Histone H3.3G34-Mutant Interneuron Progenitors Co-opt PDGFRA for Gliomagenesis.

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.

Moderately high intake of folic acid has a negative impact on mouse embryonic development.

BACKGROUND: The incidence of neural tube defects has diminished considerably since the implementation of food fortification with folic acid (FA). However, the impact of excess FA intake, particularly during pregnancy, requires investigation. In a recent study, we reported that a diet supplemented with 20-fold higher FA than the recommended intake for rodents had adverse effects on embryonic mouse development at embryonic days (E)10.5 and 14.5. In this report, we examined developmental outcomes in E14.5 embryos after administering a diet supplemented with 10-fold higher FA than recommended to pregnant mice with and without a mild deficiency of methylenetetrahydrofolate reductase (MTHFR). METHODS: Pregnant mice with or without a deficiency in MTHFR were fed a control diet (recommended FA intake of 2 mg/kg diet for rodents) or an FA-supplemented diet (FASD; 10-fold higher than the recommended intake [20 mg/kg diet]). At E14.5, mice were examined for embryonic loss and growth retardation, and hearts were assessed for defects and for ventricular wall thickness. RESULTS: Maternal FA supplementation was associated with embryonic loss, embryonic delays, a higher incidence of ventricular septal defects, and thinner left and right ventricular walls, compared to mothers fed control diet. CONCLUSIONS: Our work suggests that even moderately high levels of FA supplementation may adversely affect fetal mouse development. Additional studies are warranted to evaluate the impact of high folate intake in pregnant women. Birth Defects Research (Part A), 2013. © 2012 Wiley Periodicals, Inc.

Disturbed one-carbon metabolism causing adverse reproductive outcomes in mice is associated with altered expression of apolipoprotein AI and inflammatory mediators PPARα, interferon-γ, and interleukin-10.

Low dietary choline or deficiency of methylenetetrahydrofolate reductase (Mthfr) leads to hyperhomocysteinemia (Hhcy) and adverse reproductive outcomes. Homocysteine reduces synthesis of ApoAI, the major lipoprotein in HDL-cholesterol; ApoAI is regulated by PPARα and has antiinflammatory properties. Our aim was to determine whether pregnancy complications due to genetic or nutritional deficiencies in 1-carbon metabolism could relate to dysregulation of ApoAI and inflammatory mediators. We fed pregnant mice, with or without a deficiency of Mthfr, control or choline-deficient (ChDD) diets for 10-12 wk and examined levels of ApoAI, PPARα, IFNγ, and IL-10. ApoAI mRNA was reduced in livers of Mthfr(+/-) mice and ApoAI protein was reduced due to Mthfr deficiency or choline deficiency in liver and plasma. Placental ApoAI protein was also reduced due to Mthfr genotype or choline-deficient diet and in developmentally delayed embryos. Reduced liver PPARα expression (mRNA and protein) was observed in ChDD-fed mice and was associated with increased methylation of a CpG dinucleotide in its promoter. Hepatic IFNγ increased due to genotype, and placental IFNγ was higher in Mthfr(+/-) ChDD-fed dams compared to Mthfr(+/+) mice fed ChDD or Mthfr(+/-) mice fed CD. IL-10 was reduced in livers of ChDD-fed mice. We propose that a deficiency of dietary choline or Mthfr leads to Hhcy and reduced expression of maternal ApoAI, with reduced ApoAI transfer to embryo. Disturbances in 1-carbon metabolism also reduce maternal PPARα expression, possibly through promoter hypermethylation, and increase IFNγ and decrease IL-10 levels. This disturbance of the T helper (Th1) (IFNγ):Th2 (IL-10) ratio and the increase in inflammatory mediators may contribute to pregnancy complications.

H3.3 G34W Promotes Growth and Impedes Differentiation of Osteoblast-Like Mesenchymal Progenitors in Giant Cell Tumor of Bone.

