STEM-04. LONGITUDINAL DNA METHYLATION CHANGE FUELS DISEASE PROGRESSION THROUGH ALTERED CELL STATE DIFFERENTIATION IN IDH-MUTANT GLIOMA

Abstract IDH-mutant glioma (IDH-G) is initially slow-growing but invariably progresses to fatal disease. While IDH-G is generally characterized by global hypermethylation at CpG islands - known as the glioma CpG islands methylator phenotype (G-CIMP), recent bulk profiling studies have shown that progressed IDH-G loses the G-CIMP status (shift to G-CIMP-low). However, the impact of G-CIMP loss on glioma transcriptional cell states and their inheritance has not been elucidated. To address the question, we leveraged recent advances in single-cell multi-omics technologies to profile a longitudinal cohort of 35 IDH-G tumor samples (15 pairs), co-capturing full-length transcriptional (by Smart-Seq2) and DNA methylation (by extended-representation bisulfite sequencing (XRBS)), within the same single-nuclei. Compared to reduced-representation bisulfite sequencing (RRBS) used for single-cell DNA methylation profiling in our prior multi-omic single-cell study (Nat Genet., 2021), XRBS provided higher coverage of CpG islands (mean of 378,888 vs 198,345.1, P = 2.2 x 10-16) in this study. Transcriptional and epigenetic comparisons of cell states during IDH-G progression revealed increased stem-like states, decreased differentiation and identified potential cell states regulators. Single-cell DNA methylation analysis demonstrated methylation loss in progressed tumors, however the degree of global DNA methylation was similar between different malignant cells states within individual tumors. This suggests that lower DNA methylation is not due to a change in cell state composition, but rather that it may alter cell state architecture. To address whether decreased methylation in progressed tumors affects cellular dynamics, we applied a quantitative framework to directly measure cell state heritability and plasticity based on transcriptional annotation of high-resolution phylogenetic trees in clinical samples. The analysis suggested that decreased methylation reshapes cellular hierarchy to increased heritability of stem-like states and decreased differentiation. This study provides insight into the impact of DNA methylation on glioma progression, integrating cell states transition dynamics with epigenetic evolution.