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Phosphoproteomics Study of Leukemia Identifies Drug Repositioning Opportunity


NEW YORK (GenomeWeb) – A team of researchers at the University of Cincinnati and Cincinnati Children's Hospital Medical Center has used phosphoproteomics to investigate cell signaling linked to certain types of leukemia, obtaining results that suggest a new indication for an existing cancer drug.

In a study published last week in Leukemia, the researchers identified protein activity that was dysregulated by mutations in the gene CSF3R, which codes for the granulocyte-colony stimulating factor receptor (G-CSFR), a protein involved in the proliferation of myeloid progenitors and their differentiation into neutrophils. They also identified an existing drug, ibrutinib, sold by Pharmacyclics and Janssen as Imbruvica, that could be repurposed to treat cases of acute myeloid leukemia (AML) with CSF3R mutations.

The team was particularly interested in how patients suffering from severe congenital neutropenia (SCN) transition over time to myelodysplastic syndrome (MDS) and AML, said Kenneth Greis, director of proteomics and mass spectrometry at the University of Cincinnati, the senior author of the study.

Patients with SCN are unable to produce a normal amount of neutrophils, the most common white blood cells and a key component of the immune system. Left untreated, these patients are highly susceptible to infections, but, Greis said, their bodies can be induced to generate neutrophils by stimulating G-CSFR.

"You can get enough neutrophils to be made in most cases [so] that these patients can do quite well," he said.

However, Greis said, around 80 percent of patients ultimately develop additional mutations in CSF3R that cause them to transition to AML.

"The patients that are on this induction therapy are routinely evaluated for whether they have any cells that are expressing these mutations," he said. "But the problem is that when [mutations] start turning up, it's not clear how to address that."

To better understand this problem, Greis and his colleagues decided to investigate the effects of CSF3R mutations on cell signaling, using mass spec-based phosphoproteomics to look at how protein phosphorylation levels differed between patients with wild-type CSF3R and patients with mutated forms of the gene.

Using SILAC labeling and antibody-based phosphor-tyrosine enrichment, the researchers analyzed BaF3 cells expressing wild-type G-CSFR and two mutant forms of the protein on a Sciex TripleTOF 5600+ mass spectrometer, identifying and quantifying roughly 250 phosphotyrosine sites.

Among the proteins with differing phosphorylation patterns was a set involved in endocytosis and receptor recycling. They also identified a differentially regulated set of proteins linked to Bruton's tyrosine kinase (Btk), including Btk itself, mutated forms of which have been associated with diseases including B cell lymphomas and some cases of AML.

Using Kinase Enrichment Analysis (KEA) to look at what kinases were most likely responsible for the differentially phosphorylated proteins they observed, the researchers found that Btk activity differed significantly between the wild-type and mutant datasets, leading them to hypothesize that this protein might be an important downstream effector of mutant G-CSFR.

"It just so happens that there's already a drug on the market [ibrutinib] that targets Btk," Greis said, noting that this allowed the researchers to further evaluate the role of Btk in the transition of patients from SCN to AML.

They performed cell viability assays looking at the survival of wild-type and mutant G-CSFR cells upon treatment with varying concentrations of ibrutinib, finding that wild-type cells were significantly less sensitive to the drug. They also analyzed c-Kit+ bone marrow cells taken from wild-type and mutant G-CSFR, finding that the mutated cells were around fourfold more sensitive to ibrutinib than wild-type cells. They then repeated the experiment using a model of primary human CD34+ umbilical cord blood cells, which produced similar results.

The findings suggest that ibrutinib could be used as a sort of maintenance therapy to prevent development of AML in patients undergoing stimulation of G-CSFR as treatment for SCN, Greis said.

"We're thinking that as [doctors] do surveillance of these patients, once they start to see any [G-CSFR] mutant cells show up, they could be treated with ibrutinib to kill the mutant cells and put [the patients] back to normal where the [G-CSFR] induction therapy can continue to work," he said.

How effective this approach would be in the long term remains a question, he added, noting that "if there's anything we have learned over the years in trying to address cancer, it's that it is a bit of a whack-a-mole type approach."

"You find a target, you knock that down, and then somehow [the cancer] develops resistance, and so you have to find the next [target] in line to try to knock down," he said. "That's where we are right now. We think we have something we can knock down right now. It may be effective long term. No other mutations may develop. We don't know."

To address this question, a better understanding of the signaling pathways linked to Btk is needed, Greis said, adding that he and his colleagues would like to further explore ibrutinib-induced phosphorylation changes upstream and downstream of Btk.

"Is activation of Btk the primary way these mutant cells develop and proliferate, or are there other pathways involved that are not necessarily amenable to [ibrutinib] treatment?" he said. "That's the part that we don't know. We know that they have a rapid upregulation of the phosphorylated form of Btk. We don't know specifically how that happens. That's something we hope to sort out."

In the meantime, Greis said, he and his coauthor, Leighton Grimes, director of the cancer pathology program at Cincinnati Children's, are in discussions with clinicians about developing a clinical trial testing ibrutinib in SCN patients.

"It's not an enormous patient population," he said, noting that there are around 1,000 to 2,000 cases of patients converting from SCN to AML annually. "We've reached out to a number of cancer centers that see patients in this category, and we're actively working with our clinical colleagues at Cincinnati Children's to establish what a clinical trial would optimally look like."

The researchers have the advantage of working with a drug that has already received US Food and Drug Administration approval and has been through testing for toxicities and off-target effects, Greis said. "In that way, we're sort of ahead of the game."

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