Glioblastoma (GBM) is the most prevalent primary brain tumor in adults, with a median survival of 14 months despite multimodal treatment. Small molecule inhibitors (SMIs) targeting mutational drivers, such as the RAS/MEK/ERK-pathway, offer potential therapeutic avenues, yet clinical trials have not improved GBM survival outcomes. Using patient-derived GBM models (pdGBMs), we evaluated the cytotoxicity of SMI mono- and combi therapy and explored aberrant signaling pathways driving treatment resistance. Our analysis specifically focused on combinations with trametinib, which demonstrated the highest cytotoxic potential.

Eight BBB-penetrable and clinically approved SMIs were used to target key GBM driver oncogenes. Cell viability was measured via CellTiter-Glo and the IC50 for each inhibitor was determined for six molecularly distinct pdGBMs. Changes in phosphokinase activity after SMI monotherapy were assessed via WB and phosphokinase screening. Based upon these results, SMI combinations were made, targeting compensatory pathways driving treatment resistance. Bliss scores were established to test synergistic SMI combinations.

SMIs targeting downstream effector kinases (Trametinib, Buparlisib, Abemaciclib; IC50 70nM-1µM) or membrane-bound tyrosine kinase receptors (Afatinib, Capmatinib, Axitinib; IC50 1-15µM) reduced cell viability. However, residual cell proliferation was seen in all pdGBMs following single-agent SMI treatment, indicative for clonal expansion of treatment resistant tumor cells. Western blot analysis confirmed on-target drug activity. Subsequently, MEK-inhibition with trametinib induced compensatory signaling, including MEK/AKT-crosstalk, JNK/c-JUN upregulation and most notably MEKSer221 hyperphosphorylation. Combined trametinib and SP600125 (JNKi) mitigated JNK/c-Jun activation and synergistically reduced cell viability (Bliss >20), but failed to suppress MEKSer221 hyperphosphorylation. In a small-scale SMI drug screen, trametinib combined with VEGFRi, axitinib significantly abrogated MEKSer221 hyperphosphorylation and led to a synergistic reduction in cell viability (Bliss >20). In contrast, co-treatment with Afatinib (EGFRi) or CHIR99021 (GSK3βi) did not exhibit synergistic effects (Bliss <10).

Trametinib reduces cell viability but triggers compensatory signaling, including MEKSer221 hyperphosphorylation. This study presents a mechanistically driven selection of tumor-tailored combination treatments to overcome resistance to SMI monotherapy in pdGBM models, highlighting VEGFR co-inhibition as a promising combinatorial strategy with trametinib.