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. 2010 Nov;9(11):3065-73.
doi: 10.1158/1535-7163.MCT-10-0623. Epub 2010 Sep 3.

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

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Free PMC article

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

Ahmet Tunceroglu et al. Mol Cancer Ther. .
Free PMC article

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, imatinib and dasatinib, for patients with chronic myelogenous leukemia. Hence, even though imatinib initially produces remission in the chronic phase, ultimately these therapeutics fail via the emergence of drug resistance, in which chronic myelogenous leukemia could inevitably progress to a terminal blast phase culminating in 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-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.

Conflict of interest statement

Conflicts of Interest: The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. Design of an in vivo FRET assay to monitor Bcr-Abl activity in CML
The assay is based on the in vivo phosphorylation of the Crk II-derived Picchu biosensor on Tyr(Y)221 by Bcr-Abl. Schematic representation shows that Picchu Y221 phosphorylation by Bcr-Abl induces an intramolecular association between the SH2 domain and pTyr221, bringing the N-terminal and C-terminal ends of the molecule together in a conformation change and yielding an energy transfer from CFP to YFP.
Figure 2
Figure 2. Effect of Y221 phosphorylation by Bcr-Abl and lability to Imatinib
(A) Crk Y221 becomes tyrosine phosphorylated in wild type Bcr-Abl expressing 32D cells (lanes 1 and 2) and T315I Bcr-Abl 32D cells (lanes 3 and 4). Note that Y221 Picchu and Y245 Abl phosphorylation is labile to Imatinib in WT Bcr-Abl cells, but resistant in T315I Bcr-Abl cells (lanes 2 versus 4). As indicated by the vertical lines, lanes 1 and 4 were located on the same gel as lanes 2 and 3 but were not immediately adjacent. (B) Effect of Bcr-Abl tyrosine kinase mutations on Picchu phosphorylation. Detergent lysates were prepared as in panel A, after which equivalent amounts of lysate were immunoblotted with either anti-phosphoY221 Crk (for phosphorylated Picchu in middle panel) or anti-phosphoY245 Abl (top panel). Results demonstrate level of inhibition by 5 µM Imatinib of WT Bcr-Abl and the Y253F, E255K, and T315I mutants. Bars show percent change in Picchu phosphorylation, normalized to expression, following Imatinib. (C) Effect of Imatinib on the tyrosine phosphorylation of Picchu (left axis) and Bcr-Abl (right axis). HEK 293T cells were transiently transfected with Bcr-Abl and Picchu, and after 48 hrs, cells were treated with 5 µM Imatinib for the indicated times. After immunoblotting, gels were scanned and presented as percent of Imatinib untreated.
Figure 3
Figure 3. (A) Effect of Bcr-Abl tyrosine kinase mutations on Picchu FRET
Phosphorylation of the Picchu probe by Bcr-Abl and the corresponding changes in FRET emissions after Imatinib treatment. Bars correspond to duplicate samples, only one sample is shown for T315I mutant in this experiment. (B) Real time FRET microscopy demonstrating pseudocolor visualization of the change in FRET emission after the addition of 5 µM Imatinib for WT Bcr-Abl (panels i and ii) and the T315I mutant (panels iii and iv). See supplemental real player movie SFig 1 and SFig 2 for real-time kinetics as well as the accompanying supplemental graph, SFig3, depicting FRET tracking for each cell in SFig2. (C and D) Real-time microscopic “tracking” of FRET emissions in CosE37 cells expressing wild type Bcr-Abl (C) and T315I Bcr-Abl (D). DMSO (as control) or Imatinib was added at minute 19. Multi-cell FRET tracking can be found as supplemental data, SFig4.
Figure 4
Figure 4. Analysis of ΔFRET ratios by FACS
Flow cytometric analysis indicating the FRET YFP/CFP ratio in populations of cells with Picchu-CAAX alone (black), with Picchu-CAAX and Bcr-Abl (blue), and with Picchu-CAAX + Bcr-Abl followed by 5 µM Imatinib treatment (red). In panel B, a soluble form of Picchu was used while in panel A, a membrane targeted CAAX-fused version of Picchu was used. Note that both Picchu isoforms are highly labile to Imatinib.
Figure 5
Figure 5. Real-time microscopic tracking of dose-dependent inhibition of wild type Bcr-Abl by Imatinib (top) and Dasatinib (bottom)
CosE37 cells were transfected to co-express Picchu-CAAX and wild type Bcr-Abl as described and Imatinib (top) or Dasatinib (bottom) was applied at minute 9. Rate constants for each concentration of the kinase inhibitor are provided in the tables.
Figure 6
Figure 6
Demonstration of the utility of Picchu to detect additive or competitive effects of different therapeutics. CosE37 cells were transfected to co-express Picchu-CAAX and wild type Bcr-Abl as described and Dasatinib, of the indicated concentrations, was added at 19 minutes and Imatinib was added at minute 29.
Figure 7
Figure 7. Pharmaceutical profiling of Bcr-Abl inhibition by TKIs
Two examples of the potential utility of Picchu FRET in pharmaceutical profiling of clinically relevant Bcr-Abl mutations as to their efficient inhibition by therapeutics of varying concentrations. Inhibition is depicted as a function of TKI concentration (left), time (bottom) and ΔFRET ratios (right) for Imatinib (top) and Dasatinib (bottom) to provide a pharmaceutical signature for Bcr-Abl proteins. These data can be extrapolated to select the optimum therapeutic, or combination thereof, for different Bcr-Abl mutations for the selection of TKIs with the most desirable kinase inhibition profiles.
Figure 8
Figure 8. Schematic representation of a generic FRET based biosensor for tyrosine kinases and their inhibitors in human cancers
Adapted from Picchu, a platform of tyrosine kinase specific FRET cassettes can be generated by replacing pTyr221 and Crk SH2 domain as indicated.

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