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Sloan-Kettering Scientists Using Cancer Drug Candidate to Study Aberrant Protein Pathways


By Adam Bonislawski

A team led by scientists at Memorial Sloan-Kettering Cancer Center has devised an affinity capture-based proteomics technique that could be used to identify and map dysregulated protein pathways in a number of different cancers.

The technique, which was detailed in a paper published this week in the online edition of Nature Chemical Biology, relies on the inhibitor PU-H71 – a small molecule that selectively binds tumor-enriched Hsp90 proteins, enabling pulldown of Hsp90-bound oncogenic client proteins. According to MSK researcher Gabriela Chiosis ― one of the developers of the method ― measurement of these captured proteins combined with bioinformatic analysis could provide a better understanding of tumor biology.

Dysregulation of protein pathways through mis-expression or mutation is key to tumor development, and to maintain the stability and function of these proteins, tumor cells co-opt molecular chaperones like Hsp90. The inhibitor PU-H71 binds only weakly to the uncomplexed form of Hsp90 found in normal tissues but binds strongly to the multi-protein Hsp90 complexes found in tumor cells.

Given this, PU-H71 has potential as a cancer therapeutic and is currently in Phase I clinical trials at the National Institutes of Health Clinical Center and Memorial Sloan-Kettering. It also might prove useful, the developers of PU-H71 realized, as an affinity reagent for proteomics research.

"We designed this class of compounds ten years ago, and it has been a long process of developing it as a drug," Chiosis told ProteoMonitor. "And along the way we realized that because [PU-H71] traps these Hsp90-sheltered oncogenic complexes, we could perhaps use [it] attached to a solid support as a tool to isolate them."

To test this theory, the scientists covalently attached PU-H71 to agarose beads, which they then used to isolate Hsp90-bound complexes from K562 cells, a line of chronic myeloid leukemia. They then analyzed the captured proteins via LC-MS/MS on an AB Sciex QSTAR-Elite QTOF and a Waters Xevo QTOF and used Ingenuity Pathway Analysis to place the identified proteins into networks.

Their analysis placed the Hsp90-bound proteins into 13 networks associated with cell death, cell cycle, and cell growth and proliferation. These networks showed significant overlap with canonical CML signaling pathways, suggesting that, as the researchers had hypothesized, the captured Hsp90 complexes contained a number of proteins essential to maintaining the tumor phenotype. Among the top-scoring networks were the PI3K-AKT-mTOR, Raf-MAPK, and NfϰB-mediated signaling pathways, which Bcr-Abl uses to propagate aberrant signaling in CML.

These results, Chiosis said, demonstrated the technique's potential usefulness for elucidating cancer protein pathways.

"For the first paper, of course, you have to use a system that is well characterized and show that your method identifies what really is going awry in that cell line," she said. "Then you can move into an unknown system and identify the networks that are being altered. We used CML for validation, and of course, now we are coming out with other papers where we used other cancers to show that we can identify unknown pathways and networks."

The researchers have used the technique to examine pancreatic cancer cells, Chiosis said, identifying both pathways that overlapped with CML as well as pathways specific to pancreatic cancer.

"What we are doing is taking advantage of the Hsp90 biology and of the selectivity of the small molecule for those oncogenic works of Hsp90," she said. "So, if we do this pulldown in cancer cell A, we'll find certain networks, and if we do this pulldown in cancer cell B that has different networks driving it we'll find different things."

In addition to pancreatic cancer, the researchers are now investigating certain lymphoma cell lines, Chiosis said. They also plan to use the technique on surgically resected tumor tissue from human subjects, which they are currently in the process of obtaining.

Chiosis noted that the Sloan-Kettering team is looking into targeting additional chaperone proteins for the same purpose, calling the technique "a huge step forward in identifying the functional altered networks in cancer cells."

"Many proteomics studies right now just do an inventory of proteins, and they are so limited by the abundant proteins, even though those are not necessarily the relevant ones," she said. In contrast, she said, the PU-H71 pulldown technique isolated a number of low-abundance analytes, including a wide variety of signaling proteins.

Chiosis also cited the method's ability to pick out aberrant proteins as a key advantage, particularly compared to some antibody-based approaches.

While antibody-based phosphoproteomics may identify a large number of phosphorylated proteins in the cell, not all phosphorylated proteins detected by such methods will be aberrant, she noted. The PU-H71 technique, on the other hand, isolates primarily aberrant proteins important for driving transformation, she said.

Memorial Sloan-Kettering has patented PU-H71 for use as a drug, as a reagent for cancer research, and as a companion diagnostic, Chiosis said. It has licensed rights to PU-H71 to Samus Therapeutics, a biotech firm formed in July to develop the molecule as a cancer treatment.

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