Alexander Jucht

A subset of patients with IDH-mutant glioma respond to inhibitors of mutant IDH (IDHi), yet the molecular underpinnings of such responses are not understood. Here, we profiled by single-cell or single-nucleus RNA-sequencing three IDH-mutant oligodendrogliomas from patients who derived clinical benefit from IDHi. Importantly, the tissues were sampled on-drug, four weeks from treatment initiation. We further integrate our findings with analysis of single-cell and bulk transcriptomes from independent cohorts and experimental models. We find that IDHi treatment induces a robust differentiation toward the astrocytic lineage, accompanied by a depletion of stem-like cells and a reduction of cell proliferation. Furthermore, mutations in NOTCH1 are associated with decreased astrocytic differentiation and may limit the response to IDHi. Our study highlights the differentiating potential of IDHi on the cellular hierarchies that drive oligodendrogliomas and suggests a genetic modifier that may improve patient stratification.

Glioblastoma, the most malignant brain tumor in adults, exhibits characteristic patterns of epigenetic alterations that await elucidation. The DNA methylome of glioblastoma revealed recurrent epigenetic silencing of HTATIP2, which encodes a negative regulator of importin β-mediated cytoplasmic-nuclear protein translocation. Its deregulation may thus alter the functionality of cancer-relevant nuclear proteins, such as the base excision repair (BER) enzyme N-methylpurine DNA glycosylase (MPG), which has been associated with treatment resistance in GBM. We found that induction of HTATIP2 expression in GBM cells leads to a significant shift of predominantly nuclear to cytoplasmic MPG, whereas depletion of endogenous HTATIP2 results in enhanced nuclear MPG localization. Reduced nuclear MPG localization, prompted by HTATIP2 expression or by depletion of MPG, yielded less phosphorylated-H2AX-positive cells upon treatment with an alkylating agent. This suggested reduced MPG-mediated formation of apurinic/apyrimidinic sites, leaving behind unrepaired DNA lesions, reflecting a reduced capacity of BER in response to the alkylating agent. Epigenetic silencing of HTATIP2 may thus increase nuclear localization of MPG, thereby enhancing the capacity of the glioblastoma cells to repair treatment-related lesions and contributing to treatment resistance.

Diffusely infiltrating gliomas - including glioblastoma (GBM), isocitrate dehydrogenase (IDH) mutant gliomas, and histone 3 (H3) altered gliomas - are primary brain tumors with an invariably fatal outcome. Despite advances in the understanding of their biology, standard, targeted and immune checkpoint inhibitor immunotherapies have proven ineffective in arresting their inexorable progression and associated morbidity and mortality. Recognizing the unique aspects of the immunogenicity of cancer cells, the last decade has seen the development and evaluation of vaccine-based therapies for the treatment of solid tumors, including gliomas. Here we review the current vaccine strategies for the treatment of GBM, IDH-mutant gliomas and diffuse midline glioma H3 K27M-altered. We discuss potential benefits and challenges of vaccine therapies in these specific patient populations.

Abstract BACKGROUND Glioblastoma (GBM) is an incurable and aggressive brain cancer marked by profound intra-tumoral heterogeneity. Malignant cells exist in four core transcriptional states: stem-like (OPC-like and NPC-like) and more differentiated astrocytic (AC-like) or mesenchymal (MES-like) states. Using spatial transcriptomics, we previously identified a layered tumor structure of these states, with hypoxia emerging as an organizing driver, implicating oxygen availability as a central factor in cell state dynamics. However, how these cellular states interact with each other and with the tumor microenvironment remains incompletely understood. METHODS To uncover state-specific vulnerabilities for reducing GBM cell state diversity, we conducted a small-molecule screen in gliomasphere models using over 1,600 cysteine-reactive covalent inhibitors, assessing their effects on cell states via RNA-seq. We then employed the Sonar metabolic reporter to visualize NAD+ and NADH levels in gliomaspheres. Through experiments with orthotopic xenografts in mice and co-culture with human cortical organoids, we characterized distinct redox profiles across GBM cell states by single-cell and bulk RNA-seq. Spatial single-cell transcriptomics and mass spectrometry imaging (MSI) metabolomics on adjacent sections of frozen patient samples were used to identify preferential metabolic programs in specific cell states. RESULTS Our small-molecule screen identified compounds that promote differentiation and are enriched for NRF2 activation, a master regulator of the antioxidant response. Spatial single-cell transcriptomics and mass spectrometry metabolomics experiments confirmed that MES-like cells exhibit a heightened glycolytic signature, whereas stem-like states preferentially depend on oxidative phosphorylation (OXPHOS) for their energetic needs. Finally, pharmacologic perturbations revealed that mitochondrial and glycolysis inhibition selectively deplete different cell state populations in gliomaspheres. CONCLUSION Our findings provide deeper insight into the metabolic underpinnings of GBM cell states and support the rationale for combinatorial therapies aimed at reducing intratumoral heterogeneity that exploit their divergent dependencies.