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Dutch Researchers Combine shRNA, Proteomics to ID New Melanoma Drug Target


NEW YORK(GenomeWeb) – Using a combination of proteomics and shRNA screens, researchers at Utrecht Universityand the Netherlands Cancer Institute have identified a potential drug target in BRAF mutant melanoma.

In a study published last month in Molecular Systems Biology, the researchers identified the ROCK1 kinase as a potential drug target to be used in combination with BRAF inhibition. In preclinical validation work, targeting ROCK1 appeared to further sensitize melanoma cells to treatment with BRAF inhibitors, suggesting that such an approach could eliminate diseased cells before they have the opportunity to develop resistance to BRAF inhibition, said Maarten Altelaar, a Utrecht researcher and author on the paper.

Use of BRAF inhibitors in melanoma patients with BRAF mutations has proven highly effective, often leading to tumor remission. Most patients, however, ultimately develop resistance to this treatment and relapse.

To get around this problem, which is common to many targeted cancer treatments, researchers are pursuing combination therapies with the aim of killing off tumor cells less sensitive to BRAF inhibition before they can develop resistance or blocking pathways that enable cells to develop resistance.

In the case of the MSB work, the researchers were looking to identify targets that could increase the sensitivity of melanoma cells to BRAF inhibition with the goal of identifying drug combinations that would "kill off the cells before they have a chance to adapt and become resistant," Altelaar told GenomeWeb.

To investigate this question, Altelaar and his colleagues turned to a combination of shRNA screens against a kinome library and a mass spec-based proteomic and phosphoproteomic analysis.

The shRNA screens consisted of sensitization screens for the BRAF inhibitor PLX4720 in which they used a 4,000-shRNA library targeting 500 kinases to identify targets that, upon knockdown, would sensitize cells from the human melanoma line 04.01 to PLX4720 treatment. Because ERK inhibitors are also being developed for melanoma treatment, they performed a similar sensitization screen for the ERK inhibitor SCH772984.

Combining the results of both screens, they identified five potential targets for sensitizing cells to treatment with both BRAF and ERK inhibitors, the kinases AAK1, PLK4, IGF1R, MET, and ROCK1. As the authors noted, two of these five hits, IGFR and MET, had previously been implicated in BRAF resistance.

The researchers then turned to a proteomic analysis to identify proteins and pathways involved in early BRAF inhibitor resistance mechanisms, looking at the changes at one day and three days in melanoma cells treated with PLX4720. Running samples on Thermo Fisher Scientific Orbitrap Velos, Elite, and Q Exactive instruments, they identified roughly 5,700 proteins and 11,500 phosphosites across three biological replicates, and quantified roughly 3,800 proteins, finding, respectively, that 129, 406, and 313 proteins were significantly regulated at one day versus control, three days versus control, and three days versus one day.

Among these proteins were several associated with ROCK1, identified as a potential sensitizing target in the shRNA screen. Specifically, the researchers detected down-regulation of Rnd3, a negative regulator of ROCK1; FOXD3, a down-regulator of Rnd3; and the integrin Itgb1, which, the authors said, has in previous studies been shown to activate the RhoA-ROCK pathway in colon carcinoma cells.

The combination of the shRNA and proteomic data led Altelaar and his colleagues to identify ROCK1 as a potential target for therapy in combination with BRAF inhibitors, an idea they explored further in a series of experiments looking at whether inhibition of ROCK1 did, in fact, make cells more sensitive to treatment BRAF inhibitors.

They initially investigated this question using shRNAs to silence ROCK1, finding, as their shRNA and proteomic data suggested, that decreased levels of ROCK1 led to fewer cells surviving treatment with PLX4720. They then repeated the experiment using the ROCK1 inihibitor GSK269962A, finding again that treatment with the ROCK1 inhibitor significantly improved PLX4720's ability to kill melanoma cells. Repeating the experiments with the ERK inhibitor SCH772984, they found that targeted ROCK1 similarly sensitized melanoma cells to ERK inhibition. 

Altelaar said that the study represented his lab's first effort at combining shRNA screens and proteomic analysis and that, while this combination is not particular common in the literature, it was key to identifying ROCK1 as potential target.

The proteomic analysis, he noted, identified a large number of potentially interesting signaling events, expression changes, and protein-protein interactions, while the shRNA screen identified a small set of potential targets that helped focus the analysis.

"It really helps to have the information from the shRNA screen so you can do a more targeted mining of your [proteomic] data," Altelaar said.

At the same time, while the shRNA screen targeted only kinases, the proteomic analysis is able to identify elements like downstream substrates that might also prove interesting as potential drug targets.

This, Altelaar noted, is particularly relevant given the goal of using combinations of therapies to stop resistance before it starts. Because kinases are often important in a variety of cell functions, targeting multiple such proteins raises the potential for toxicity and side effects. However, with the proteomic data "you might be able to find some substrates downstream that are less toxic but still have the effect that you want," Altelaar said.

In the case of ROCK1, however, the researchers found that inhibition of this kinase alone did not increase cell death, suggesting, he said, that it might not be particularly toxic as an addition to BRAF inhibitors.

The MSB paper was primarily a discovery effort, and, Altelaar said, he and his co-authors currently have no firm plans for any sort of clinical trial. However, he noted, that his collaborators at the Netherlands Cancer Institute have a "direct link to the clinic," making such future work a possibility.