Development of a cell-based assay for Bcr-Abl kinase activity and application to a TKI-resistant chronic myeloid leukemia cellular model

Steven Bradley Ouellette, Purdue University

Abstract

Protein kinases (PKs) are an important class of enzymes that control critical cellular processes and responses to the environment. PKs propagate extracellular or intracellular molecular signals through phosphorylation of specific endogenous protein substrates. Mutations that occur in protein kinase genes often result in dysregulation of signaling pathways that control cell proliferation, differentiation, apoptosis, or survival. Mutated PKs are a primary cause carcinogenesis through uncontrolled signaling, and so cancer is widely considered a signaling disease. Targeted therapies have been designed to inhibit specific kinases implicated in a variety of cancer types. Tyrosine kinase inhibitors (TKIs) target tyrosine kinases, and have had success in improving cancer patient outcome. Imatinib is one of the first approved TKIs to target cancer. Imatinib is used to inhibit the constitutively active tyrosine kinase, Bcr-Abl, which is known to drive chronic myeloid leukemia. Patient intolerance or acquired resistance to imatinib has motivated the development of additional TKIs that target Bcr-Abl. The ability to measure PK activity has a central role in the development of TKIs through screening libraries of chemical entities against a target kinase. PK activity assays are also important for understanding PKs in disease biology. In the clinic, measuring kinase activity may assist in diagnosis and monitoring TKI effectiveness. The current cell-based PK activity assays are not well suited for maximizing the biological relevance of results, assay throughput, or ease of use, which can hinder progress in TKI discovery and PK activity assays for clinical applications. In this work we show the optimization of a cell-based kinase activity assay using a multi-functional peptide substrate for Bcr-Abl and demonstrate its use for drug discovery and for characterizing TKI-resistant human chronic myeloid leukemia cells. The results indicate that the assay is an appropriate cell-based platform for identifying Bcr-Abl kinase inhibitors in high-throughput screens. Additionally, we show that the assay can predict drug-resistance in a panel of human chronic myeloid leukemia cell lines. Overall, these findings suggest that our approach can be applied in drug discovery and, with further development, in the clinic for predicting response to Bcr-Abl inhibitors. Moreover, as a platform technology this approach could be adapted to additional kinases being pursued in drug discovery or targeted in the clinic.

Degree

Ph.D.

Advisors

Parker, Purdue University.

Subject Area

Pharmacy sciences

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