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.

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Glioblastoma is the most malignant primary brain tumor for which the prognosis remains dismal even with aggressive surgical, medical, and radiation therapies. Glioblastoma stem cells (GSCs) promote therapeutic resistance and cellular heterogeneity due to their self-renewal properties and capacity for plasticity. To understand the molecular processes essential for maintaining GSCs, we performed an integrative analysis comparing active enhancer landscapes, transcriptional profiles, and functional genomics profiles of GSCs and non-neoplastic neural stem cells (NSCs). We identified sorting nexin 10 (SNX10), an endosomal protein sorting factor, as selectively expressed in GSCs compared to NSCs and essential for GSC survival. Targeting SNX10 impaired GSC viability and proliferation, induced apoptosis, and reduced self-renewal capacity. Mechanistically, GSCs utilized endosomal protein sorting to promote platelet-derived growth factor receptor β (PDGFRβ) proliferative and stem cell signaling pathways through post-transcriptional regulation of the PDGFR tyrosine kinase. Targeting SNX10 expression extended survival of orthotopic xenograft-bearing mice, and high SNX10 expression correlated with poor glioblastoma patient prognosis, suggesting its potential clinical importance. Thus, our study reveals an essential connection between endosomal protein sorting and oncogenic receptor tyrosine kinase signaling and suggests that targeting endosomal sorting may represent a promising therapeutic approach for glioblastoma treatment.

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In the version of this article initially published, the Acknowledgements statement should have included the following: "S.C.M. is

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.

In previous studies, we demonstrated that panobinostat, a histone deacetylase inhibitor, and bortezomib, a proteasomal inhibitor, displayed synergistic therapeutic activity against pediatric and adult high‐grade gliomas. Despite the remarkable initial response to this combination, resistance emerged. Here, in this study, we aimed to investigate the molecular mechanisms underlying the anticancer effects of panobinostat and marizomib, a brain‐penetrant proteasomal inhibitor, and the potential for exploitable vulnerabilities associated with acquired resistance. RNA sequencing followed by gene set enrichment analysis (GSEA) was employed to compare the molecular signatures enriched in resistant compared with drug‐naïve cells. The levels of adenosine 5′‐triphosphate (ATP), nicotinamide adenine dinucleotide (NAD)+ content, hexokinase activity, and tricarboxylic acid (TCA) cycle metabolites required for oxidative phosphorylation to meet their bioenergetic needs were analyzed. Here, we report that panobinostat and marizomib significantly depleted ATP and NAD+ content, increased mitochondrial permeability and reactive oxygen species generation, and promoted apoptosis in pediatric and adult glioma cell lines at initial treatment. However, resistant cells exhibited increased levels of TCA cycle metabolites, which required for oxidative phosphorylation to meet their bioenergetic needs. Therefore, we targeted glycolysis and the electron transport chain (ETC) with small molecule inhibitors, which displayed substantial efficacy, suggesting that resistant cell survival is dependent on glycolytic and ETC complexes. To verify these observations in vivo, lonidamine, an inhibitor of glycolysis and mitochondrial function, was chosen. We produced two diffuse intrinsic pontine glioma (DIPG) models, and lonidamine treatment significantly increased median survival in both models, with particularly dramatic effects in panobinostat‐ and marizomib‐resistant cells. These data provide new insights into mechanisms of treatment resistance in gliomas.