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MD Anderson RPPA Study Reveals New Potential Drug Targets in Small Cell Lung Cancer

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Researchers from the MD Anderson Cancer Center have identified PARP1 and several other proteins as potential drug targets in small cell lung cancer using reverse-phase protein arrays to compare proteins in small cell and non-small cell lung cancer cell lines.

The group reported its results in the journal Cancer Discovery this month. While the researchers identified several potentially actionable targets, they chose PARP1 as the first to investigate further because several PARP inhibitors are already in development for other cancers. Testing two PARP inhibitors in cell lines and patient samples, the group found they could slow the growth of SCLC cells.

Lauren Byers, the study's first author and an assistant professor of oncology at MD Anderson, told ProteoMonitor in an email that she and her teammate John Heymach are now developing two clinical trials to look at the action of PARP inhibitors against small cell lung cancer.

"Although the findings from our study need further investigation in animal models and clinical trials, we are very excited about the possibility of PARP inhibitors for the treatment of SCLC," she wrote.

According to Byers, the team expects to open the first of these trials "soon" in collaboration with Memorial Sloan Kettering Cancer Center oncologist Catherine Pietanza as principal investigator, supported by the National Institutes of Health's Cancer Therapy Evaluation Program. The trial will be a study of chemotherapy with or without the PARP inhibitor ABT-888, she said.

In the group's proteomics discovery study, Byers and Heymach's team profiled approximately 34 SCLC lines and 74 NSCLC lines.

The team used a reverse phase array platform built at MD Anderson by researchers in the department of systems biology that Byers said she and Heymach have adapted to the study of lung cancer by developing an extended panel of validated antibodies that are tailored to the study of the disease. "We have a robotic arrayer and staining system that lets us produce our own RPPAs in our lab," she wrote.

The RPPA technique, which uses cell lysates spotted in array formats that can then be probed with antibodies to multiple proteins of interest, was originally developed by George Mason University researchers Emanuel Petricoin and Lance Liotta, who have collaborated with MD Anderson researchers in the past on cancer marker studies using the method (PM 3/26/2010).

By comparing the non-small cell and small cell lung cancers, Byers and her colleagues hoped to identify proteins specific to small cell cancers that might be useful as drug targets.

Comparing proteins in the two sets of cell lines, the researchers discovered higher expression of several proteins in SCLC than NSCLC, including EZH2 and PARP1, and lower expression of other targets, including EGFR, and downstream targets in the PI3K/Akt/mTOR pathway and the RAS/Raf/MEK pathway.

Byers said the team's reverse-phase protein array approach has several advantages. "[It] let us look in detail at protein expression and activation levels in cancer cells," she wrote. "For example … to look directly at the levels of phosphorylated proteins and certain post-translational modifications, which, in turn, gives us clues to what signaling pathways or other cellular machinery may be more active within a certain subset of lung cancers."

Byers said the group can print up to 1000 samples on a single array, and for each project, prints around 200 of these arrays, using each one to measure a different protein of interest. "This allows us to quantify protein levels in each sample and compare them between different groups — in this case SCLC and NSCLC — in a highly precise manner," she said.

"Looking at proteins' levels has specific advantages, as compared to looking at the RNA or DNA level," she added. "In addition to being able to measure the activation state of different proteins, we also know that most drugs act on proteins, and so identifying targets at the protein levels may have more direct clinical application."

After identifying proteins differentially expressed between SCLC and NSCLC, the researchers then compared mRNA levels in cell lines and tumor samples to validate the SCLC proteins. Among the group of DNA repair proteins, PARP1 had the greatest differential mRNA expression, the group wrote. Other potentially druggable targets included EZH2, Bcl-2, PRKDC, and PCNA.

Because the team approached the study by comparing SCLC and NSCLC, it's possible they may have missed important pathways or targets that are highly expressed in both cancer types, the authors reported. Future studies comparing SCLC with normal lung samples, or a larger group of tumor types may help identify additional targets.

Of the targets Byers and Heymach did identify, they decided to focus first on PARP1. "For our PARP experiments, we selected drugs that were already fairly advanced in their clinical development, [like] AZD2281, [which] had been in Phase III testing in breast cancer, with the goal of being able to rapidly translate findings from the lab into clinical trials, and eventually into standard patient care," Byers said.

In the Cancer Discovery study, the researchers followed their RPPA experiments by testing the effect of PARP inhibition with AZD2281 – AstraZeneca's olaparib – in several cell lines. In all the cell lines the team treated with the drug, poly ADP-ribose levels fell significantly in a "dose-dependant manner, indicating inhibition of PARP1 activity," the authors wrote.

As controls, the team also tested two breast cancer cell lines — one with a BRCA1 mutation and the other with a PTEN mutation — with AZD2281. Although the lines were sensitive to the drug and another PARP inhibitor, AG014699, SCLC cell lines were either as sensitive or more sensitive, the researchers reported.

Byers said that in addition to moving forward with clinical trials combining PARP inhibitors with chemotherapy in SCLC, the team is also further investigating the other potential targets identified in the RPPA profiling.

In addition, Byers said, the group plans to further investigate the mechanism of PARP inhibitor sensitivity in SCLC and look for biomarkers in patient tumors from the planned clinical trials that could help "identify upfront which patients are most likely to benefit from therapy — or how tumors might develop resistance."

She said this work will be funded by a recent $100,000 award she received from the LUNGevity Foundation.

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