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

Abstract

Background: Glioblastoma 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). Methods: Live-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. Results: Collectively 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. Conclusion: The 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.

Inhibition of adaptive therapy tolerance in cancer: is triplet mitochondrial targeting the key?

“…Our own lab's contribution to uncovering the role of mitochondria in cancer therapy tolerance also comes from studies of brain tumor cells. Following previous findings demonstrating potent synthetic lethality in GB cells by multikinase inhibition and PP2A reactivation [14], we recently demonstrated that the critical kinase targets for this synthetic lethal phenotype were AKT and mitochondrial pyruvate dehydrogenase kinases (PDKs) [15]. Across heterogenous GB and medulloblastoma (MB) cell models, none of the combinations of two compounds from AKT inhibitor (AKTi), PDK inhibitor (PDKi), or PP2A reactivator (PP2Aa) induced cell killing across all cell models, whereas this was observed in all cell models with the AKTi + PDKi + PP2Aa triplet therapy.…”

“…These findings highlighted that triplet targeting is required to overcome both therapy‐induced adaptation but also the heterogeneity of therapy responses across brain tumor cell populations. Mechanistically, PP2A reactivation blocked compensatory OXPHOS activation by PDKi, and induced mitochondrial proton leakage, whereas the combination of AKTi + PDKi caused widespread BH3‐mediated mitochondrial apoptosis priming [ 15 ]. The significant therapeutic impact of the orally dosed mitochondrial triplet therapy was demonstrated in both GB and MB intracranial models.…”

“…Collectively these studies strongly indicate mitochondrial adaptation as an important mechanism behind nongenetic drug tolerance [ 3 , 7 , 8 , 10 , 11 , 13 , 15 ]. Although the combination strategies used across these studies are seemingly different, they do share certain aspects that might be key for their superior activity over the doublet or monotherapies.…”

“…1 ). At the mechanistic level, compensatory OXPHOS induction and BH3 protein regulation emerged as two interlinked themes related to adaptive mitochondrial therapy tolerance [ 7 , 8 , 10 , 11 , 15 ]. This was revealed by the results that VEN treatment, in addition to its well‐established function in blocking BCL‐2 [ 2 ], inhibits OXPHOS [ 8 , 10 ], and that pharmacological PP2A reactivation both dephosphorylates BH3 proteins [ 11 ] and inhibits OXPHOS [ 15 ].…”

“…At the mechanistic level, compensatory OXPHOS induction and BH3 protein regulation emerged as two interlinked themes related to adaptive mitochondrial therapy tolerance [ 7 , 8 , 10 , 11 , 15 ]. This was revealed by the results that VEN treatment, in addition to its well‐established function in blocking BCL‐2 [ 2 ], inhibits OXPHOS [ 8 , 10 ], and that pharmacological PP2A reactivation both dephosphorylates BH3 proteins [ 11 ] and inhibits OXPHOS [ 15 ]. Together with the evidence that ActD targets mitochondria [ 3 ], these results indicate that drugs previously described to have selective target activities may be more promiscuous/have a wider reach and also impact mitochondria function.…”