Glycine 34-to-tryptophan (G34W) substitutions in H3.3 arise in approximately 90% of giant cell tumor of bone (GCT). Here, we show H3.3 G34W is necessary for tumor formation. By profiling the epigenome, transcriptome, and secreted proteome of patient samples and tumor-derived cells CRISPR-Cas9-edited for H3.3 G34W, we show that H3.3K36me3 loss on mutant H3.3 alters the deposition of the repressive H3K27me3 mark from intergenic to genic regions, beyond areas of H3.3 deposition. This promotes redistribution of other chromatin marks and aberrant transcription, altering cell fate in mesenchymal progenitors and hindering differentiation. Single-cell transcriptomics reveals that H3.3 G34W stromal cells recapitulate a neoplastic trajectory from a SPP1+ osteoblast-like progenitor population toward an ACTA2+ myofibroblast-like population, which secretes extracellular matrix ligands predicted to recruit and activate osteoclasts. Our findings suggest that H3.3 G34W leads to GCT by sustaining a transformed state in osteoblast-like progenitors, which promotes neoplastic growth, pathologic recruitment of giant osteoclasts, and bone destruction. SIGNIFICANCE: This study shows that H3.3 G34W drives GCT tumorigenesis through aberrant epigenetic remodeling, altering differentiation trajectories in mesenchymal progenitors. H3.3 G34W promotes in neoplastic stromal cells an osteoblast-like progenitor state that enables undue interactions with the tumor microenvironment, driving GCT pathogenesis. These epigenetic changes may be amenable to therapeutic targeting in GCT.See related commentary by Licht, p. 1794.This article is highlighted in the In This Issue feature, p. 1775.

Hyperhomocysteinemia is associated with hypertriglyceridemia in mice with methylenetetrahydrofolate reductase deficiency.

Hyperhomocysteinemia (HHcy) can result from genetic or nutritional disturbances in folate metabolism. The most common genetic cause of mild HHcy is methylenetetrahydrofolate reductase (MTHFR) deficiency. To explore interactions between HHcy and lipid metabolism in atherogenesis, we measured plasma homocysteine (Hcy), triglycerides and cholesterol in Mthfr(+/+) and Mthfr(+/-) mice on C57BL/6 and BALB/c backgrounds, fed control or folate-deficient diets. We also crossed ApoE(C57)(-/-) mice with Mthfr(C57)(+/-) and Mthfr(BALB/c)(+/-) mice, and examined the same plasma variables as well as lipid accumulation in aortic sinus and whole aorta. Mthfr(+/-) mice had significantly higher plasma Hcy and plasma triglycerides relative to Mthfr(+/+) mice. A significant positive correlation was observed between plasma Hcy and plasma triglycerides in all mice. Mthfr(+/-) mice had lower plasma ApoA-IV protein levels which could reduce clearance of triglyceride-rich lipoproteins from the circulation. In the double mutant experiments, plasma Hcy was higher in Mthfr(+/-) compared with Mthfr(+/+) in ApoE(C57)(-/-)/Mthfr(C57) and ApoE(C57)(-/-)/Mthfr(BALB/c) mice. Triglycerides in female ApoE(C57)(-/-)/Mthfr(BALB/c)(+/-) mice were higher than those in ApoE(C57)(-/-)/Mthfr(BALB/c)(+/-) mice and correlated positively with Hcy. ApoE(C57)(-/-)/Mthfr(C57)(+/-) male mice had more lipid deposition in aortic sinus and whole aorta compared with ApoE(C57)(-/-)/Mthfr(c57)(+/+) mice. Our results suggest that HHcy is associated with hypertriglyceridemia and that MTHFR deficiency may exacerbate lipid accumulation in ApoE deficiency.

Methylenetetrahydrofolate reductase deficiency and low dietary folate increase embryonic delay and placental abnormalities in mice.

