Methylation array of cultured GBM models to create surrogate CNV profiles

We therefore investigated whether EGFR inhibition in GBM induces broad adaptive kinase responses that could be co-targeted to overcome resistance.

Methods

We molecularly profiled 29 patient-derived GBM cell lines for EGFR status and selected five representative models spanning distinct EGFR amplification states for functional analyses. Cells were treated with EGFR inhibitors and responses were assessed using 72-hour viability assays, time-resolved immunoblotting, and phosphoproteomics (LC-MS/MS) with kinase activity inference. Results: EGFR inhibitors preferentially impaired viability in EGFR-driven models and transiently reduced EGFR phosphorylation during the initial response.

However, partial restoration of EGFR phosphorylation and downstream signaling occurred after 24 hours of continuous inhibitor exposure. Phosphoproteomics revealed no evidence of broad kinome rewiring within this timeframe but instead identified increased EGFR protein levels associated with restored EGFR pathway activity. The phosphorylated-to-total EGFR ratio remained relatively stable, consistent with sustained target engagement despite signaling rebound.

Conclusions: Early adaptive responses to EGFR inhibition in GBM were not characterized by broad kinome rewiring but by restoration of EGFR signaling associated with increased EGFR abundance. These findings suggest that adaptive signaling remains largely EGFR-dependent despite continued inhibitor exposure, identifying regulation of EGFR abundance as a potential contributor to therapeutic resistance.