaction. Previously, we demonstrated that exogenous expression of H2B-E76K disrupted nucleosome stability, altered chromatin accessibility, and gene expression. We developed a new model utilizing CRISPR “knock-in” technology to integrate the E76K mutation into the endogenous H2BC4 gene of the lung epithelial cell line BEAS-2B to create a homozygous and heterozygous H2B-E76K cell line. Although the mutant histone only accounts for about 2-5% of total histone H2B, expression of H2B-E76K catalyzed distinct changes in cell migration, proliferation, colony formation and gene expression. CUT&RUN technology revealed mutant histones broadly incorporate throughout the genome. Gene set enrichment analysis (GSEA) revealed that H2B-E76K homozygous cells displayed expression changes similar to polycomb repressive complex 1 (PRC1) disruption or oncogenic KRAS expression. Using available datasets, we discovered that expression of H2B-E76K caused increased expression of PTN, PKIB, and CDH2, genes important in migration, epithelial-mesenchymal transition (EMT), and adhesion pathways. Furthermore, analysis of chromatin accessibility changes indicated that increased accessibility was significant in and around genes governing cell migration. Notably, H2B-E76K significantly increased heterogeneity of gene expression as measured using scRNA-seq. Together these results indicate that mutations disrupting histone structure may create millions of dysfunctional nucleosomes and new sites of “open” chromatin, resulting in loss of nucleosome-mediated gene repression and/or altered interactions with regulators of chromatin, fundamentally changing cell growth and migration properties to favor tumorigenesis. Citation Format: Kimberly Espinoza Pereira, Richard L. Bennett, Jixiu Shan, Jay Sarthy, Jonathan D. Licht. Oncohistone H2B-E76K alters gene expression & cellular phenotype in a bronchial epithelial cell line [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 3484.

Abstract 3562: Dissecting mechanisms underlying FOXR2-mediated gliomagenesis in diffuse midline gliomas

Abstract Background: Diffuse midline gliomas (DMGs) are a universally fatal brain tumor of childhood. While histone mutations are a critical tumor initiating event, they are insufficient to drive gliomagenesis. Histone mutations co-occur with somatic alterations in other pathways including TP53, MAPK, and MYC signaling. However, the mechanisms through which these pathways are activated have not been fully elucidated. Methods: We applied an integrative approach using transcriptomics, epigenetics, proteomics, in vitro cancer models, and in vivo mouse models to systematically evaluate how FOXR2 mediates gliomagenesis. Results: We have recently found that a subset of DMGs aberrantly express FOXR2, a forkhead transcription factor. FOXR2 is both sufficient to enhance tumor formation, and necessary for FOXR2-expressing DMGs. While FOXR2 indeed enhances MYC protein stability, FOXR2 exerts oncogenesis through MYC-independent functions and specifically hijacks E26-transformation specific (ETS) transcriptional circuits and FOXR2 DNA-binding is highly enriched at ETS motifs. We have performed proteomic and phospho-proteomic analysis of FOXR2-expressing human neural stem cells to identify proteins and phospho-sites that are highly enriched in FOXR2-expressing cells. Conclusion: Taken together, this study elucidates how FOXR2 interacts with ETS transcription factors to mediate oncogenesis, and further highlights a role for FOXR2 in activating ETS and MAPK signaling. Citation Format: Jessica W. Tsai, Paloma Cejas, Marissa Coppola, Dayle K. Wang, Smruti Patel, David W. Wu, Phonepasong Arounleut, Xin Wei, Ningxuan Zhou, Sudeepa Syamala, Frank P. Dubois, Kristine Pelton, Jayne Vogelzang, Cecilia Sousa, Audrey Baguette, Xiaolong Chen, Alexandra L. Condurat, Sarah E. Dixon-Clarke, Annarah Charles, Kevin N. Zhou, Sophie D. Lu, Elizabeth M. Gonzalez, Madison S. Chacon, Jeromy J. Digiacomo, Rushil Kumbhani, Dana Novikov, Maria Tsoli, David S. Ziegler, Uta Dirksen, Natalie Jager, Gnana Prakash Balasubramanian, Christof M. Kramm, Michaela Nathrath, Stefan Bielack, Suzanne J. Baker, Jinghui Zhang, James M. McFarland, Gad Getz, Francois Aguet, Nada Jabado, Olaf Witt, Stefan M. Pfister, Keith L. Ligon, Volker Hovestadt, Claudia Kleinman, Henry Long, David T. Jones, Pratiti Bandopadhayay, Timothy N. Phoenix. Dissecting mechanisms underlying FOXR2-mediated gliomagenesis in diffuse midline gliomas. [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 3562.