BACKGROUND: Despite extensive research on mild methylenetetrahydrofolate reductase (MTHFR) deficiency and low dietary folate in different disorders, the association of these metabolic disturbances with a variety of congenital defects and pregnancy complications remains controversial. In this study we investigated the effects of MTHFR and dietary folate deficiency at 10.5 days post coitum (dpc) in our mouse model of mild MTHFR deficiency. METHODS: Mthfr +/+ and +/- female mice were fed a control or folic acid-deficient diet for 6 weeks, then mated with Mthfr +/- males. At 10.5 dpc, embryos were examined and placentae were collected for histologic evaluation. RESULTS: Maternal MTHFR and folate deficiencies resulted in increased developmental delays and smaller embryos. We also observed a low frequency of a variety of embryonic defects in the experimental groups, such as neural tube, heart looping, and turning defects; these results mimic the low incidence and multifactorial nature of these anomalies in humans. Folate-deficient mice also had increased embryonic losses and severe placental defects, including placental abruption and disturbed patterning of placental layers. Folate-deficient placentae had decreased ApoA-I expression, and there was a trend toward a negative correlation between ApoA-I expression with maternal homocysteine concentrations. CONCLUSIONS: Our study provides biological evidence linking maternal MTHFR and dietary folate deficiencies to adverse pregnancy outcomes in mice. It underscores the importance of folate not only in reducing the incidence of early embryonic defects, but also in the prevention of developmental delays and placental abnormalities that may increase susceptibility to other defects and to reproductive complications.

Homocysteine modulates the effect of simvastatin on expression of ApoA-I and NF-kappaB/iNOS.

AIMS: Statins can ameliorate atherosclerosis by inhibition of cholesterol biosynthesis or by modulation of inflammation. In earlier work, we showed that homocysteine (Hcy) reduced synthesis of apolipoprotein A-I (ApoA-I). Our goal in this study was to determine whether Hcy could interfere with the ability of simvastatin to increase ApoA-I synthesis or to modify statin-dependent regulation of inflammatory factors. METHODS AND RESULTS: Human HepG2 hepatocarcinoma cells and murine RAW264.7 macrophages were treated with simvastatin, with and without Hcy, to examine the expression of ApoA-I and nuclear factor-kappaB (NF-kappaB) or the NF-kappaB target, inducible nitric-oxide synthase (iNOS), respectively. Mice with methylenetetrahydrofolate reductase (Mthfr) deficiency, an animal model of hyperhomocysteinemia, were administered simvastatin (in diets or by injection) for in vivo assessment of these interactions. In HepG2 cells, Hcy reduced the statin-dependent increases in ApoA-I protein, mRNA, and ApoA-I promoter activity. In RAW264.7 macrophages, simvastatin decreased, whereas Hcy increased, the expression of pro-inflammatory NF-kappaB protein; in the presence of both Hcy and simvastatin, the pro-inflammatory effect of Hcy prevailed. Hcy increased mRNA levels of iNOS in the macrophage line; the combination of Hcy and simvastatin resulted in a trend towards greater induction. In mouse studies, simvastatin decreased cholesterol levels, but levels of ApoA-I in Mthfr-deficient mice remained lower than those in Mthfr(+/+) mice. Simvastatin injection increased iNOS protein and mRNA levels in peripheral blood of hyperhomocysteinemic Mthfr-deficient mice, but not in Mthfr(+/+) mice. The drug also increased MTHFR protein in cells and mouse liver, an effect that was modified by Hcy. CONCLUSION: These findings provide a link between statins and folate-dependent Hcy metabolism, and suggest that Hcy interferes with some anti-atherogenic and anti-inflammatory properties of simvastatin. Our work may have clinical relevance for hyperhomocysteinemic individuals on statin therapy.

H3K27M induces defective chromatin spread of PRC2-mediated repressive H3K27me2/me3 and is essential for glioma tumorigenesis.

Lys-27-Met mutations in histone 3 genes (H3K27M) characterize a subgroup of deadly gliomas and decrease genome-wide H3K27 trimethylation. Here we use primary H3K27M tumor lines and isogenic CRISPR-edited controls to assess H3K27M effects in vitro and in vivo. We find that whereas H3K27me3 and H3K27me2 are normally deposited by PRC2 across broad regions, their deposition is severely reduced in H3.3K27M cells. H3K27me3 is unable to spread from large unmethylated CpG islands, while H3K27me2 can be deposited outside these PRC2 high-affinity sites but to levels corresponding to H3K27me3 deposition in wild-type cells. Our findings indicate that PRC2 recruitment and propagation on chromatin are seemingly unaffected by K27M, which mostly impairs spread of the repressive marks it catalyzes, especially H3K27me3. Genome-wide loss of H3K27me3 and me2 deposition has limited transcriptomic consequences, preferentially affecting lowly-expressed genes regulating neurogenesis. Removal of H3K27M restores H3K27me2/me3 spread, impairs cell proliferation, and completely abolishes their capacity to form tumors in mice.

