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UCLA Study Identifies Leukemia Pathways Potentially Involved in Gleevec Resistance

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By Adam Bonislawski

University of California, Los Angeles, scientists have completed a global phosphoproteomic analysis of Bcr-Abl-transformed cells that identified several signaling mechanisms potentially tied to drug resistance in leukemia patients.

The study, which will be published next week in the March 29 edition of Science Signaling, measured proteome-wide changes in hematopoietic BaF3 cells in response to perturbation of several Src family kinases, or SFKs, identifying four SFK negative feedback mechanisms that appear to be suppressed in cell lines resistant to the Novartis leukemia drug Gleevec.

According to Thomas Graeber, professor of molecular and medical pharmacology at UCLA and lead author on the paper, identification of these mechanisms could help doctors select better therapies for leukemia patients, allowing them to identify those likely to be drug-resistant.

"Therapeutically, by measuring the levels of these feedback pathways in cells upon first presentation [of leukemia] you might be able to a priori predict whether the patient is going to be [Gleevec] resistant or not," he told ProteoMonitor.

For such patients, a drug like dasatinib – sold as Sprycel by Bristol-Myers Squibb – might be a better first option, Graeber said, noting that "if you were to find patients that looked like they were going to have SFK-mediated resistance, you could argue that those patients should go on dasatinib," which targets both Bcr-Abl and Src kinases.

Having identified these pathways as leads, the researchers now plan to look at this "clinical angle," he said, adding that the main challenge currently is lining up enough patients exhibiting the resistance mechanisms under investigation.

"You need to prescreen each case to make sure it's not explained by some other category of resistance," Graeber said. "But other than gathering enough of the right patient samples, I think we're ready to start looking at that [clinical] question."

Currently his team is working with University of California, San Francisco, researcher Markus Muschen – a co-author on the study – on mouse xenograft systems that could expand the number of patient samples they have to work with.

Aside from these near-term clinical leads, the research also helped delineate the structures of the Bcr-Abl and SFK signaling networks and suggested patterns of crosstalk between the two pathways that could provide further insight into the development of drug resistance, Graeber said.

"Although these [negative feedback] mechanisms seem to be responsive in particular to the Src activity, their negative regulation affects not just Src substrates but even Bcr-Abl substrates that are mediated through the non-Src-branch," he said. "So there's an interesting motif for cross-talk between pathways where a highly activated pathway may invoke a negative feedback [pathway] that cross-talks onto other pathways."

This balance between negative and positive feedback pathways could be key to cancer patients' development of drug resistance, Graeber added.

"Depending on that balance [between the positive and negative signaling] what are the kinetics when you inhibit a main pathway?" he said. "Does that negative signaling continue to keep the other pathways quiet and the cell dies? Or does [the negative] signaling fade away quickly enough that the other pathways can be re-elevated and keep the cell alive?"

The study marked a change in approach for Graeber's team, he said. While in previous work they've typically tried to identify pathways of potential interest by linking phosphoproteomic profiles to patient phenotypes, in this case they decided to begin by perturbing the Bcr-Abl and SFK networks and tracing the effects outward.

"In the past we've said, 'Let's just look at the global picture and correlate that with phenotype to see if we can learn what signaling events in that global network may be important,'" he said. "In this study we're saying, 'We know this is an important area, so let's focus some attention on it and see what we can learn.'"

The researchers quantified the phosphoproteins under study using label-free mass spec on a Thermo Fisher Scientific LTQ Orbitrap instrument, analyzing the results with a variety of bioinformatic tools incorporating data from literature-based protein function and interaction databases.

The study is "a really good example of the use of a manipulatable cell line-based system and high resolution mass spectrometry," Emanuel Petricoin, co-director of George Mason University's Center for Applied Proteomics and Molecular Medicine, told ProteoMonitor. "How cells rewire around therapy is of critical importance to understanding mechanisms of drug resistance, and this is a great example of how you use mass spectrometry to start to develop hypotheses about that."

He noted that because the study used transformed cell lines as opposed to actual patient samples, it remains to be seen "how much these specific signaling networks that were uncovered actually recapitulate what happens in patients." However, he added, "it does form a good basis of hypotheses" that can now be investigated in patient samples.

Graeber's lab has in the past developed several research tools that could be useful for this sort of work. In November, his team published a study in Cancer Medicine that used an integrated chip-based microfluidic and imaging platform they'd devised to measure ABL kinase activity in BCR-ABL-positive leukemia patient samples (PM 11/19/2010).

The microfluidic system allows the direct measurement of a kinase's activity which, Graeber noted, makes it a potentially useful complement to approaches like reverse-phase protein microarrays or mass spec-based phosphoproteomics that measure protein phosphorylation levels as a proxy for kinase activity.

Also potentially useful, he said, is a microfluidic image-cytometry platform for the single-cell quantitation of proteins in samples as small as 1,000 cells that his lab developed and licensed in August to biotech start-up CytoScale Diagnostics (PM 08/06/2010), which plans to release the device as a research platform and, eventually, for diagnostic use.

"This all fits in with that [instrument development]," he said. "The kinase activity platform could be used to look at Bcr-Abl and Src activity, letting us ask who are the patients on [Gleevec] who still have elevated Src activity. And now that through this unbiased global approach we've identified candidates we think are informative about the state of the cell, we can use antibodies directed against those specific events [to measure them] on the cytometry platform."


Have topics you'd like to see covered in ProteoMonitor? Contact the editor at abonislawski [at] genomeweb [.] com.

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