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Triplet kinase-phosphatase targeting to overcome kinase inhibitor tolerance in brain tumor cells

Kinase inhibitor tolerance of human glioblastoma is an unmet clinical challenge and a mechanistic enigma. Here, we demonstrate that glioblastoma cell tolerance to multi-kinase inhibition can be reverted by reactivation of Protein Phosphatase 2A (PP2A). To characterize kinase targets of clinical stage multi-kinase inhibitor UCN-01 synergizing with PP2A reactivation, we established a strategy, named Actionable Targets of Multi-kinase Inhibitors (AToMI). AToMI revealed AKT and mitochondrial pyruvate dehydrogenase kinases (PDK1-4) as the co-targets for UCN-01-elicited synthetic lethality with PP2A reactivation. Notably, heterogeneous glioblastoma and medulloblastoma models were tolerant to AKT and PDK1-4 inhibitor monotherapies, and their combinations, but were effectively inhibited by triplet therapy including pharmacological PP2A reactivation. Mechanistically, overcoming the kinase therapy tolerance by the triplet therapy could be explained by combinatorial effects on signaling rewiring between AKT and PDK1-4, decrease in mitochondrial oxidative phosphorylation, and BH3-only protein mediated apoptosis priming. The brain-penetrant triplet combination had a significant in vivo efficacy in intracranial glioblastoma and medulloblastoma models. Collectively, we present a generalizable approach to identify actionable co-targets of multi-kinase inhibitors and demonstrate that overcoming of the kinase inhibitor tolerance in brain tumor cells requires triplet targeting of AKT, PDK1-4, and PP2A.