Abstract
The protein kinase Bcr-Abl plays a major role in the pathogenesis of chronic myelogenous leukemia (CML), and is the target of the breakthrough drug imatinib (Gleevec™). While most patients respond well to imatinib, approximately 30% never achieve remission or develop resistance within 1–5 years of starting imatinib treatment. Evidence from clinical studies suggests that achieving at least 50% inhibition of a patient’s Bcr-Abl kinase activity (relative to their level at diagnosis) is associated with improved patient outcomes, including reduced occurrence of resistance and longer maintenance of remission. Accordingly, sensitive assays for detecting Bcr-Abl kinase activity compatible with small amounts of patient material are desirable as potential companion diagnostics for imatinib. Here we report the detection of Bcr-Abl activity and inhibition by imatinib in the human CML cell line K562 using a cell-penetrating peptide biosensor and multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer. MRM enabled reproducible, selective detection of the peptide biosensor at fmol levels from aliquots of cell lysate equivalent to ∼15,000 cells. This degree of sensitivity will facilitate the miniaturization of the entire assay procedure down to cell numbers approaching 15,000, making it practical for translational applications in patient cells in which the limited amount of available patient material often presents a major challenge.
Highlights
Kinase inhibitor drugs represent an approximately $10 billion market in the pharmaceutical industry, and this is anticipated to expand even further over the coming decade. [1] The classic example and breakthrough drug for this therapeutic strategy is GleevecH
Imatinib inhibits the Bcr-Abl kinase, an oncogene encoded on the Philadelphia chromosome on which the disease process of chronic myelogenous leukemia (CML) depends
90% of CML patients achieve initial remission with imatinib, and for,70% of patients, that remission remains stable for a long period of time; a significant proportion (,30%) either never respond or develop recurrent and/or resistant disease and experience relapse within a few years. [2,3] Evidence from monitoring the relative inhibition of Bcr-Abl in CML patients beginning therapy suggests that failure to achieve at least 50% relative inhibition of the kinase’s activity is associated with poorer short- and long-term outcomes
Summary
Kinase inhibitor drugs represent an approximately $10 billion market in the pharmaceutical industry, and this is anticipated to expand even further over the coming decade. [1] The classic example and breakthrough drug for this therapeutic strategy is GleevecH (imatinib). We set out to establish an assay that would provide higher sensitivity for detecting Bcr-Abl kinase activity using the peptide biosensor and demonstrate the method in a more clinically relevant model (the human CML cell line K562).
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