Adaptive kinase signaling enables escape from small molecule inhibition in glioblastoma

OBJECTIVE: Patients with glioblastoma (GB) have a median survival of 15 months, despite intensive treatment with surgical resection, radiotherapy, and chemotherapy. A major challenge in overcoming treatment resistance is intratumoral heterogeneity, characterized by molecular, phenotypic, and clinical diversity. Small-molecule inhibitors (SMIs) target specific oncogenic signaling pathways and have become successful therapies in several molecularly defined cancers. In GB, however, SMIs have thus far failed to substantially improve patient outcomes. Here, we used a primary patient-derived glioblastoma organoid (PGO) platform to investigate adaptive bypass signaling mechanisms that drive resistance to targeted therapy. METHODS: Six primary PGOs were treated with nine FDA-approved SMIs selected for their blood-brain barrier permeability and ability to target key GB driver genes. Drug sensitivity was assessed by cell viability measurements and IC₅₀ determination. To identify compensatory signaling mechanisms, kinase activity profiling was performed following monotherapy treatment. RESULTS: Although inhibition of downstream effector proteins reduced cell viability more effectively (IC₅₀: 70 nM-1 μM) than inhibition of upstream receptor tyrosine kinases (IC₅₀: 1-15 μM), residual proliferation persisted in all PGOs. Kinase profiling revealed compensatory pathway activation following monotherapy, enabling the identification of effective SMI combinations. The most prominent combination targeted glycogen synthase kinase-3 (GSK-3) and mitogen-activated protein kinase kinase (MEK) using CHIR99021 and trametinib, respectively. CONCLUSION: These findings demonstrate that adaptive bypass signaling limits single-agent SMI efficacy in GB. PGO-based kinase profiling enables the identification of resistance mechanisms and rational combination therapies, supporting its application in personalized treatment development.