Single-cell multimodal glioma analyses reveal epigenetic regulators of cellular plasticity and environmental stress response AUTHORS: Kevin C. Johnson1,12¥, Kevin J. Anderson1,12, Elise T. Courtois1, Floris P. Barthel1,2, Frederick S. Varn1, Diane Luo1, Martine Seignon1, Eunhee Yi1, Hoon Kim1, Marcos RH Estecio3, Ming Tang4, Nicholas E. Navin5, Rahul Maurya1, Chew Yee Ngan1, Niels Verburg6, Philip C De Witt Hamer6, Ketan Bulsara7, Michael L. Samuels1, Sunit Das8,9,10, Paul Robson1,11, Roel GW Verhaak1¥ AUTHOR AFFILIATIONS: 1The Jackson Laboratory for Genomic Medicine, Farmington, CT, 06032, USA. 2Amsterdam UMC, Vrije Universiteit Amsterdam, Department of Pathology, Brain Tumor Center Amsterdam, Amsterdam UMC, Vrije Universiteit, Amsterdam, The Netherlands 3Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Houston, TX, US 4Department of Data Science, Dana-Farber Cancer Institute, Cambridge, MA, US 5Department of Genetics, The University of Texas MD Anderson Cancer Center, Houston, TX, US 6Amsterdam UMC, Vrije Universiteit Amsterdam, Department of Neurosurgery, Brain Tumor Center Amsterdam, de Boelelaan 1117, Amsterdam, The Netherlands 7Division of Neurosurgery, The University of Connecticut Health Center, Farmington, CT, US 8Arthur and Sonia Labatt Brain Tumour Research Centre, Hospital for SickKids, University of Toronto. 9Institute of Medical Science, University of Toronto. 10Division of Neurosurgery, Li Ka Shing Knowledge Institute, St. Michael’s Hospital, University of Toronto. 11Genetics and Genome Sciences, University of Connecticut School of Medicine 12Co-first author ABSTRACT: Glioma intratumoral heterogeneity enables adaptation to challenging microenvironments and contributes to universal therapeutic resistance. Here, we integrated 914 single-cell DNA methylomes, 55,284 single-cell transcriptomes, and bulk multi-omic profiles across 11 adult IDH-mutant or IDH-wild-type gliomas to delineate sources of intratumoral heterogeneity. We found that local DNA methylation instability, or epimutation burden, was elevated in more aggressive tumors, reflected intratumoral variability, linked with transcriptional disruption, and associated with environmental stress response. We show that the activation of cell-state specific transcription factors is impacted by epimutations and that loosened epigenetic control may facilitate cellular plasticity. Our analyses support that somatic copy number alterations (SCNAs) promote epigenetic instability and that SCNAs largely precede epigenetic and transcriptomic diversification during glioma evolution. We confirmed the link between genetic and epigenetic instability by analyzing larger cohorts of bulk longitudinally collected and spatially separated DNA methylation data. Increased DNA methylation instability was associated with accelerated disease progression, and recurrently selected DNA methylation changes were enriched for environmental stress response pathways. Our work provides an integrative framework to better understand glioma evolution and highlights the importance of epigenetic heterogeneity in shaping therapeutic response.