Correction to: Integrative study of EZH2 mutational status, copy number, protein expression and H3K27 trimethylation in AML/MDS patients

An amendment to this paper has been published and can be accessed via the original article.

Integrative study of EZH2 mutational status, copy number, protein expression and H3K27 trimethylation in AML/MDS patients

Background Mutations in the EZH2 gene are recurrently found in patients with myeloid neoplasms and are associated with a poor prognosis. We aimed to characterize genetic and epigenetic alterations of EZH2 in 58 patients (51 with acute myeloid leukemia and 7 with myelodysplastic or myeloproliferative neoplasms) by integrating data on EZH2 mutational status, co-occurring mutations, and EZH2 copy number status with EZH2 protein expression, histone H3K27 trimethylation, and EZH2 promoter methylation. Results EZH2 was mutated in 6/51 acute myeloid leukemia patients (12%) and 7/7 patients with other myeloid neoplasms. EZH2 mutations were not overrepresented in patients with chromosome 7q deletions or losses. In acute myeloid leukemia patients, EZH2 mutations frequently co-occurred with CEBPA (67%), ASXL1 (50%), TET2 and RAD21 mutations (33% each). In EZH2-mutated patients with myelodysplastic or myeloproliferative neoplasms, the most common co-mutations were in ASXL1 (100%), NRAS, RUNX1, and STAG2 (29% each). EZH2 mutations were associated with a significant decrease in EZH2 expression (p = 0.0002), which was similar in patients with chromosome 7 aberrations and patients with intact chromosome 7. An association between EZH2 protein expression and H3K27 trimethylation was observed in EZH2-unmutated patients (R2 = 0.2, p = 0.01). The monoallelic state of EZH2 was not associated with EZH2 promoter hypermethylation. In multivariable analyses, EZH2 mutations were associated with a trend towards an increased risk of death (hazard ratio 2.51 [95% confidence interval 0.87–7.25], p = 0.09); similarly, low EZH2 expression was associated with elevated risk (hazard ratio 2.54 [95% confidence interval 1.07–6.04], p = 0.04). Conclusions Perturbations of EZH2 activity in AML/MDS occur on different, genetic and non-genetic levels. Both low EZH2 protein expression and, by trend, EZH2 gene mutations predicted inferior overall survival of AML patients receiving standard chemotherapy.

ETMM-08 METABOLIC REGULATION OF THE EPIGENOME DRIVES LETHAL INFANTILE EPENDYMOMA

Ependymomas are malignant glial tumours that occur throughout the central nervous system. Of the nine distinct molecular subgroups of ependymoma, Posterior Fossa A (PFA), is the most prevalent, occurring in the hindbrain of infants and young children. Lacking highly recurrent somatic mutations, PFAs are thought to be a largely epigenetically driven entity, defined by hypomethylation at the histone 3 lysine 27 residue. Previous transcriptional analysis of PFAs revealed an enrichment of hypoxia signaling genes. Thus, we hypothesized that hypoxic signaling, in combination with a unique metabolic milieu, drive PFA oncogenesis through epigenetic regulation. In this study, we identified that PFA cells control the availability of specific metabolites under hypoxic conditions, resulting in diminished H3K27 trimethylation and increased H3K27 acetylation in vitro and in vivo. Unique to PFA cells, transient exposure to ambient oxygen results in irreversible cellular toxicity. Furthermore, perturbation of key metabolic pathways is sufficient to inhibit growth of PFA primary cultures in vitro. PFA cells sequester s-adenosylmethionine while upregulating EZHIP, a polycomb repressive complex 2 (PRC2) inhibitor, resulting in decreased H3K27 trimethylation. Furthermore, hypoxia fine-tunes the abundance of alpha-ketoglutarate and acetyl-CoA to fuel demethylase and acetyltransferase activity. Paradoxically, a genome-wide CRISPR knockout screen identified the core components of PRC2 as uniquely essential in PFAs. Our findings suggest that PFAs thrive in a narrow “Goldilocks” zone, whereby they must maintain a unique epigenome and deviation to increased or decreased H3K27 trimethylation results in diminished cellular fitness. Previously, we showed that PFAs have a putative cell of origin arising in the first trimester of development. Using single-cell RNAseq and metabolomics, we demonstrate that PFAs resemble the natural metabolic-hypoxic milieu of normal development. Therefore, targeting metabolism and/or the epigenome presents a unique opportunity for rational therapy for infants with PFA ependymoma.

