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, Amit Gujar1, 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 associated with cell-to-cell DNA methylation differences, elevated in more aggressive tumors, linked with transcriptional disruption, and associated with environmental stress response. We demonstrate via in vitro perturbation experiments coupled with single cell RNA sequencing that the stressors, hypoxia and irradiation, increase local DNA methylation instability and are associated with cell state transitions. We identified a positive association between genetic and epigenetic instability that was supported by analyzing larger cohorts of bulk longitudinally collected 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.