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139th National Cancer Advisory Board

Gleevec® yet allow the kinase to function; the Bcr-Abl can still have its oncogenic effect, and Gleevec® can no longer inhibit it. Mutations on the Abl kinase P loop, an important regulatory domain, are particularly significant. A second generation of Bcr-Abl inhibitor drugs to replace Gleevec in resistant patients is in development or approved, but there are no predictive assays underway to help in selecting which if any should be used. Clinical challenges include: the need for rapid testing for Imatinib resistance, the selection of second-line therapy, the identification of effective dosage, and determining the failure of STI therapy.

The measurement of Bcr-Abl activity poses a technology challenge, particularly in the desire to understand whether the kinase is on when Gleevec or the approved alternative, AMN or Dasatinib, are present. To do this, researchers need to be able to detect Bcr-Abl activity in a whole cell lysate directly from the patient’s blood. The process needs to be rapid, robust, and simple, and amenable to the clinical laboratory or it will not be useful. It also must be adaptable to high throughput for it to be used in the screening for new drugs and to accelerate the process.

An approach that has been employed is to use small peptides that have been selected through library screening. Dr. Kron mentioned a short peptide called Abltide as being an Abl substrate that has a tyrosine that is phosphorylated by the Abl or Bcr-Abl kinase. The goal of a kinase assay is to detect ADP, the phosphotyrosine, or the phosphopeptide, and ignore the cell lysate, other kinases, and phosphatases. The project started with a proof-of-principle bead-based assay of Bcr-Abl in cell lysates. Dr. Kron exploited a molecular recognition trick to add a second peptide to the Abltide to make it stick better to the Bcr-Abl and Abl kinases. A super substrate that was very highly phosphorylated by Abl or Bcr-Abl resulted; when Imatinib was added, the phosphorylation went away. Dr. Kron showed a peripheral blood ficoll-paque extracted sample from an Imatinib-resistant patient compared to the wild type Bcr-Abl from a leukemia cell line, K562; the IC50 in this simple assay was about 10 µM for the K562 cells but there was no response to Imatinib in the patient sample.

Dr. Kron next described how a new technology called Luminex was adapted to a new purpose. This involved tiny plastic beads with glutathione on them which allows glutathione-S transferase protein fused to Abltide to be stuck onto the beads. The beads were reacted with cell extract, plus or minus Imatinib, and analyzed using anti-phosphotyrosine antibody and anti-IgG with phycoerythrin (fluorescent protein). The samples were run through a small bench-top machine, the Luminex reader, to sip each well of a 96 well plate and analyze the fluorescence. Dr. Kron showed work of an undergraduate at the University of Chicago who found that, in the absence of Imatinib, there are about 1,000 units of fluorescence while, with Imatinib, the activity is only 50 units, with an IC50 of about 20 µM. Additionally, Dr. Kron worked with Dr. Sean Palacek, a chemical engineer at the University of Wisconsin, Madison, to develop an acrylic super glue for proteins. A polyacrylamide hydrogel carries a Bcr-Abl substrate; when Bcr-Abl, plus or minus Imatinib, is added and detected, more added Imatinib results in less signal (IC50) of about 20 µM. This simple technology can be used to screen for new inhibitors.

The next generation of technology uses matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF). In a vacuum, a laser desorbs proteins and peptides from the surface, which then enter the time-of-flight tube of a mass spectrometer where they are separated by mass over charge, yielding a tool to measure molecular weight. Phosphorylation causes a mass shift of 80 units and the two peaks both display impressive signal to noise. Thus a computer can analyze the amount of peptide and phosphopeptide present, and the ratio of these two reveals the amount of phosphorylation. Adding Imatinib affects this by up to 80 units, which is an amazing signal to noise. This technology has been adapted to chips as well. To move further toward an integrated assay system, the Cotter laboratory at Johns Hopkins University is making mini-MALDI-TOFs, where the time-of-flight tube is 4 cm rather


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