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. 
The classic example and breakthrough drug for this therapeutic strategy is Gleevec® (imatinib). Imatinib inhibits the Bcr-Abl kinase, an oncogene encoded on the Philadelphia chromosome (a translocation of chromosomes 9 and 22; tumor cells with this translocation are known as Ph+) on which the disease process of chronic myelogenous leukemia (CML) depends. Approximately 90% of CML patients achieve initial remission with imatinib, and for ~70% of patients, that remission remains stable for a long period of time; however, a significant proportion (~30%) either never respond or develop recurrent and/or resistant disease and experience relapse within a few years. 
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. 
A similar relationship has been observed for other promising kinase inhibitor drugs. 
Relatedly, failure of clinical trials for inhibitors targeted at other kinases due to heterogeneity of patient response (unlike the relatively homogenous response of CML patients to imatinib) is a major risk in kinase inhibitor drug development, and often arises due to a lack of pharmacodynamic assessment of the response of the drug target and downstream signaling pathways. 
The Food and Drug Administration has become increasingly involved in efforts to include companion diagnostics as a part of the drug approval process. 
Kinase assays developed as companion diagnostics could assist with earlier decision making on the potential for a drug to be successful. This could also enable more effective selection of patients who are likely to benefit, refining the population for defining successful response in a clinical trial. 
Additionally, the patent on Gleevec has recently expired; however, imatinib is currently first-line therapy for CML, and still holds the biggest market. 
The still-branded inhibitors Tasigna® (nilotinib) and Sprycel® (dasatinib) are being promoted as first-line therapies for newly-diagnosed CML patients, but may not be necessary in most cases where imatinib is effective but just not reaching the relative inhibition required for the best clincial outcomes. Accordingly, stakeholders including patients, doctors and insurance companies would benefit from information on whether less costly, generic imatinib could be effective for a given individual. Pharmaceutical companies may also find methods to detect sensitivity to branded drugs useful to support the need for their branded drugs, for example in cases where imatinib is found to be ineffective regardless of dose.
A relevant companion diagnostic for kinase inhibitor pharmacodynamics needs to measure enzymatic activity, and thus substrate phosphorylation, for a targeted kinase. Protein and peptide phosphorylation by kinases has traditionally been detected using 32
P-radiolabeled ATP or antibody-based methods (such as ELISA and Western blot). These methods are reliable and well-characterized, but often are limited by concerns over waste generation (radiolabeled assays) or the requirement for phosphosite-specific antibodies, which are not always available at the scales necessary for clinical tests at a reasonable cost. To overcome these limitations, several approaches (e.g. microarray, bead-based and targeted mass spectrometry (MS) methods) have been described that detect kinase activity from relatively small amounts of cell lysate (down to ~10 µg). 
The number of Ph+, Bcr-Abl expressing CML cells per ml of blood is highly variable from patient to patient, reaching several hundreds of millions during chronic phase (during which diagnosis typically occurs) and dropping to 0.15–5×106
upon hematological remission (when patients would likely benefit from monitoring for maintenance of kinase inhibition and potential for disease recurrence). Typical total protein yields from CML model cells such as K562 average approximately 50–250 µg per 106
cells, so assays that can achieve reproducible signal with ~10 µg of sample would potentially be appropriate for translational assays on patient material. However, kinase signaling is highly dependent on factors such as subcellular localization and scaffolding–therefore, assays that can measure kinase activity in intact cells are desirable for achieving the most biologically-relevant measurements of activation and inhibition. Furthermore, there is evidence from proteomic studies that downstream phosphorylation sites (such as Y207 of CrkL) may not be accurate reporters of drug sensitivity and pharmacodynamics in leukemias including CML. 
Accordingly, exogenous sensors that report kinase activation states (rather than the phosphorylation of downstream substrates) with high sensitivity may provide useful information about individual patient response to kinase inhibitors and facilitate personalized therapeutic decisions in the clinic.
Genetically-engineered Förster resonance energy transfer (FRET) protein constructs for detecting Bcr-Abl activity in intact cells have been reported, however transfecting patient cells with sensor constructs for clinical assays is very challenging and not practical for a typical clinical laboratory. We previously reported a cell-permeable peptide biosensor for Abl kinase and its application for detecting DNA damage-related Abl kinase activation in intact cells (). 
Because it can be applied in a relatively simple workflow (involving straightforward, reproducible incubation and lysis steps), this peptide biosensor strategy has the potential to be compatible with clinical laboratories. Our previous applications of the assay used Western blot and MALDI-TOF MS to detect peptide phosphorylation and required 2–5×106
cells per experiment, which corresponds to 6–15×106
cells for triplicate analyses. While these cell numbers are trivial to achieve in cultured cell lines and may be available from patient material in some cases at diagnosis, they are not optimal for application to a clinical setting in which the yield of CML cells per ml of whole blood is typically lower than those levels once treatment with imatinib is initiated. Moreover, our previous demonstrations of the assay technology were performed in model cell lines genetically engineered to overexpress constructs of Abl kinase that were not necessarily relevant to the CML disease model. Accordingly, we set out to establish an assay that would provide higher sensitivity for detecting Bcr-Abl kinase activity using the peptide biosensor and also demonstrate the method in a more clinically relevant model (the human CML cell line K562). We chose LC/MS with multiple reaction monitoring (MRM) as an analysis strategy based on its excellent sensitivity and signal to noise. Lower detection limits for MRM can reach the fmol-amol range depending on the specific analytes and instrumentation involved. We and others
have successfully detected and quantified peptides and their phosphorylated derivatives using this technique. Here we describe the development of the MRM method and demonstrate that the biosensor/MRM strategy can be employed to detect Bcr-Abl activity and inhibition in CML cells as a model for monitoring drug sensitivity in human leukemia.
Peptide-based biosensor for Abl kinase.