Pharmacological PP2A reactivation overcomes multikinase inhibitor tolerance across brain tumor cell models

Abstract

BackgroundGlioblastoma is characterized by hyperactivation of kinase signaling pathways. Regardless, most glioblastoma clinical trials targeting kinase signaling have failed. We hypothesized that overcoming the glioblastoma kinase inhibitor tolerance requires efficient shut-down of phosphorylation-dependent signaling rewiring by simultaneous inhibition of multiple critical kinases combined with reactivation of Protein Phosphatase 2A (PP2A). MethodsLive-cell imaging and colony growth assays were used to determine long-term impact of therapy effects on ten brain tumor cell models. Immunoblotting, MS-phosphoproteomics, and Seahorse metabolic assay were used for analysis of therapy-induced signaling rewiring. BH3 profiling was used to understand the mitochondrial apoptosis mechanisms. Medulloblastoma models were used to expand the importance to other brain cancer. Intracranial xenografts were used to validate the in vivo therapeutic impact of the triplet therapy. ResultsCollectively all tested ten glioblastoma and medulloblastoma cell models were effectively eradicated by the newly discovered triplet therapy combining inhibition of AKT and PDK1-4 kinases with pharmacological PP2A reactivation. Mechanistically, the brain tumor cell selective lethality of the triplet therapy could be explained by its combinatorial effects on therapy-induced signaling rewiring, OXPHOS, and apoptosis priming. The brain-penetrant triplet combination had a significant in vivo efficacy in intracranial glioblastoma and medulloblastoma models. ConclusionThe results confirm highly heterogenous responses of brain cancer cells to mono - and doublet combination therapies targeting phosphorylation-dependent signaling. However, the brain cancer cells cannot escape the triplet therapy targeting of AKT, PDK1-4, and PP2A. The results encourage evaluation of brain tumor PP2A status for design of future kinase inhibitor combination trials. Key PointsO_LIDevelopment of triplet kinase-phosphatase targeting therapy strategy for overcoming therapy tolerance across brain tumor models. C_LIO_LIIdentification of interplay between therapy-induced signaling rewiring, OXPHOS, and BH3 protein-mediated apoptosis priming as a cause for kinase inhibitor tolerance in brain cancers. C_LIO_LIValidation of the results in intracranial in vivo models with orally bioavailable and brain penetrant triplet therapy combination. C_LI Importance of the StudyBased on current genetic knowledge, glioblastoma should be particularly suitable target for kinase inhibitor therapies, However, in glioblastoma alone over 180 clinical trials with kinase inhibitors have failed. In this manuscript, we recapitulate this clinical observation by demonstrating broad tolerance of brain cancer cell models to kinase inhibitors even when combined with reactivation of PP2A. However, we discover that the therapy-induced signaling rewiring, and therapy tolerance, can be overcome by triplet targeting of AKT, PDK1-4 and PP2A. We provide strong evidence for the translatability of the findings by orally dosed brain penetrant triplet therapy combination in intracranial brain cancer models. The results encourage biomarker profiling of brain tumors for their PP2A status for clinical trials with combination of AKT and PDK1-4 inhibitors. Further, the results indicate that rapidly developing PP2A reactivation therapies will constitute an attractive future therapy option for brain tumors when combined with multi-kinase inhibition.