Abstract 5328: Protein phosphatase 2A activity is a major determinant of therapy response in cancer cells
Authors: Kauko Otto , Imanishi Susumu , Kaur Amanpreet , Laajala Daniel , Kulesskiy Evgeny , Jumppanen Mikael , Corthals Garry , Aittokallio Tero , Wennerberg Krister , Westermarck Jukka 
Published: August 2015

Abstract
Protein phosphatase 2A (PP2A) dephosphorylates majority of Ser/Thr phosphorylated proteins. Consequently, PP2A is an antagonist of multiple oncogenic pathways and PP2A reactivation may provide an alternative route to target these pathways. Importantly, because PP2A reactivation would result in simultaneous dephosphorylation of both collateral and downstream effectors of kinase pathways, it might circumvent commonly encountered kinase inhibitor resistance mechanisms.
To systematically study the role of PP2A in cancer therapy response we used RNAi targeting against PP2A inhibitor proteins CIP2A, PME-1 and SET (PP2A reactivation), and PP2A structural subunits (PP2A inhibition), together with high throughput drug sensitivity screen covering 300 clinical cancer drugs and investigational compounds. Changes in phosphorylation status of PP2A targets in response to RNAi perturbation, was studied by LC-MS/MS-based label-free quantitative phosphoproteomics analysis. Importantly, we show that cancer cell drug sensitivity across 300 compounds correlates with PP2A activity profile so that PP2A inhibition made cells on average resistant to therapies, whereas their sensitization by PP2A inhibitor protein siRNAs correlated with changes in phosphoprotein regulation. In particular, cancer cell response to kinase inhibitors followed very closely PP2A activity regulation and PP2A reactivation resulted in increased average sensitivity to 105 kinase inhibitors. Furthermore, we show that PP2A reactivation results in convergent phosphorylation patterns with targeting of kinase pathways by RAS inhibition. We also identify novel PP2A regulated phosphorylation sites in target proteins of these kinases. As a validation study, we demonstrate that PP2A inhibitor protein PME-1 mediates resistance of glioma cells to various types of survival pathway kinase inhibitors, as well as to temozolomide. Co-treatment with PME-1 siRNA and kinase inhibitors eradicates several GBM cell lines, and glioma stem cells, in vitro, and this combination strategy shows pronounced efficacy also in in vivo models. The PME-1-elicited resistance mechanism is mediated by reactivation of specific PP2A complexes and involves regulation of PP2A target HDAC4.
Together our data combines 4500 drug response profiles, and more than 5000 quantitative phosphopeptide idenitifications to draw first systemic map of relevance of PP2A biology in cancer therapy responses. In particular the data reveals an unprecedented importance of PP2A activity for kinase inhibitor responses. Based on these data we propose that in order to efficiently inhibit phosphorylation-dependent signaling in cancer cells, and thus provide better therapeutic index for the kinase inhibitors, they should be combined with emerging small molecule PP2A reactivating compounds.
Citation Format: Otto Kauko, Susumu Imanishi, Amanpreet Kaur, Daniel Laajala, Evgeny Kulesskiy, Mikael Jumppanen, Garry Corthals, Tero Aittokallio, Krister Wennerberg, Jukka Westermarck. Protein phosphatase 2A activity is a major determinant of therapy response in cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5328. doi:10.1158/1538-7445.AM2015-5328