BACKGROUND: Diffuse midline glioma, H3 K27-altered (DMG) is a fatal tumour that arises in the midline structures of the brain. When located in the pons, it is more commonly referred to as diffuse intrinsic pontine glioma (DIPG). DMG/DIPG is usually diagnosed when children are < 10 years, and it has a median overall survival of < 12 months after diagnosis. Radiological imaging is still the gold standard for DIPG diagnosis while the use of biopsy procedures led to our knowledge on its biology, such as with the identification of the canonical histone H3K27M mutation. However, the need to improve survival encourages the development of non-invasive, fast and inexpensive assays on biofluids for optimizing molecular diagnoses in DMG/DIPG. Here, we propose a rapid, new, imaging and epigenetics-based approach to diagnose DMG/DIPG in the plasma of paediatric patients. METHODS: A total of 20 healthy children (mean age: 10.5 years) and 24 children diagnosed with DMG/DIPG (mean age: 8.5 years) were recruited. Individual histones (H2A, H2B, H3, H4, macroH2A1.1 and macroH2A1.2), histone dimers and nucleosomes were assayed in biofluids by means of a new advanced flow cytometry ImageStream(X)-adapted method. RESULTS: We report a significant increase in circulating histone dimers and tetramers (macroH2A1.1/H2B versus control: p value < 0.0001; macroH2A1.2/H2B versus control: p value < 0.0001; H2A/H2B versus control: p value < 0.0001; H3/H4 versus control: p value = 0.008; H2A/H2B/H3/H4 versus control: p value < 0.0001) and a significant downregulation of individual histones (H2B versus control: p value < 0.0001; H3 versus control: p value < 0.0001; H4 versus control: p value < 0.0001). Moreover, histones were also detectable in the cerebrospinal fluid (CSF) of patients with DMG/DIPG and in the supernatant of SF8628, OPBG-DIPG002 and OPBG-DIPG004 DMG/DIPG cell lines, with patterns mostly similar to each other, but distinct compared to blood plasma. CONCLUSIONS: In summary, we identified circulating histone signatures able to detect the presence of DMG/DIPG in biofluids of children, using a rapid and non-invasive ImageStream(X)-based imaging technology, which may improve diagnosis and benefit the patients.

• MeSH
• difuzní intrinsický pontinní gliom * genetika   diagnóza   krev   MeSH
• dítě MeSH
• gliom * genetika   diagnóza   krev   MeSH
• histony * genetika   krev   metabolismus   MeSH
• lidé MeSH
• mladiství MeSH
• mutace MeSH
• nádorové biomarkery krev   MeSH
• nádory mozkového kmene * genetika   diagnóza   krev   MeSH
• nukleozomy metabolismus   MeSH
• předškolní dítě MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• histony * MeSH
• nádorové biomarkery MeSH
• nukleozomy MeSH

Introduction: Pediatric brain tumours (PBT) are one of the most common malignancies during childhood, with variable severity according to the location and histological type. Certain types of gliomas, such a glioblastoma and diffuse intrinsic pontine glioma (DIPG), have a much higher mortality than ependymoma and medulloblastoma. Early detection of PBT is essential for diagnosis and therapeutic interventions. Liquid biopsies have been demonstrated using cerebrospinal fluid (CSF), mostly restricted to cell free DNA, which display limitations of quantity and integrity. In this pilot study, we sought to demonstrate the detectability and robustness of cell free histones in the CSF. Methods: We collected CSF samples from a pilot cohort of 8 children with brain tumours including DIPG, medulloblastoma, glioblastoma, ependymoma and others. As controls, we collected CSF samples from nine children with unrelated blood malignancies and without brain tumours. We applied a multichannel flow imaging approach on ImageStream(X) to image indiviual histone or histone complexes on different channels. Results: Single histones (H2A, macroH2A1.1, macroH2A1.2 H2B, H3, H4 and histone H3 bearing the H3K27M mutation), and histone complexes are specifically detectable in the CSF of PBT patients. H2A and its variants macroH2A1.1/macroH2A1/2 displayed the strongest signal and abundance, together with disease associated H3K27M. In contrast, mostly H4 is detectable in the CSF of pediatric patients with blood malignancies. Discussion: In conclusion, free histones and histone complexes are detectable with a strong signal in the CSF of children affected by brain tumours, using ImageStream(X) technology and may provide additive diagnostic and predictive information.

