Abstract Cancer evolution constitutes a foremost obstacle to effective treatment, driving malignant cells to adapt and overcome therapy. Diffuse glioma illustrates the quandary of cancer evolution: despite maximal treatment, the disease invariably recurs. Recent single-cell RNA-sequencing (scRNAseq) profiling of gliomas in the Suvà laboratory showed that defined cellular states, developmental hierarchies and plasticity coordinate to fuel glioma growth, evolution and resistance to therapy. However, this raises the critical question of the determinants of glioma cell states. We hypothesize that genetic, epigenetic and micro-environmental determinants govern the biology of key cellular states that drive gliomas and their plasticity, calling for an integrative model of cancer evolution, encompassing all sources of intra-tumoral heritable variations. To address this challenge, the Landau laboratory developed novel multi-modality single-cell sequencing technologies that enable direct integration across genetic, epigenetic, and transcriptional dimensions of cell-to-cell variation, and have applied them to study clonal evolution in hematological malignancies. In this R01 co-PI collaborative grant, we seek to apply this multi-layered single-cell approach to human gliomas in order to define how cellular phenotypic plasticity and clonal evolution enable tumor cell fitness. Specifically, we will: define intrinsic and extrinsic determinants of cellular states in glioma (aim1), map the epigenetic encoding of cell state programs in glioma (aim2), and reconstruct high-resolution lineage histories of glioma single-cells to measure the heritability and plasticity of cell states (aim3). Altogether, this research proposal seeks to pioneer the integrative analysis of epigenetic identity with genetic and transcriptional information to systematically dissect drivers of cellular states that underlie glioma differentiation and evolution.