Multiple myeloma is the second most common hematologic malignancy in the United States. While the disease remains incurable, there are widespread translational efforts to improve chemotherapeutic options in this disease. Molecularly targeted therapies, particularly those directed at the proteasome, but more recently kinase targets as well, are showing clinical efficacy. Therapeutically inhibiting specific enzymes creates a critical need for assays capable of measuring the activities of these proteins in myeloma disease models and in patient samples. The ability to measure relevant enzyme activity in primary tumor samples at baseline and/or after treatment would provide the ability to tailor patient therapy based on aberrant signal transduction, validate mechanisms of resistance in patients, and would offer an invaluable pharmacodynamic tool to assess whether resistance is associated with inadequate target inhibition. An interdisciplinary collaboration is proposed to create the analytical and chemical tools needed to directly measure the enzymatic activities of protein kinases and the proteasome in cells taken directly from patients with multiple myeloma. The investigators will develop unique fluorescent reagents to report the activity of the protein targets of molecular-based therapies currently in use or in clinical trials. Kinase substrates will be modified to create long- lived compounds by attachment of stably folded beta-hairpin structures or """"""""protectides"""""""". These beta-hairpins will also be used to create substrates with secondary structure for metabolism by the proteasome. Membrane-permeant forms of these protectide-substrates will be capable of accurately reporting the enzymatic activity within a living cell. Microelectrophoretic separations combined with low-level fluorescence detection will enable the quantitative analysis of these molecules from single mammalian cells. This capability will resolve three major issues currently faced in the biochemical analysis of clinical myeloma samples: lack of direct measurement of the enzymatic activity of target proteins;sample size requirements that are impractically large for clinical implementation;and sample heterogeneity that can mask pertinent aspects related to patient response. Protein kinase B, protein kinase C, and the chymotrypsin-like activity of the proteasome were chosen for this work by virtue of their pertinence to both research and clinical practice in multiple myeloma.
The goal of the proposed work is to develop an assay to study active signal transduction pathways in the tumor cells of patients. This would enhance diagnostics and prognostics as well aid therapeutic planning.
The final aim of the work focuses on multiple myeloma, a disease with a very poor survival rate and the second most common hematologic malignancy in the United States where advances in therapy are being aggressively sought.
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