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PKC and PI3 Kinase/ AKT pathway activation has been implicated in human cancer. Activation of these signaling pathways can enhance cell motility, promote cell cycle progression, suppress apoptosis, enable angiogenesis and impart resistance to numerous chemotherapeutics. The bisindolylmaleimide, Enzastaurin (LY317615.HCl), was developed as an ATP-competitive inhibitor of PKCβ with an IC50 of 6nM. At higher concentrations approximating plasma concentrations achieved in clinical trials patients, we now show that Enzastaurin suppresses signaling through the PKC and AKT pathways in HCT116 human colorectal carcinoma cells. Enzastaurin treatment, at concentrations reflecting plasma concentrations achieved in clinical trials, blocks phosphorylation of AKT at Threonine 308 and phosphorylation of ribosomal protein S6 at ser240/244 in a time-dependent manner. Enzastaurin treatment also rapidly suppresses phosphorylation of GSK3β at serine 9 in these colon cancer cells. Accordingly, Enzastaurin induces apoptosis in human carcinoma cells and suppresses growth of human cancer xenografts. As in cultured cells, xenografts harvested from mice treated by gavage with Enzastaurin show a marked, time-dependent reduction in GSK3βser9 phosphorylation. GSK3βser9 phosphorylation is reduced to the same extent and with the same timecourse in peripheral blood mononuclear cells harvested from these xenograft-bearing mice, suggesting that GSK3βser9 phosphorylation may serve as a reliable pharmacodynamic marker for Enzastaurin activity. To assess the relationship of GSK3βser9 phosphorylation to disease progression and the plausibility of using this marker for patient stratification, we are now evaluating GSK3βser9 phosphorylation by immunohistochemistry and ELISA in primary human cancer tissues. To determine which molecular events are most critical to Enzastaurin-induced apoptosis, we have now established and are characterizing resistance variants of the HCT116 colorectal cancer cell line for alterations in the AGC kinases targeted by Enzastaurin. Further, we have begun to explore the mechanism of Enzastaurin-induced apoptosis in colorectal cancer cells. Both caspase 8 and caspase 9 inhibitors diminish Enzastaurin-induced apoptosis, suggesting a role for both intrinsic and extrinsic cell death pathways in Enzastaurin-induced apoptosis. Collectively, these data show that Enzastaurin blocks signaling through the PKC and AKT pathways, inducing apoptosis in human tumor cells, blocking angiogenesis and suppressing cancer xenograft growth. These data also show that GSK3βser9 phosphorylation can provide the pharmacodynamic marker for Enzastaurin activity in both PBMCs and tumor tissues and may provide a foundation for patient stratification as Enzastaurin advances in clinical development.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]