New approaches are needed in the molecular analyses of the growth-promoting signal transduction pathways involved in the development and maintenance of cancer. The Laser Micropipet System (LMS), a new technology for the biochemical assay of kinases, holds the promise of directly assaying the activities of protein tyrosine kinases in primary patient cells. The LMS was developed for the assay of serine/threonine kinases in individual cells grown in tissue culture. This R21/R33 application proposes to expand the applications of this instrumentation to directly determine the activity of oncogenic tyrosine kinases in primary cells from patients. Assay of Bcr-Abl in chronic myelogenous leukemia (CML) will demonstrate the power of this tool for research, clinical, and pharmacologic applications. While this assay will be of value in the study of the biology and resistance mechanisms in CML, the studies will lay the groundwork for a new paradigm in the study of molecular mechanisms in cancer signal transduction and will have general applicability. The R21 phase of this proposal is designed to establish the feasibility of assaying Bcr-Abl activity and to demonstrate the necessary assay conditions for measurements in cells derived from a patient with CML.
The specific aims are to: 1) identify and characterize a reporter of intracellular Bcr-Ab1 kinase activity, 2) define and optimize conditions for measuring Bcr-Ab1 activity in human cells, 3) compare reporter phosphorylation in cells expressing Bcr-Ab1 vs. cells lacking this kinase, and 4) demonstrate the feasibility of using primary patient cells in kinase assays. The goal of the R33 phase is to demonstrate the potential for the LMS-based kinase assay in applications for the study of the biology of pharmacologic resistance in CML, including from primary patient cells. The R33 specific aims are to: 1) develop a membrane-permeant Bcr-Ab1 kinase reporter, 2) demonstrate the ability to detect imatinib resistance in CML cell lines, 3) determine Bcr-Ab1 activity in primary cells from patients with CML and their response to pharmacologic inhibition, and 4) determine if the assay can detect the emergence of imatinib resistance in CML patients and can differentiate the mechanisms of drug resistance.
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