Utilization of phospho-GSK3b as a cellular and in vivo pharmacodynamic marker to evaluate the activity of AKT pathway inhibitors

288 The PI3 Kinase/AKT pathway plays a critical role in cell survival signaling and the activation of this pathway has been linked to tumorigenesis. Upregulation of AKT (PKB) and PI3 Kinase is seen in many tumor types and the negative regulator of this pathway PTEN is deleted in a number of tumor types. Therefore, this pathway remains an attractive target for cancer drug discovery. Glycogen Synthase Kinase-beta (GSK3b) is characterized to be phosphorylated at its Ser 9 site by AKT. Phosphorylation of GSK3b by AKT results in inactivation of the kinase activity of GSK3b and downstream activation of glycogen synthesis and metabolic events leading to and promoting cell proliferation. Therefore, phosphorylation of GSK3b at the Ser 9 site can be utilized as a pharmacodynamic marker for evaluating the in vivo PK/PD effects of AKT/PI3K inhibitors. We developed a novel in cell ELISA to measure the phosphorylation of GSK3b in U87MG cells. In order to link cellular target inhibition to in vivo target inhibition, we utilized a commercially available sandwich ELISA to measure the in vivo activity of PI3 Kinase/AKT pathway inhibitors quantitating the phosphorylation level of GSK3b in native U87MG glioblastoma cells. We treated these cells and mice bearing U87MG xenografts with the PI3 Kinase inhibitor wortmannin and an Isoquinoline Sulfonamide (IS) AKT inhibitor. The ELISA analysis showed that PI3 Kinase inhibitor Wortmannin and IS AKT inhibitor inhibits GSK3b phosphorylation U87MG cells potently in vitro and in vivo. The inhibition correlates well with the inhibition of phosphorylation of other known AKT substrates such as phospho-Forkhead and phospho-TSC2. In vivo pGSK3b inhibition shows excellent correlation with serum exposure of compound. These results show that phospho-GSK3b can be utilized as a in vitro and in vivo PD marker to develop inhibitors of AKT and PI3 Kinase as potential targeted cancer therapeutic agents.