RBFOX3 is associated with neuronal-like phenotypic transition in recurrent glioblastoma

Emerging evidence links neuronal-like phenotypic transition to glioblastoma (GB) recurrence and therapy resistance, yet the underlying mechanisms remain incompletely understood. We hypothesized that dysregulated alternative splicing leads to a neuronal-like state and chemoresistance at tumor recurrence. We reanalysed longitudinal bulk RNA-seq data from paired primary and recurrent GBs to quantify dynamic alternative splicing during tumor evolution. By integrating single-cell and spatial transcriptomics, we characterized shifts in cellular composition and spatial interactions linked to these splicing changes. We then prioritized splicing regulators and validated their effects on tumor cells using in vitro cell lines and co-culture assays. A subset of recurrent GBs exhibits neuronal programs and chemoresistance during tumor evolution, accompanied by pronounced differential splicing patterns. Single-cell analyses link these splicing changes to the co-enrichment of neurons and a neuronal-like tumor subpopulation at recurrence. Spatial transcriptomics further indicates close synaptic interactions between this tumor state and adjacent neurons in tumor-infiltrative regions. Through integrative computational ranking and experimental validation, we find that the neuron-specific splicing factor RBFOX3 promotes the neuronal-like GB tumor state by dysregulating splicing. In tumor-neuron co-culture systems, RBFOX3 overexpression is linked to the upregulation of presynaptic markers. Dysregulated alternative splicing mediated by RBFOX3 is associated with a neuronal-like tumor state and might contribute to chemoresistance at GB relapse, highlighting the potential role of splicing regulation in GB evolution.