Glioblastoma (GB) is the most common malignant primary brain tumor in adults and carries a median survival of ~14 months despite surgery, chemoradiation, and adjuvant chemotherapy. Small-molecule inhibitors (SMIs) targeting oncogenic pathways such as RAS/RAF/MEK/ERK (MAPK) have shown efficacy in several solid tumors. However, clinical trials in GB have failed to demonstrate survival benefit. This limited efficacy is attributed to marked intra-tumoral heterogeneity, promoting resistant subclones, and compensatory kinase signaling that bypasses MEK inhibition. Using patient-derived GB models (pdGBs), we investigated treatment-induced signaling adaptations contributing to MEK inhibitor resistance.

Phosphokinase activity within the MAPK pathway and related RTK and cell cycle signaling networks following MEK inhibition was evaluated by Western blot in U1242, U87, and two patient-derived GB organoïds. Based on these findings, SMI combination treatments were conducted to target compensatory signaling responses. Treatment efficacy was assessed in 2D using Alamar Blue and crystal violet assays, and in 3D spheroid models, with Bliss scores calculated to determine drug synergy.

MEK inhibition with trametinib or selumetinib induced adaptive kinase network remodeling across all GB models, characterized by MEK–AKT crosstalk and enhanced JNK/c-JUN activity. Within the MAPK cascade, MEK inhibition increased c-RAF phosphorylation together with pronounced MEKSer221 hyperphosphorylation consistent with a feedback activation response. Interestingly, ERK blockade with ulixertinib produced a comparable compensatory response. Combining MEK with JNK inhibition (SP600125) to block parallel activated JNK signaling synergistically reduced cell viability, but did not prevent MEK inhibitor induced MEKSer221 hyperphosphorylation. In a focused screen of clinically relevant SMIs, co-treatment with the VEGFR inhibitor axitinib markedly decreased MEKSer221 phosphorylation and demonstrated strong synergy in 3D spheroid models. Similar effects were observed with the SHP-2 inhibitor batoprotafib, whereas combinations with the EGFR inhibitor afatinib or the GSK3β inhibitor CHIR99021 did not result in additional growth inhibition.

This study outlines a mechanistic strategy for selecting SMIs to overcome MEK-inhibitor resistance in pdGB models.

The authors have no funding to disclose.