A new IDH-independent hypermethylation phenotype is associated with astrocyte-like cell state in glioblastoma

DNA methylation plays a crucial role in cancer development and progression and has been linked to genetically and clinically distinct tumor classes, including IDH-mutated and IDH-wildtype adult-type diffuse gliomas. Here, we identified a CpG-island methylator phenotype (CIMP) that characterizes the receptor tyrosine kinase 2 (RTK2) subtype of IDH-wildtype glioblastoma. This RTK2-CIMP affects genomic locations and cell functions distinct from those of IDH mutation-associated IDH-CIMP and suppresses the expression of its target genes. The RTK2-CIMP-region chromatin is characterized by a combination of repressive and activating marks, including polycomb-associated H3K27me3 and enhancer-associated H3K4me1, consistent with DNA methylation-mediated silencing of genes with bivalent-state promoters in neural progenitor cells. Functionally, RTK2-CIMP affects neuronal lineage genes and is significantly associated with astrocyte (AC)-like glioblastoma, suggesting that RTK2-CIMP is an epigenetic signature of the AC-like cell state. Furthermore, we demonstrated that RTK2-CIMP can be induced by genetic manipulation in glioblastoma cells, suggesting that RTK2-CIMP is a key contributor to cell-state plasticity in glioblastoma. In mGB1 cells, Sox10 was stably knocked down using short hairpin RNA (shRNA). Briefly, nonoverlapping target sequences of shRNA sequences (sh1: TRCN0000018985; sh2: TRCN0000244290; Table EV1) were cloned and inserted into pLKO.1-puro (Sigma, SHG002). For the delivery of shRNAs, lentiviruses of pLKO.1-non target control, sh1 and sh2 were produced and transduced into mGB1 cells. Transduced cells were selected with 1 µg/ml puromycin for 48 h, and the K.D. efficiency was checked by both qPCR and Western blot.