Introduction.

Deregulation of protein phosphatase PP2A by overexpression of PP2A inhibitor proteins (PIPs) such as protein phosphatase methylesterase 1 (PME‑1), Cancerous Inhibitor of PP2A (CIP2A), SET nuclear proto-oncogene (SET) have been proposed as a potential general cause for kinase inhibitor resistance in cancer. While overexpression of PIPs has been studied in various adult cancers, their relevance in pediatric brain cancer is poorly understood. Here, we highlight the implications of PME-1 in the pediatric brain tumor medulloblastoma tumor growth and present a novel combination therapy approach based on PP2A activation.

Material and method.

In vitro and in vivo studies were done on established medulloblastoma cell lines, DAOY (SHH group) and D283-Med (Group 3). CRISPR/Cas9 approach was used for PME-1 inhibition. All animal experiments were authorized by the National Animal Experiment Board of Finland and studies were performed according to the instructions provided by the Institutional Animal Care and Use Committees of the University of Turku, Turku, Finland.

Results and discussion.

Overall survival analysis of medulloblastoma patients revealed that mRNA expression of several PIPs is associated with poor patient prognosis. Interrogation of CRISPR/Cas9 loss-of-function data showed many PP2A and PIP genes among the 10 top preferentially essential genes, especially in DAOY cells. Intracranial in vivo experiments validated the importance of PME-1 for medulloblastoma growth, with significantly prolonged survival in mice carrying intracranial PME-1 knockout DAOY and D283-Med cells.

To validate the role of PP2A inhibition in kinase inhibitor resistance in medulloblastoma, we next tested the impact of recently developed triplet kinase/phosphatase combination therapy on DAOY cells. A triplet combination of AKT inhibitor, mitochondrial PDK1-4 inhibitor, and PP2A reactivating compounds (SMAPs) induced very robust cell killing in long-term colony growth assays. Moreover, orally dosed triplet therapy significantly increased the survival of mice carrying intracranial DAOY xenograft. Using mass spectrometry phosphoproteomics analysis, we molecularly profiled the triplet therapy effect in tumors validating its impact on both apoptosis and the cell cycle. Furthermore, triplet therapy inhibited phosphorylation of the activation loop of AKT1, 2, and 3, and impacted several phosphoproteins linked to mTOR signaling.

Conclusion. These results highlight PP2A inhibitor protein PME-1 as a novel potential medulloblastoma oncoprotein and therapy resistance driver. Further, the triplet kinase/phosphatase therapy is a promising lead for the development of novel combination therapy approaches for medulloblastoma.