Pervasive H3K27 Acetylation Leads to ERV Expression and a Therapeutic Vulnerability in H3K27M Gliomas.

High-grade gliomas defined by histone 3 K27M driver mutations exhibit global loss of H3K27 trimethylation and reciprocal gain of H3K27 acetylation, respectively shaping repressive and active chromatin landscapes. We generated tumor-derived isogenic models bearing this mutation and show that it leads to pervasive H3K27ac deposition across the genome. In turn, active enhancers and promoters are not created de novo and instead reflect the epigenomic landscape of the cell of origin. H3K27ac is enriched at repeat elements, resulting in their increased expression, which in turn can be further amplified by DNA demethylation and histone deacetylase inhibitors providing an exquisite therapeutic vulnerability. These agents may therefore modulate anti-tumor immune responses as a therapeutic modality for this untreatable disease.

Mutant H3 histones drive human pre-leukemic hematopoietic stem cell expansion and promote leukemic aggressiveness.

Our ability to manage acute myeloid leukemia (AML) is limited by our incomplete understanding of the epigenetic disruption central to leukemogenesis, including improper histone methylation. Here we examine 16 histone H3 genes in 434 primary AML samples and identify Q69H, A26P, R2Q, R8H and K27M/I mutations (1.6%), with higher incidence in secondary AML (9%). These mutations occur in pre-leukemic hematopoietic stem cells (HSCs) and exist in the major leukemic clones in patients. They increase the frequency of functional HSCs, alter differentiation, and amplify leukemic aggressiveness. These effects are dependent on the specific mutation. H3K27 mutation increases the expression of genes involved in erythrocyte and myeloid differentiation with altered H3K27 tri-methylation and K27 acetylation. The functional impact of histone mutations is independent of RUNX1 mutation, although they at times co-occur. This study establishes that H3 mutations are drivers of human pre-cancerous stem cell expansion and important early events in leukemogenesis.

Stalled developmental programs at the root of pediatric brain tumors.

Childhood brain tumors have suspected prenatal origins. To identify vulnerable developmental states, we generated a single-cell transcriptome atlas of >65,000 cells from embryonal pons and forebrain, two major tumor locations. We derived signatures for 191 distinct cell populations and defined the regional cellular diversity and differentiation dynamics. Projection of bulk tumor transcriptomes onto this dataset shows that WNT medulloblastomas match the rhombic lip-derived mossy fiber neuronal lineage and embryonal tumors with multilayered rosettes fully recapitulate a neuronal lineage, while group 2a/b atypical teratoid/rhabdoid tumors may originate outside the neuroectoderm. Importantly, single-cell tumor profiles reveal highly defined cell hierarchies that mirror transcriptional programs of the corresponding normal lineages. Our findings identify impaired differentiation of specific neural progenitors as a common mechanism underlying these pediatric cancers and provide a rational framework for future modeling and therapeutic interventions.

Author Correction: Germline HAVCR2 mutations altering TIM-3 characterize subcutaneous panniculitis-like T cell lymphomas with hemophagocytic lymphohistiocytic syndrome.

In the version of this article originally published, the main-text sentence "In three patients of European ancestry, we identified the germline variant encoding p.Ile97Met in TIM-3, which was homozygous in two (P12 and P13) and heterozygous in one (P15) in the germline but with no TIM-3 plasma membrane expression in the tumor" misstated the identifiers of the two homozygous individuals, which should have been P13 and P14. The error has been corrected in the HTML, PDF and print versions of the paper.

Elevated homocysteine reduces apolipoprotein A-I expression in hyperhomocysteinemic mice and in males with coronary artery disease.