• Klíčová slova
• epigenetics, histones, imaging, liquid biopsy, pediatric brain tumors,
• Publikační typ
• časopisecké články MeSH

Histone 3 lysine27-to-methionine (H3-K27M) mutations most frequently occur in diffuse midline gliomas (DMGs) of the childhood pons but are also increasingly recognized in adults. Their potential heterogeneity at different ages and midline locations is vastly understudied. Here, through dissecting the single-cell transcriptomic, epigenomic and spatial architectures of a comprehensive cohort of patient H3-K27M DMGs, we delineate how age and anatomical location shape glioma cell-intrinsic and -extrinsic features in light of the shared driver mutation. We show that stem-like oligodendroglial precursor-like cells, present across all clinico-anatomical groups, display varying levels of maturation dependent on location. We reveal a previously underappreciated relationship between mesenchymal cancer cell states and age, linked to age-dependent differences in the immune microenvironment. Further, we resolve the spatial organization of H3-K27M DMG cell populations and identify a mitotic oligodendroglial-lineage niche. Collectively, our study provides a powerful framework for rational modeling and therapeutic interventions.

• MeSH
• dítě MeSH
• gliom * genetika   MeSH
• histony genetika   MeSH
• lidé MeSH
• methionin MeSH
• mutace MeSH
• nádorové mikroprostředí genetika   MeSH
• Racemethionin MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• histony MeSH
• methionin MeSH
• Racemethionin MeSH

Medulloblastoma (MB) comprises a group of heterogeneous paediatric embryonal neoplasms of the hindbrain with strong links to early development of the hindbrain1-4. Mutations that activate Sonic hedgehog signalling lead to Sonic hedgehog MB in the upper rhombic lip (RL) granule cell lineage5-8. By contrast, mutations that activate WNT signalling lead to WNT MB in the lower RL9,10. However, little is known about the more commonly occurring group 4 (G4) MB, which is thought to arise in the unipolar brush cell lineage3,4. Here we demonstrate that somatic mutations that cause G4 MB converge on the core binding factor alpha (CBFA) complex and mutually exclusive alterations that affect CBFA2T2, CBFA2T3, PRDM6, UTX and OTX2. CBFA2T2 is expressed early in the progenitor cells of the cerebellar RL subventricular zone in Homo sapiens, and G4 MB transcriptionally resembles these progenitors but are stalled in developmental time. Knockdown of OTX2 in model systems relieves this differentiation blockade, which allows MB cells to spontaneously proceed along normal developmental differentiation trajectories. The specific nature of the split human RL, which is destined to generate most of the neurons in the human brain, and its high level of susceptible EOMES+KI67+ unipolar brush cell progenitor cells probably predisposes our species to the development of G4 MB.

• MeSH
• antigen Ki-67 metabolismus   MeSH
• buněčná diferenciace * genetika   MeSH
• buněčný rodokmen MeSH
• histondemethylasy MeSH
• lidé MeSH
• meduloblastom * klasifikace   genetika   patologie   MeSH
• metencephalon * embryologie   patologie   MeSH
• mozeček embryologie   patologie   MeSH
• mutace MeSH
• nádory mozečku * klasifikace   genetika   patologie   MeSH
• proteiny hedgehog metabolismus   MeSH
• proteiny T-boxu metabolismus   MeSH
• represorové proteiny MeSH
• svalové proteiny MeSH
• transkripční faktory Otx nedostatek   genetika   MeSH
• transkripční faktory PEBP2A genetika   MeSH
• transkripční faktory MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• antigen Ki-67 MeSH
• CBFA2T2 myeloid-transforming gene-related protein MeSH Prohlížeč
• CBFA2T3 protein, human MeSH Prohlížeč
• core binding factor alpha MeSH Prohlížeč
• EOMES protein, human MeSH Prohlížeč
• histondemethylasy MeSH
• KDM6A protein, human MeSH Prohlížeč
• OTX2 protein, human MeSH Prohlížeč
• PRDM6 protein, human MeSH Prohlížeč
• proteiny hedgehog MeSH
• proteiny T-boxu MeSH
• represorové proteiny MeSH
• svalové proteiny MeSH
• transkripční faktory Otx MeSH
• transkripční faktory PEBP2A MeSH
• transkripční faktory MeSH

