Abstract Diffuse midline glioma (DMG) is a highly invasive and fatal pediatric brain tumor that arises from glial cells within the pons of the brainstem. Despite focal radiation treatment, DMG continues to have a poor prognosis, with a median survival of less than 10 months. A significant challenge in treating DMG is the blood-brain barrier (BBB), which tightly restricts access to therapeutic agents, preventing effective drug concentrations from reaching the brain. Glycogen synthase kinase-3 (GSK3) has emerged as a promising therapeutic target due to its critical roles in regulating cell migration, angiogenesis, and endothelial barrier function. We investigated the effects of GSK3 inhibition on pediatric glioma models using the indirubin derivative 6-bromoindirubin-3′-acetoxime (BIA) and the selective GSK3 inhibitor CHIR99021 (C91) in various assays. In a 2D scratch migration assay, both BIA (1µM) and C91 (3µM) significantly slowed glioma cell migration over 24 hours. In 3D BBB spheroid models both compounds altered tight junction integrity, enhancing barrier permeability and antibody uptake. Immunofluorescence analysis revealed that BIA treatment led to decreased expression of tight junction proteins in endothelial monolayers. Additionally, both BIA and C91 impaired tube formation and altered endothelial migration patterns, demonstrating anti-angiogenic activity. To assess potential combination strategies, a high-throughput screen of 176 FDA-approved drugs was performed on a panel of pediatric glioma cell lines in combination with BIA identifying several compounds that are being further investigated. Our hypothesis is that GSK3 inhibition could be an effective therapeutic approach for DMG by targeting tumor invasion, angiogenesis, and improving drug delivery through modulation of endothelial cell tight junctions. Unlike BIA’s broader kinase inhibition profile, selective GSK3 inhibitors like CHIR99021 may provide similar effects to BIA with reduced off-target toxicity. Future studies will focus on elucidating the underlying mechanisms and developing drug formulations for in vivo studies to assess its translational potential.
Clark et al. (2025) studied this question.