EPEN-52. METABOLIC REGULATION OF THE EPIGENOME DRIVES LETHAL INFANTILE EPENDYMOMA

PFA ependymomas are a lethal glial malignancy of the hindbrain found in infants and toddlers. Lacking any highly recurrent somatic mutations, PFAs have been proposed to be a largely epigenetically driven entity, defined by hypomethylation at the histone 3 lysine 27 residue. Unfortunately, an almost complete lack of model systems has limited the discovery of novel PFA therapies. In this study, we have identified that the PFA hypoxic microenvironment controls the availability of specific metabolites, resulting in diminished H3K27 trimethylation and increased H3K27 acetylation in vitro and in vivo. Unique to PFA cells, transient exposure to ambient oxygen results in irreversible cellular toxicity. Furthermore, perturbation of key metabolic pathways is sufficient to inhibit growth of PFA primary cultures in vitro. Although PFA tumors exhibit a low basal level of H3K27me3, inhibition of H3K27 methylation paradoxically demonstrates significant and specific activity against PFA. Thus, we propose a “Goldilocks Model” of metabolic-epigenetic regulation in PFA ependymoma, whereby increased or decreased H3K27 trimethylation results in cell death. Mapping of PFA ependymoma tumours suggests a cell of origin arising in the first trimester of human development where there is a known hypoxic microenvironment. Therefore, targeting metabolism and/or the epigenome presents a unique opportunity for rational therapy for infants with PFA ependymoma.

DIPG-58. HISTONE H3 WILD-TYPE DIPG/DMG OVEREXPRESSING EZHIP EXTEND THE SPECTRUM OF DIFFUSE MIDLINE GLIOMAS WITH PRC2 INHIBITION BEYOND H3-K27M MUTATION

Diffuse midline gliomas (DMG) H3 K27M-mutant were introduced in the 2016 WHO Classification unifying diffuse intrinsic pontine gliomas (DIPG) and gliomas from the thalamus and spinal cord harboring a histone H3-K27M mutation leading to Polycomb Repressor Complex 2 (PRC2) inhibition. However, few cases of DMG tumors presenting a H3K27 trimethylation loss, but lacking an H3-K27M mutation were reported. To address this question, we combined a retrospective cohort of 10 patients biopsied for a DIPG at the Necker Hospital or included in the BIOMEDE trial (NCT02233049) and extended our analysis to H3-wildtype (WT) diffuse gliomas from other midline locations presenting either H3K27 trimethylation loss or ACVR1 mutation from Necker, ICR, the HERBY trial, the INFORM registry study and the St. Jude PCGP representing 9 additional cases. Genomic profiling identified alterations frequently found in DMG, but none could explain the observed loss of H3K27 trimethylation. Similar observations were previously made in the PF-A subgroup of ependymoma, where the H3K27me3 loss resulted from EZHIP/CXorf67 overexpression rather than H3-K27M mutations. We thus analyzed EZHIP expression and observed its overexpression in all but one H3-WT DMGs compared to H3-K27M mutated tumors (EZHIP negative). Strikingly, based on their DNA methylation profiles, all H3-WT DMG samples analyzed clustered close to H3-K27M DIPG, rather than EZHIP overexpressing PF-A ependymomas. To conclude, we described a new subgroup of DMG lacking H3-K27M mutation, defined by H3K27 trimethylation loss and EZHIP overexpression that can be detected by IHC. We propose that these EZHIP/H3-WT DMGs extend the spectrum of DMG with PRC2 inhibition beyond H3-K27M mutation.