Abstract 2115: Zero-waste molecular diagnostics from biopsy needle wash water

Abstract Background: Over the past decade, the WHO classification of brain tumors has increasingly integrated molecular diagnostics into official classification criteria. However, biopsy tissue is precious due to high morbidity associated with repeat biopsies, and usually earmarked for histology, leaving little or no tissue for clinical molecular diagnostics. We have identified that the fluid used to wash biopsy needles after cores contains excess tumor DNA and cells that can be diagnostically useful. Additional research of tumor DNA and tumor tissue recovered from biopsy needle washes provides an opportunity for improvement in the diagnosis and treatment for brain tumor patients, while utilizing material that is otherwise thrown away. Methods: In two cases, surgeons were instructed to save 5-15ml of fluid used to wash biopsy needles. Isothermal targeted amplification of H3.3 and H3.1 histone genes was performed on raw wash fluid. Resulting amplicons were sequenced using an Oxford Nanopore MinION. DNA extracted from wash fluid was subjected to NGS sequencing via the Illumina TruSight Oncology 500 panel and compared to a CLIA certified NGS sequencing results from clinically indicated biopsy tissue. Results: For case 1, 5ul of raw wash fluid was subjected to rapid Loop-mediated Isothermal Amplification (LAMP) targeting the H3.1 and H3.3 K27M histone mutations and immediately sequenced using a MinION sequencer via rapid library preparation. Sequencing revealed a H3.3 K27M histone mutation present in wash fluid at 12% variant allele fraction (VAF). The variant call (99.99% CI) was available 1hr5mins after acquisition of material. For case 2, 5ml of wash fluid was processed using a cell-free DNA extraction kit resulting in ~250ng of recovered DNA. A LAMP assay targeting the H3.3 K27M mutation was performed on extracted DNA from wash fluid. Sequencing revealed an H3.3 K27M histone mutation present at 55% VAF, which was later corroborated using digital droplet PCR. Clinically indicated tissue from both biopsies was submitted for cancer panel sequencing in a CLIA certified setting. DNA extracted from wash fluid from both cases was subjected to Illumina TruSight Oncology 500 panel sequencing. In both cases, the panel was able to corroborate all SNV mutations reported by clinical sequencing that were also covered by the panel. Accrual of needle wash specimens is ongoing, with multiple monthly procedures. Conclusions: Large amounts of diagnostically useful material is present in biopsy needle wash fluid, which is usually discarded. Isothermal assays coupled with MinION sequencing can rapidly identify clinically relevant point mutations directly from wash fluid (e.g. IDH1), and enough DNA was recovered from wash fluid from both case-studies to submit for targeted cancer panel sequencing. These results suggest biopsy needle wash fluid should be considered a first-class diagnostic resource in any cancer. Citation Format: Jack Wadden, Vishal John, Seongbae Kong, Kait Verbal, Amy K. Bruzek, Wajd Al-Holou, Jason A. Heth, Hugh Garton, Carl Koschmann. Zero-waste molecular diagnostics from biopsy needle wash water [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 2115.

A screen for histone mutations that affect quiescence in S. cerevisiae.

