Adaptive Kinase Signaling Enables Escape from Small-Molecule Inhibition in Glioblastoma

Patients with glioblastoma (GBM) have a median survival of 15 months. Despite an intensive treatment schedule with resection, radio- and chemotherapy, recurrence is inevitable. A significant challenge to overcome GBM treatment resistance involves intratumoral heterogeneity, characterized by molecular, phenotypic, and clinical distinctive GBM subtypes. Different small molecule inhibitors (SMI) have been designed to inhibit signalling proteins in oncogenic driver pathways in GBM. However, SMIs have been unsuccessful in improving patient outcomes. Here, we investigate whether crosstalk between signalling pathways and signalling pathway redundancy are responsible for single-agent resistance using a primary patient-derived glioblastoma organoid (PGO) platform. This study used nine FDA-approved small-molecule inhibitors, based on their ability to cross the blood-brain barrier, targeting key GBM driver genes. Although inhibition of downstream effector proteins reduced cell viability more effectively (IC50 70nM-1M) than inhibiting upstream membrane-bound tyrosine kinase receptors (IC50 1-15M), remaining cell proliferation was seen in all six PGOs. To uncover resistance mechanisms, we analysed phosphokinase activity following monotherapy in various PGOs and identified compensatory pathway activation, leading to the discovery of effective small-molecule inhibitor combinations, most notably CHIR99021 (GSK-3 inhibitor) with trametinib (MEK inhibitor). In conclusion, our findings highlight the potential of combination therapy targeting compensatory pathways to overcome single-agent resistance in GBM, emphasizing the utility of patient-derived glioblastoma organoids as a platform for personalized therapeutic development.