A platelet biomarker for assessing phosphoinositide 3-kinase inhibition during cancer chemotherapy.

Thrombin cleavages of selective proteinase-activated receptors (PAR) as well as PAR-activating peptide ligands can initiate the phosphoinositide 3-kinase (PI3K) signaling cascade in platelets. Downstream to this event, fibrinogen receptors on platelets undergo conformational changes that enhance fibrinogen binding. In our study, we used this phenomenon as a surrogate biomarker for assessing effects on PI3K activity. Our method, using flow cytometric measurement of fluorescent ligand and antibody binding, uncovered a 16- to 45-fold signal window after PAR-induced platelet activation. Pretreatment (in vitro) with the PI3K inhibitors wortmannin and LY294002 resulted in concentration-dependent inhibition at predicted potencies. In addition, platelets taken from mice treated with wortmannin were blocked from PAR-induced ex vivo activation concomitantly with a decrease in phosphorylation of AKT from excised tumor xenografts. This surrogate biomarker assay was successfully tested (in vitro) on blood specimens received from volunteer cancer patients. Our results indicate that measurement of platelet activation could serve as an effective drug activity biomarker during clinical evaluation of putative PI3K inhibitors.

Development and validation of a drug activity biomarker that shows target inhibition in cancer patients receiving enzastaurin, a novel protein kinase C-beta inhibitor.

PURPOSE: To evaluate the effects of the novel protein kinase C (PKC) inhibitor enzastaurin on intracellular phosphoprotein signaling using flow cytometry and to use this approach to measure enzastaurin effects on surrogate target cells taken from cancer patients that were orally dosed with this agent. EXPERIMENTAL DESIGN: The activity of PKC was assayed in intact cells using a modification of published techniques. The U937 cell line and peripheral blood mononuclear cells were stimulated with phorbol ester, fixed, permeabilized, and reacted with an antibody specific for the phosphorylated forms of PKC substrates. The processed samples were quantitatively analyzed using flow cytometry. The assay was validated for selectivity, sensitivity, and reproducibility. Finally, blood was obtained from volunteer cancer patients before and after receiving once daily oral doses of enzastaurin. These samples were stimulated ex vivo with phorbol ester and were assayed for PKC activity using this approach. RESULTS: Assay of U937 cells confirmed the selectivity of the antibody reagent and enzastaurin for PKC. Multiparametric analysis of peripheral blood mononuclear cells showed monocytes to be the preferred surrogate target cell. Day-to-day PKC activity in normal donors was reproducible. Initial results showed that five of six cancer patients had decreased PKC activity following enzastaurin administration. In a following study, a group of nine patients displayed a significant decrease in PKC activity after receiving once daily oral doses of enzastaurin. CONCLUSION: An inhibition of surrogate target cell PKC activity was observed both in vitro and ex vivo after exposure to the novel kinase inhibitor, enzastaurin.