Quiescence or G0 is a reversible state in which cells cease division but retain the ability to resume proliferation. Quiescence occurs in all organisms and is essential for stem cell maintenance and tissue renewal. It is also related to chronological lifespan - the survival of post-mitotic quiescent cells over time - and thus contributes to longevity. Important questions remain regarding the mechanisms that control entry into quiescence, maintenance of quiescence, and re-entry of quiescent cells into the cell cycle. S. cerevisiae has emerged as an excellent organism in which to address these questions because of the ease in which quiescent cells can be isolated. Following entry into G0, yeast cells remain viable for an extended period and can re-enter the cell cycle when exposed to growth-promoting signals. Histone acetylation is lost during the formation of quiescent cells and chromatin becomes highly condensed. This unique chromatin landscape regulates quiescence-specific transcriptional repression and has been linked to the formation and maintenance of quiescent cells. To ask if other chromatin features regulate quiescence, we conducted two comprehensive screens of histone H3 and H4 mutants, and identified mutants that show either altered quiescence entry or chronological lifespan. Examination of several quiescence entry mutants found that none of the mutants retain histone acetylation in quiescent cells, but show differences in chromatin condensation. A comparison of H3 and H4 mutants with altered chronological lifespan to those with altered quiescence entry found that chromatin plays both overlapping and independent roles in the continuum of the quiescence program.

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.

Post-Translation Modifications and Mutations of Human Linker Histone Subtypes: Their Manifestation in Disease.

Linker histones (LH) are a critical component of chromatin in addition to the canonical histones (H2A, H2B, H3, and H4). In humans, 11 subtypes (7 somatic and 4 germinal) of linker histones have been identified, and their diverse cellular functions in chromatin structure, DNA replication, DNA repair, transcription, and apoptosis have been explored, especially for the somatic subtypes. Delineating the unique role of human linker histone (hLH) and their subtypes is highly tedious given their high homology and overlapping expression patterns. However, recent advancements in mass spectrometry combined with HPLC have helped in identifying the post-translational modifications (PTMs) found on the different LH subtypes. However, while a number of PTMs have been identified and their potential nuclear and non-nuclear functions explored in cellular processes, there are very few studies delineating the direct relevance of these PTMs in diseases. In addition, recent whole-genome sequencing of clinical samples from cancer patients and individuals afflicted with Rahman syndrome have identified high-frequency mutations and therefore broadened the perspective of the linker histone mutations in diseases. In this review, we compile the identified PTMs of hLH subtypes, current knowledge of the relevance of hLH PTMs in human diseases, and the correlation of PTMs coinciding with mutations mapped in diseases.

Leptomeningeal disease in histone-mutant gliomas.

The 2016 WHO classification described a subtype of midline gliomas harboring histone 3 (H3) K27M alterations, and the 2021 edition added a new subtype of hemispheric diffuse gliomas with H3 G34R/V mutations. The incidence and clinical behavior of leptomeningeal disease (LMD) in these patients is not well defined. Retrospective study of patients with H3-altered gliomas diagnosed from 01/2012 to 08/2021; histone mutations were identified through next-generation sequencing (NGS) of tumor biopsy and/or cerebrospinal fluid (CSF). We identified 42 patients harboring H3 mutations (K27M mutations in 33 patients, G34R/V in 8, and both in one). Median age was 21 (4-70); 27 were male. LMD was diagnosed in 21/42 (50%) patients, corresponding to a 3-year cumulative incidence of 44.7% (95% confidence interval (CI): 26.1%-63.4%) for the K27-mutant group and a 1-year cumulative incidence of 37.5% in the G34-mutant group (95% CI: 0.01%-74.4%; no events after 1 year). Median time from tumor diagnosis to LMD was 12.9 months for H3-K27 patients and 5.6 months for H3-G34 patients. H3 mutation was detected in CSF in all patients with LMD who had NGS (8 H3-K27-mutant patients). In the H3-K27-mutant group, modeled risk of death was increased in patients who developed LMD (hazard ratio: 7.37, 95% CI: 2.98-18.23, P < .0001). In our cohort, 50% of patients developed LMD. Although further studies are needed, CSF ctDNA characterization may aid in identifying molecular tumor profiles in glioma patients with LMD, and neuroaxis imaging and CSF NGS should be considered for early LMD detection.

