Glioblastoma (GBM), the most lethal primary brain tumor, often develops resistance to temozolomide (TMZ) through drug-tolerant persister cells (TTPCCs). Super-enhancers (SEs) have been implicated in such resistance. This study investigated the role of PPP1R15B, an SE-associated gene, in regulating the PPP1R15B/EIF2A axis in GBM TTPCCs.

TTPCCs were derived from patient-derived GBM organoids. RNA sequencing and ChIP-seq (GSE229600) confirmed PPP1R15B as SE-associated. The functional impact of PPP1R15B knockdown or RAPHIN-1 inhibition on EIF2A signaling, oxidative phosphorylation, mitochondrial function, and cell viability was assessed. Xenograft models were used to evaluate in vivo efficacy.

TTPCCs exhibited clinical-grade TMZ resistance, and RNA sequencing identified PPP1R15B as a key player. Correlation analysis showed that PPP1R15B levels positively associated with EIF2A expression (r² = 0.128) and negatively with MGMT (r² = –0.189). Across glioma stages, higher PPP1R15B/EIF2A expression correlated with reduced TMZ sensitivity. ChIP-seq revealed heightened SE activity at the PPP1R15B locus in TTPCCs, while functional assays indicated that disrupting PPP1R15B/EIF2A axis using RAPHIN-1 enhanced TMZ sensitivity, diminished cell viability, and promoted apoptosis. Mechanistically, PPP1R15B inhibition impaired OXPHOS and induced mitochondrial dysfunction, intensifying metabolic stress. In vivo, RAPHIN-1 combined with TMZ significantly reduced tumor growth and depleted TTPCCs by reducing the PPP1R15B/EIF2A axis expression.

The PPP1R15B super-enhancer critically drives the PPP1R15B/EIF2A axis and TMZ resistance in GBM TTPCCs. Targeting this axis may offer a promising therapeutic approach to overcome TMZ resistance.