Real-time Fluorescent Resonance Energy Transfer Analysis to Monitor Drug Resistance in Chronic Myelogenous Leukemia

Abstract

Despite the initial effectiveness of oncogene-directed cancer therapeutics, acquired drug resistance remains the ultimate “Achilles’ heel” for long-term durable remission in cancer patients. Acquisition of drug resistance is not more evident elsewhere than in the use of tyrosine kinase inhibitors (TKIs), Imatinib and Dasatinib, for patients with Chronic Myelogenous Leukemia (CML). Hence, while Imatinib initially produces remission in the chronic phase, ultimately these therapeutics fail via emergence of drug resistance, where CML can inevitably progress to a terminal blast phase culminating in a fatal outcome. Technically, it is challenging to predict the onset of drug resistance in a small number of oncogene-transformed cells, making the decision of when and how to employ second generation tyrosine kinase inhibitors, or employ novel compounds that would be of benefit in treating drug resistant Bcr-Abl mutants mainly retrospective. Here, we characterize a rapid and sensitive real-time Fluorescent Resonance Energy Transfer (FRET) based assay that is able to detect the in vivo activity of Bcr-Abl and its inhibition by small molecule compounds. Due to its real-time and in vivo nature, such an approach has the potential to monitor a drug-resistant phenotype, as well as to identify pharmaceutical agents that inhibit drug-resistant Bcr-Abl oncoproteins in vivo.

Biosensors and their applications – A review

“…This probe was further employed to regulate response to therapy, and to observe the onset of drug-resistant cells, permitting prediction for alternative therapeutics. [54][55][56]…”

Genetically encoded fluorescent biosensors illuminate kinase signaling in cancer

“…As such, there is an emerging need for in vivo tools that can rapidly and sensitively monitor Bcr-Abl activity and evaluate drug resistance as well as guide clinical therapies. With this in mind, Tunceroglu et al (49) utilized Picchu (26) to profile inhibitor efficacy and thus monitor drug resistance in CML cells. However, because Picchu can also respond to both Abl and EGFR activity, Mizutani et al (50) instead developed a more specific and sensitive FRET-based biosensor based on CrkL, which is the most characteristic Bcr-Abl substrate.…”

Fluorescent Reporters and Biosensors for Probing the Dynamic Behavior of Protein Kinases

“…They are equally a source of inspiration for biomedical applications, including diagnostic assays, enabling development of complementary assays or serving as alternatives to the more traditional antigenic and/or genomic approaches [23]. This is of particular relevance in the case of PK dysregulation associated with disease, since fluorescent biosensors can provide a readout of target activity, and thereby highlight alterations associated with the onset of disease, monitor changes in PK activity throughout disease progression and assess response to therapeutics, as exemplified by application of the Bcr-Abl biosensor to CML [24,25,26]. …”

“…Following these first successful developments, optimized derivatives were developed, such as the Picchu sensor to probe cAbl, and the Pickles sensor to probe Bcr-Abl activity, which exploits ECFP and Venus (variant of YFP) [24,89]. Pickles has more recently been applied to assess Bcr-Abl activity in cells from patients, response to drugs and development of resistance to drugs [24,25,26]. …”