Diffuse midline gliomas (DMGs) bearing driver mutations of histone 3 lysine 27 (H3K27M) are incurable brain tumors with unique epigenomes. Here, we generated a syngeneic H3K27M mouse model to study the amino acid metabolic dependencies of these tumors. H3K27M mutant cells were highly dependent on methionine. Interrogating the methionine cycle dependency through a short-interfering RNA screen identified the enzyme methionine adenosyltransferase 2A (MAT2A) as a critical vulnerability in these tumors. This vulnerability was not mediated through the canonical mechanism of MTAP deletion; instead, DMG cells have lower levels of MAT2A protein, which is mediated by negative feedback induced by the metabolite decarboxylated S-adenosyl methionine. Depletion of residual MAT2A induces global depletion of H3K36me3, a chromatin mark of transcriptional elongation perturbing oncogenic and developmental transcriptional programs. Moreover, methionine-restricted diets extended survival in multiple models of DMG in vivo. Collectively, our results suggest that MAT2A presents an exploitable therapeutic vulnerability in H3K27M gliomas.

• MeSH
• epigenom MeSH
• gliom * genetika   MeSH
• histony genetika   MeSH
• methionin genetika   MeSH
• methioninadenosyltransferasa metabolismus   MeSH
• myši MeSH
• nádory mozku * genetika   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• histony MeSH
• Mat2a protein, mouse MeSH Prohlížeč
• methionin MeSH
• methioninadenosyltransferasa MeSH

Histone H3.3 glycine 34 to arginine/valine (G34R/V) mutations drive deadly gliomas and show exquisite regional and temporal specificity, suggesting a developmental context permissive to their effects. Here we show that 50% of G34R/V tumors (n = 95) bear activating PDGFRA mutations that display strong selection pressure at recurrence. Although considered gliomas, G34R/V tumors actually arise in GSX2/DLX-expressing interneuron progenitors, where G34R/V mutations impair neuronal differentiation. The lineage of origin may facilitate PDGFRA co-option through a chromatin loop connecting PDGFRA to GSX2 regulatory elements, promoting PDGFRA overexpression and mutation. At the single-cell level, G34R/V tumors harbor dual neuronal/astroglial identity and lack oligodendroglial programs, actively repressed by GSX2/DLX-mediated cell fate specification. G34R/V may become dispensable for tumor maintenance, whereas mutant-PDGFRA is potently oncogenic. Collectively, our results open novel research avenues in deadly tumors. G34R/V gliomas are neuronal malignancies where interneuron progenitors are stalled in differentiation by G34R/V mutations and malignant gliogenesis is promoted by co-option of a potentially targetable pathway, PDGFRA signaling.

• Klíčová slova
• GSX2, H3.3 G34R/V, PDGFRA, cell-of-origin, chromatin conformation, gliomas, interneuron progenitors, oncohistones, pediatric cancer, single-cell transcriptome,
• MeSH
• astrocyty metabolismus   patologie   MeSH
• biologické modely MeSH
• buněčný rodokmen MeSH
• chromatin metabolismus   MeSH
• embryo savčí metabolismus   MeSH
• epigeneze genetická MeSH
• genetická transkripce MeSH
• gliom genetika   patologie   MeSH
• histony genetika   metabolismus   MeSH
• interneurony metabolismus   MeSH
• karcinogeneze genetika   patologie   MeSH
• lysin metabolismus   MeSH
• mutace genetika   MeSH
• myši inbrední C57BL MeSH
• nádory mozku genetika   patologie   MeSH
• nervové kmenové buňky metabolismus   MeSH
• oligodendroglie metabolismus   MeSH
• přední mozek embryologie   MeSH
• přeprogramování buněk genetika   MeSH
• promotorové oblasti (genetika) genetika   MeSH
• regulace genové exprese u nádorů MeSH
• růstový faktor odvozený z trombocytů - receptor alfa genetika   metabolismus   MeSH
• stupeň nádoru MeSH
• transkriptom genetika   MeSH
• umlčování genů MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• chromatin MeSH
• histony MeSH
• lysin MeSH
• růstový faktor odvozený z trombocytů - receptor alfa MeSH