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.

Broad Institute of MIT and Harvard Cambridge MA USA

Cancer Signalling Research Group School of Biomedical Sciences and Pharmacy College of Health Medicine and Wellbeing University of Newcastle Callaghan New South Wales Australia

Center for Data Driven Discovery in Biomedicine Children's Hospital of Philadelphia Philadelphia PA USA

Center for Neuropathology Ludwig Maximilians University Munich Germany

Central European Institute of Technology Masaryk University Brno Czech Republic

Department of Biology Faculty of Medicine Masaryk University Brno Czech Republic

Department of Clinical Cell Biology and FACS Core Unit St Anna Children's Cancer Research Institute Vienna Austria

Department of Neuro Oncology Brain Tumor Center The University of Texas MD Anderson Cancer Center Houston TX USA

Department of Neurological Surgery University of California San Francisco San Francisco CA USA

Department of Neurological Surgery University of Pittsburgh School of Medicine Pittsburgh PA USA

Department of Neurology and Neurological Sciences Stanford University School of Medicine Stanford CA USA

Department of Neurosurgery Medical University of Vienna Vienna Austria

Department of Oncologic Pathology Dana Farber Cancer Institute Boston MA USA

Department of Pathology and Laboratory Medicine Children's Hospital Los Angeles Keck School of Medicine of University of Southern California Los Angeles CA USA

Department of Pathology Boston Children's Hospital Boston MA USA

Department of Pathology Brigham and Women's Hospital Boston MA USA

Department of Pathology Center for Cancer Research Massachusetts General Hospital Boston MA USA

Department of Pediatric Oncology Dana Farber Boston Children's Cancer and Blood Disorders Center Boston MA USA

Department of Pediatrics and Adolescent Medicine Comprehensive Center for Pediatrics and Comprehensive Cancer Center Medical University of Vienna Vienna Austria

Department of Urology Comprehensive Cancer Center Medical University of Vienna Vienna Austria

Division of Neuropathology and Neurochemistry Department of Neurology Medical University of Vienna Vienna Austria

Division of Pediatric Hematology Oncology Department of Pediatrics Michigan Medicine Ann Arbor MI USA

Hopp Children's Cancer Center Heidelberg Heidelberg Germany

Howard Hughes Medical Institute Stanford CA USA

John G Rangos Sr Research Center Children's Hospital of Pittsburgh Pittsburgh PA USA

Massachusetts General Hospital Cancer Center Boston MA USA

Pediatric Oncology Department University Hospital Brno Faculty of Medicine Masaryk University ICRC Brno Czech Republic

Precision Medicine Program Hunter Medical Research Institute New Lambton Heights New South Wales Australia

Science for Life Laboratory Department of Biochemistry and Biophysics Stockholm University Stockholm Sweden

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