Characterization of low-grade epilepsy-associated tumor from implanted stereoelectroencephalography electrodes.

Low-grade epilepsy-associated tumors (LEATs) are a common cause of drug-resistant epilepsy in children. Herein, we demonstrate the feasibility of using tumor tissue derived from stereoelectroencephalography (sEEG) electrodes upon removal to molecularly characterize tumors and aid in diagnosis. An 18-year-old male with focal epilepsy and MRI suggestive of a dysembryoplastic neuroepithelial tumor (DNET) in the left posterior temporal lobe underwent implantation of seven peri-tumoral sEEG electrodes for peri-operative language mapping and demarcation of the peri-tumoral ictal zone prior to DNET resection. Using electrodes that passed through tumor tissue, we show successful isolation of tumor DNA and subsequent analysis using standard methods for tumor classification by DNA, including Glioseq targeted sequencing and DNA methylation array analysis. This study provides preliminary evidence for the feasibility of molecular diagnosis of LEATs or other lesions using a minimally invasive method with microscopic tissue volumes. The implications of sEEG electrodes in tumor characterization are broad but would aid in diagnosis and subsequent targeted therapeutic strategies.

An ERK5-PFKFB3 axis regulates glycolysis and represents a therapeutic vulnerability in pediatric diffuse midline glioma.

Metabolic reprogramming in pediatric diffuse midline glioma is driven by gene expression changes induced by the hallmark histone mutation H3K27M, which results in aberrantly permissive activation of oncogenic signaling pathways. Previous studies of diffuse midline glioma with altered H3K27 (DMG-H3K27a) have shown that the RAS pathway, specifically through its downstream kinase, extracellular-signal-related kinase 5 (ERK5), is critical for tumor growth. Further downstream effectors of ERK5 and their role in DMG-H3K27a metabolic reprogramming have not been explored. We establish that ERK5 is a critical regulator of cell proliferation and glycolysis in DMG-H3K27a. We demonstrate that ERK5 mediates glycolysis through activation of transcription factor MEF2A, which subsequently modulates expression of glycolytic enzyme PFKFB3. We show that in vitro and mouse models of DMG-H3K27a are sensitive to the loss of PFKFB3. Multi-targeted drug therapy against the ERK5-PFKFB3 axis, such as with small-molecule inhibitors, may represent a promising therapeutic approach in patients with pediatric diffuse midline glioma.

Targeting mitochondrial energetics reverses panobinostat- and marizomib-induced resistance in pediatric and adult high-grade gliomas.

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.

The landscape of tumor cell states and spatial organization in H3-K27M mutant diffuse midline glioma across age and location.

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.

Loss of MAT2A compromises methionine metabolism and represents a vulnerability in H3K27M mutant glioma by modulating the epigenome.

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.