Development of a comparative high-throughput screening platform for identifying glioblastoma subtype specific inhibitors

Glioblastoma (GBM) is the most lethal primary malignant brain tumor in adults, with the development of effective therapeutic agents largely hampered by vast tumor heterogeneity and the impedance of efficient drug delivery by the blood-brain barrier (BBB). Our prior research has demonstrated that adult neural stem cells (NSCs) and oligodendrocyte precursor cells (OPCs) can act as cells of origin for two distinct GBM subtypes (Type 1and Type 2) in mice, with significant conservation to human Type I and Type II GBM in functional properties and distinct responses to the inhibition by Tucatinib and Dasatinib. Based on these findings, we have established a robust high-throughput screening (HTS) assay to identify lineage-dependent subtype-specific as well as lineage-independent small molecule inhibitors for therapeutic development. Reported in the current study, we conducted a HTS using a kinase inhibitor library (900 compounds) in Type 1 and Type 2 GBM cells. Our primary screen identified 84 common inhibitors, 11 Type 1-specific inhibitors, and 18 Type 2-specific inhibitors. The confirmation screen verified R406 and Ponatinib as selective inhibitors of Type 2 GBM cells, and this was further validated in dose-dependent assays. Additionally, R406 exhibited a synergistic effect with Tucatinib in Type 2 GBM cells, providing a rationale for combination therapy. Our study demonstrated the feasibility of identifying subtype-specific therapeutic vulnerabilities using cell-lineage based GBM models and laid the foundation for expanded HTS studies in larger scale screens in both mouse and human GBM subtypes.