Hyperhomocysteinemia, a risk factor for cardiovascular disease, is caused by nutritional or genetic disturbances in homocysteine metabolism. A polymorphism in methylenetetrahydrofolate reductase (MTHFR) is the most common genetic cause of mild hyperhomocysteinemia. To examine mechanisms by which an elevation in plasma homocysteine leads to vascular disease, we first performed microarray analyses in livers of Mthfr-deficient mice and identified differentially expressed genes that are involved in lipid and cholesterol metabolism. Microarrays and RT-PCR showed decreased mRNA for apolipoprotein A (ApoA)-IV and for ApoA-I and increased mRNA for cholesterol 7alpha hydroxylase (Cyp7A1) in Mthfr(+/-) mice compared with Mthfr(+/+) mice. Western blotting revealed that ApoA-I protein levels in liver and plasma of Mthfr(+/-) mice were 52% and 62% of levels in the respective tissues of Mthfr(+/+) mice. We also performed Western analysis for plasma ApoA-I protein levels in 60 males with coronary artery disease and identified a significant (P<0.01) negative correlation (-0.33) between ApoA-I and plasma homocysteine levels. This cohort also displayed a negative correlation (-0.24, P=0.06) between high-density lipoprotein cholesterol and plasma homocysteine. Treatment of HepG2 cells with supraphysiological levels of 5 mmol/L homocysteine reduced peroxisome proliferator-activated receptor (PPAR) alpha and ApoA-I protein levels and decreased ApoA-I promoter activity. Transfection with a PPARalpha construct upregulated ApoA-I and MTHFR. Our results suggest that hyperhomocysteinemia may increase risk of atherosclerosis by decreasing expression of ApoA-I and increasing expression of CYP7A1.

TRPV4 and KRAS and FGFR1 gain-of-function mutations drive giant cell lesions of the jaw.

Giant cell lesions of the jaw (GCLJ) are debilitating tumors of unknown origin with limited available therapies. Here, we analyze 58 sporadic samples using next generation or targeted sequencing and report somatic, heterozygous, gain-of-function mutations in KRAS, FGFR1, and p.M713V/I-TRPV4 in 72% (42/58) of GCLJ. TRPV4 p.M713V/I mutations are exclusive to central GCLJ and occur at a critical position adjacent to the cation permeable pore of the channel. Expression of TRPV4 mutants in HEK293 cells leads to increased cell death, as well as increased constitutive and stimulated channel activity, both of which can be prevented using TRPV4 antagonists. Furthermore, these mutations induce sustained activation of ERK1/2, indicating that their effects converge with that of KRAS and FGFR1 mutations on the activation of the MAPK pathway in GCLJ. Our data extend the spectrum of TRPV4 channelopathies and provide rationale for the use of TRPV4 and RAS/MAPK antagonists at the bedside in GCLJ.

Germline HAVCR2 mutations altering TIM-3 characterize subcutaneous panniculitis-like T cell lymphomas with hemophagocytic lymphohistiocytic syndrome.

Subcutaneous panniculitis-like T cell lymphoma (SPTCL), a non-Hodgkin lymphoma, can be associated with hemophagocytic lymphohistiocytosis (HLH), a life-threatening immune activation that adversely affects survival1,2. T cell immunoglobulin mucin 3 (TIM-3) is a modulator of immune responses expressed on subgroups of T and innate immune cells. We identify in ~60% of SPTCL cases germline, loss-of-function, missense variants altering highly conserved residues of TIM-3, c.245A>G (p.Tyr82Cys) and c.291A>G (p.Ile97Met), each with specific geographic distribution. The variant encoding p.Tyr82Cys TIM-3 occurs on a potential founder chromosome in patients with East Asian and Polynesian ancestry, while p.Ile97Met TIM-3 occurs in patients with European ancestry. Both variants induce protein misfolding and abrogate TIM-3's plasma membrane expression, leading to persistent immune activation and increased production of inflammatory cytokines, including tumor necrosis factor-α and interleukin-1ß, promoting HLH and SPTCL. Our findings highlight HLH-SPTCL as a new genetic entity and identify mutations causing TIM-3 alterations as a causative genetic defect in SPTCL. While HLH-SPTCL patients with mutant TIM-3 benefit from immunomodulation, therapeutic repression of the TIM-3 checkpoint may have adverse consequences.

