This phased innovation award proposal is focused on developing a robust approach to quantitative assay of specific protein tyrosine kinase activities from cancer cells. Our model is the oncogenic BCR-ABL fusion protein, the gene product of the t(9;22) Philadelphia chromosome (Ph1) translocation observed in the vast majority of Chronic Myelogenous Leukemia (CML) and in up to 30% of adults with Acute Lymphoblastic Leukemia and in other hematological neoplastic diseases. The activation of Abl kinase by fusion to BCR that is inferred to underly the malignant transformation of Phl positive CML is effectively opposed by the orally administered tyrosine kinase inhibitor (TKI) Imatinib Mesylate (IM, STI-571, Gleevec). The activity of IM as an Abl kinase inhibitor in vitro is thought to be the critical determinant of its efficacy in vivo. Nonetheless, a clinically useful assay for IM inhibition of BCR-ABL kinase activity in circulating CML leukemia cells is lacking. We propose to develop a protein/peptide chip-based assay for BCR-ABL that can detect the degree of inhibition by IM to evaluate dosing and drug resistance. Insofar as other activated tyrosine kinases may be critical mediators of malignancy in both leukemias and solid tumors, developing such an assay would be a powerful tool in evaluating other TKI drug candidates targeting these kinases for their efficacy in vivo. Thus, this project is directed at two major discovery objectives and three development objectives. First, in the initial project year, we intend to use our established methods for anti-phosphotyrosine antibody-based detection of purified Abl kinase activity on a peptide chip to 1) Recapitulate our Abl kinase assay with undiluted whole cell extracts from cell lines expressing BCR-ABL and 2) Use this assay to measure the inhibition of BCR-ABL by IM both in extracts and intact cells. During the development phase, we intend to use both BCR-ABL expressing cancer cell lines and circulating leukemic cells from treated patients as samples to 1) Optimize the BCR-ABL substrate and reaction conditions to enhance sensitivity and specificity of phosphorylation, 2) Examine alternative detection methods for BCR-ABL activity based on phosphospecific antibodies and thiophosphate targeted chemistry, and 3) Evaluate different chemistries for immobilizing BCR-ABL substrates on a surface and geometries for detection of phosphorylation. By these aims we intend to develop a highly versatile kinase assay system which can be applied to monitoring of patient response to IM and as a tool for discovery and testing of new TKI cancer drugs.
