Abstract PO-019: Deciphering differentiation hierarchies, heritability and plasticity in human gliomas via single-cell multi-omics

Human diffuse gliomas are incurable brain tumors, where cellular state diversity fuels tumor progression and resistance to therapy. Single-cell RNA-sequencing (scRNAseq) studies recently charted the cellular states of the two major categories of human gliomas, IDH-mutant gliomas (IDH-MUT) and IDH-wildtype glioblastoma (GBM), showing that malignant cells partly recapitulate neurodevelopmental trajectories. This raises the central questions of how cell states are encoded epigenetically and whether unidirectional hierarchies or more plastic state transitions govern glioma cellular architectures. To address these questions, we generated multi-omics single-cell profiling, integrating DNA methylation (DNAme), transcriptome and genotyping of 1,728 cells from 11 GBM and IDH-MUT primary patient samples. The assessment of DNAme intra-tumoral heterogeneity of malignant cells revealed that single-cell DNAme profiles within tumors span multiple bulk subtypes, are associated with important biological features of malignant cells, and may be confounded by the tumor micro-environment. Such sources of intra-tumoral heterogeneity in bulk profiles are important to recognize, as DNAme profiling is increasingly being utilized for bulk clinical brain tumor classification. The direct comparison of the methylomes of distinct glioma cellular states revealed Polycomb repressive complex 2 (PRC2) targets DNAme as a key switch in the differentiation of malignant GBM cells. In contrast, dissecting aberrant circuits of hypermethylation and gene expression in IDH-MUT gliomas, we observed a decoupling of the promoter methylation-expression relationship, with disruption of CTCF insulation and enhancer vulnerability which increases with cellular differentiation. To define cell state transition dynamics directly in patient samples, we generated high-resolution lineage histories of glioma cells using heritable DNAme changes, and projected the scRNAseq-derived cell states onto the lineage trees. This analysis demonstrated that cell states are heritable across malignant gliomas and, while in IDH-MUT differentiation far outpaces de-differentiation, GBM harbors a higher degree of cellular state plasticity allowing reversion of GBM cells from a differentiated to a stem-like state. Overall, our work provides detailed insights into gliomagenesis, dissecting the epigenetic encoding, regulatory programs, and dynamics of the cellular states that drive human gliomas. Importantly, it also carries significant translational implication, as the high degree of de-differentiation in GBM challenges the paradigm of therapeutically targeting glioma stem-like cells to deprive tumors of their ability to regenerate. Citation Format: Federico Gaiti, Ronan Chaligne, Dana Silverbush, Joshua S. Schiffman, Hannah R. Weisman, Lloyd Kluegel, Simon Gritsch, Sunil D. Deochand, Alyssa R. Richman, Johanna Klughammer, Tommaso Biancalani, Christoph Muus, Caroline Sheridan, Alicia Alonso, Franco Izzo, Orit Rozenblatt-Rosen, Aviv Regev, Mario L. Suva, Dan A. Landau. Deciphering differentiation hierarchies, heritability and plasticity in human gliomas via single-cell multi-omics [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-019.