H3.3K27M Cooperates with Trp53 Loss and PDGFRA Gain in Mouse Embryonic Neural Progenitor Cells to Induce Invasive High-Grade Gliomas.

Gain-of-function mutations in histone 3 (H3) variants are found in a substantial proportion of pediatric high-grade gliomas (pHGG), often in association with TP53 loss and platelet-derived growth factor receptor alpha (PDGFRA) amplification. Here, we describe a somatic mouse model wherein H3.3K27M and Trp53 loss alone are sufficient for neoplastic transformation if introduced in utero. H3.3K27M-driven lesions are clonal, H3K27me3 depleted, Olig2 positive, highly proliferative, and diffusely spreading, thus recapitulating hallmark molecular and histopathological features of pHGG. Addition of wild-type PDGFRA decreases latency and increases tumor invasion, while ATRX knockdown is associated with more circumscribed tumors. H3.3K27M-tumor cells serially engraft in recipient mice, and preliminary drug screening reveals mutation-specific vulnerabilities. Overall, we provide a faithful H3.3K27M-pHGG model which enables insights into oncohistone pathogenesis and investigation of future therapies.

Characterizing temporal genomic heterogeneity in pediatric high-grade gliomas.

Pediatric high-grade gliomas (pHGGs) are aggressive neoplasms representing approximately 20% of brain tumors in children. Current therapies offer limited disease control, and patients have a poor prognosis. Empiric use of targeted therapy, especially at progression, is increasingly practiced despite a paucity of data regarding temporal and therapy-driven genomic evolution in pHGGs. To study the genetic landscape of pHGGs at recurrence, we performed whole exome and methylation analyses on matched primary and recurrent pHGGs from 16 patients. Tumor mutational profiles identified three distinct subgroups. Group 1 (n = 7) harbored known hotspot mutations in Histone 3 (H3) (K27M or G34V) or IDH1 (H3/IDH1 mutants) and co-occurring TP53 or ACVR1 mutations in tumor pairs across the disease course. Group 2 (n = 7), H3/IDH1 wildtype tumor pairs, harbored novel mutations in chromatin modifiers (ZMYND11, EP300 n = 2), all associated with TP53 alterations, or had BRAF V600E mutations (n = 2) conserved across tumor pairs. Group 3 included 2 tumors with NF1 germline mutations. Pairs from primary and relapsed pHGG samples clustered within the same DNA methylation subgroup. ATRX mutations were clonal and retained in H3G34V and H3/IDH1 wildtype tumors, while different genetic alterations in this gene were observed at diagnosis and recurrence in IDH1 mutant tumors. Mutations in putative drug targets (EGFR, ERBB2, PDGFRA, PI3K) were not always shared between primary and recurrence samples, indicating evolution during progression. Our findings indicate that specific key driver mutations in pHGGs are conserved at recurrence and are prime targets for therapeutic development and clinical trials (e.g. H3 post-translational modifications, IDH1, BRAF V600E). Other actionable mutations are acquired or lost, indicating that re-biopsy at recurrence will provide better guidance for effective targeted therapy of pHGGs.

Impaired H3K36 methylation defines a subset of head and neck squamous cell carcinomas.

Human papillomavirus (HPV)-negative head and neck squamous cell carcinomas (HNSCCs) are deadly and common cancers. Recent genomic studies implicate multiple genetic pathways, including cell signaling, cell cycle and immune evasion, in their development. Here we analyze public data sets and uncover a previously unappreciated role of epigenome deregulation in the genesis of 13% of HPV-negative HNSCCs. Specifically, we identify novel recurrent mutations encoding p.Lys36Met (K36M) alterations in multiple H3 histone genes. histones. We further validate the presence of these alterations in multiple independent HNSCC data sets and show that, along with previously described NSD1 mutations, they correspond to a specific DNA methylation cluster. The K36M substitution and NSD1 defects converge on altering methylation of histone H3 at K36 (H3K36), subsequently blocking cellular differentiation and promoting oncogenesis. Our data further indicate limited redundancy for NSD family members in HPV-negative HNSCCs and suggest a potential role for impaired H3K36 methylation in their development. Further investigation of drugs targeting chromatin regulators is warranted in HPV-negative HNSCCs driven by aberrant H3K36 methylation.