Regulation of telomere silencing by the core histones-autophagy-Sir2 axis.

Telomeres contain compacted heterochromatin, and genes adjacent to telomeres are subjected to transcription silencing. Maintaining telomere structure integrity and transcription silencing is important to prevent the occurrence of premature aging and aging-related diseases. How telomere silencing is regulated during aging is not well understood. Here, we find that the four core histones are reduced during yeast chronological aging, leading to compromised telomere silencing. Mechanistically, histone loss promotes the nuclear export of Sir2 and its degradation by autophagy. Meanwhile, reducing core histones enhances the autophagy pathway, which further accelerates autophagy-mediated Sir2 degradation. By screening the histone mutant library, we identify eight histone mutants and one histone modification (histone methyltransferase Set1-catalyzed H3K4 trimethylation) that regulate telomere silencing by modulating the core histones-autophagy-Sir2 axis. Overall, our findings reveal core histones and autophagy as causes of aging-coupled loss of telomere silencing and shed light on dynamic regulation of telomere structure during aging.

Functional Analysis of Histone ADP-Ribosylation In Vitro and in Cells.

Gene regulation in the nucleus requires precise control of the molecular processes that dictate how, when, and which genes are transcribed. The posttranslational modification (PTM) of histones in chromatin is an effective means to link cellular signaling to gene expression outcomes. The repertoire of histone PTMs includes phosphorylation, acetylation, methylation, ubiquitylation, and ADP-ribosylation (ADPRylation). ADPRylation is a reversible PTM that results in the covalent transfer of ADP-ribose units derived from NAD+ to substrate proteins on glutamate, aspartate, serine, and other amino acids. Histones were the first substrate proteins identified for ADPRylation, over five decades ago. Since that time, histone ADPRylation has been shown to be a widespread and critical regulator of chromatin structure and function during transcription, DNA repair, and replication. Here, we describe a set of protocols that allow the user to investigate site-specific histone ADPRylation and its functional consequences in biochemical assays and in cells in a variety of biological systems. With the recent discovery that some cancer-causing histone mutations (i.e., oncohistone mutations) occur at functional sites of regulatory ADPRylation, these protocols may have additional utility in studies of oncology.

Abstract IA007: Transforming chromatin: Oncohistone mutations in pediatric high-grade glioma

Abstract Pediatric diffuse high-grade gliomas (HGGs) are a heterogeneous spectrum of disease with abysmal survival rates. Approximately half of diffuse high-grade gliomas in children arise in midline structures predominantly the brainstem, but also thalamus, cerebellum and spinal cord. Approximately 80% of these tumors harbor H3 K27M mutations, which result in dramatic depletion of the post-translational modification H3K27me3. Alternative mutations in diffuse midline gliomas can result in similar reduction of H3K27me3, leading to a redefined classification of this collection of tumors as diffuse midline glioma, H3 K27-altered. In contrast, distinct histone H3 mutations, H3.3 G34R/V, are found in approximately 30% of diffuse gliomas arising in the cerebral hemispheres of older adolescents and young adults, defining the tumor subgroup of diffuse hemispheric glioma, H3 G34-mutant. The striking spatiotemporal pattern of these histone mutations, termed oncohistones, indicates an intimate association between epigenetic dysregulation, brain development, and tumorigenesis. We will discuss use of genetically engineered and patient-derived models to investigate the contribution of oncohistone mutations to disrupted development, epigenetic dysregulation and gliomagenesis. Citation Format: Kaitlin Budd, Chang-Hyuk Kwon, Lawryn H. Kasper, Christopher Roberts, Jordan Roach, Jennifer Ocasio-Adorno, Jon D. Larson, Suzanne J. Baker. Transforming chromatin: Oncohistone mutations in pediatric high-grade glioma. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr IA007.