(639d) Quantifying Tyrosine Kinase Activity In Cancer Cell Lysates Via Phosphorylation of Hydrogel-Immobilized Substrates

 Many types of cancer are caused by overactive or overexpressed kinases.  Over the past decade, the use of kinase inhibitors has offered a new approach for treating cancers associated with increased or disregulated kinase activity.  Many patients, however, are initially resistant to these inhibitors or develop resistance during treatment.  Additionally, diagnosis of a particular type of cancer does not provide insight into what kinase therapies may be effective.  This research seeks to develop a kinase activity assay to determine which kinase inhibitors will be most effective for individual patients and to quickly identify if a patient has developed resistance to a particular inhibitor.  Specifically, we are currently working to detect Met tyrosine kinase activity in lung cancer as well as to multiplex the assay to simultaneously detect the activity of multiple kinases, including Bcr-Abl kinase.  To accomplish these goals, we immobilize spots of a phosphorylation substrate for Met kinase in a microchannel on a glass slide.  We then expose the substrate to a kinase reaction mixture containing lysate from a cancer cell line.  A kinase inhibitor may also be included in the reaction mixture.  A phosphotyrosine antibody followed by a fluorescent secondary antibody is then used to quantify substrate phosphorylation and kinase activity.  Our results have demonstrated increases in substrate phosphorylation with increasing lysate and immobilized substrate concentrations.  Further results also show that a Met kinase inhibitor decreases substrate phosphorylation. Immunodepleting Met kinase from the cell lysate also decreases substrate phosphorylation.   Together, these results show that this assay can detect specific kinase activity in cancer cell lines.  This assay offers promise as a way to quickly and relatively cheaply determine which kinase inhibitors will be effective for which patients, allowing patients to more quickly receive an effective treatment.