Spatial and temporal homogeneity of driver mutations in diffuse intrinsic pontine glioma.

Diffuse Intrinsic Pontine Gliomas (DIPGs) are deadly paediatric brain tumours where needle biopsies help guide diagnosis and targeted therapies. To address spatial heterogeneity, here we analyse 134 specimens from various neuroanatomical structures of whole autopsy brains from nine DIPG patients. Evolutionary reconstruction indicates histone 3 (H3) K27M--including H3.2K27M--mutations potentially arise first and are invariably associated with specific, high-fidelity obligate partners throughout the tumour and its spread, from diagnosis to end-stage disease, suggesting mutual need for tumorigenesis. These H3K27M ubiquitously-associated mutations involve alterations in TP53 cell-cycle (TP53/PPM1D) or specific growth factor pathways (ACVR1/PIK3R1). Later oncogenic alterations arise in sub-clones and often affect the PI3K pathway. Our findings are consistent with early tumour spread outside the brainstem including the cerebrum. The spatial and temporal homogeneity of main driver mutations in DIPG implies they will be captured by limited biopsies and emphasizes the need to develop therapies specifically targeting obligate oncohistone partnerships.

High folic acid consumption leads to pseudo-MTHFR deficiency, altered lipid metabolism, and liver injury in mice.

BACKGROUND: Increased consumption of folic acid is prevalent, leading to concerns about negative consequences. The effects of folic acid on the liver, the primary organ for folate metabolism, are largely unknown. Methylenetetrahydrofolate reductase (MTHFR) provides methyl donors for S-adenosylmethionine (SAM) synthesis and methylation reactions. OBJECTIVE: Our goal was to investigate the impact of high folic acid intake on liver disease and methyl metabolism. DESIGN: Folic acid-supplemented diet (FASD, 10-fold higher than recommended) and control diet were fed to male Mthfr(+/+) and Mthfr(+/-) mice for 6 mo to assess gene-nutrient interactions. Liver pathology, folate and choline metabolites, and gene expression in folate and lipid pathways were examined. RESULTS: Liver and spleen weights were higher and hematologic profiles were altered in FASD-fed mice. Liver histology revealed unusually large, degenerating cells in FASD Mthfr(+/-) mice, consistent with nonalcoholic fatty liver disease. High folic acid inhibited MTHFR activity in vitro, and MTHFR protein was reduced in FASD-fed mice. 5-Methyltetrahydrofolate, SAM, and SAM/S-adenosylhomocysteine ratios were lower in FASD and Mthfr(+/-) livers. Choline metabolites, including phosphatidylcholine, were reduced due to genotype and/or diet in an attempt to restore methylation capacity through choline/betaine-dependent SAM synthesis. Expression changes in genes of one-carbon and lipid metabolism were particularly significant in FASD Mthfr(+/-) mice. The latter changes, which included higher nuclear sterol regulatory element-binding protein 1, higher Srepb2 messenger RNA (mRNA), lower farnesoid X receptor (Nr1h4) mRNA, and lower Cyp7a1 mRNA, would lead to greater lipogenesis and reduced cholesterol catabolism into bile. CONCLUSIONS: We suggest that high folic acid consumption reduces MTHFR protein and activity levels, creating a pseudo-MTHFR deficiency. This deficiency results in hepatocyte degeneration, suggesting a 2-hit mechanism whereby mutant hepatocytes cannot accommodate the lipid disturbances and altered membrane integrity arising from changes in phospholipid/lipid metabolism. These preliminary findings may have clinical implications for individuals consuming high-dose folic acid supplements, particularly those who are MTHFR deficient.