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Assays for Heat Shock Protein Complexes Could Serve as Companion Dx for Inhibitor Drugs

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NEW YORK (GenomeWeb) – New assays detecting complexes of heat shock proteins in cancer cells could be used to predict patient response to inhibitors of those proteins.

The protein complexes involve chaperone proteins, specifically so-called heat-shock proteins that are also involved in general response to cellular stress, among other functions. Led by Gabriela Chiosis, a team of Memorial Sloan Kettering Cancer Center (MSKCC) scientists found that while they're not universally essential to cancer, for the cancers that have them, the complexes are essential for survival.

"In certain cancers these proteins are not just floating around in the cell and performing the normal function, they band together and team up. This networking we describe makes [these kinds of cancer cells] different," Chiosis told GenomeWeb.

While different complex-presenting cancer cells have varied networks, the researchers used quantitative mass spectrometry to identify a core "team" of heat shock proteins. Inhibiting even just one of the core proteins can cause the whole network to fall apart, leading the cancer cell to die.

Thus, cancers featuring the chaperone complexes might be more susceptible to heat shock protein inhibitor drugs.

"What we have developed [are] ways of finding these bound up chaperones," Chiosis said. The researchers described three assays — an imaging assay, a flow cytometry assay, and a protein separation assay — that could be used to detect the complexes and select patients who might respond better to that therapy for clinical trials.

The MSKCC team published their study last week in Nature.

The finding could help explain why heat shock protein inhibitors, which have been under clinical investigation for years, have failed to live up to their promise.

While Chiosis has successfully used the inhibitors to better understand tumor biology, numerous clinical candidates have failed to gain FDA approval. In 2011, Chiosis told GenomeWeb, "We designed this class of compounds 10 years ago, and it has been a long process of developing it as a drug."

Chiosis is also co-founder of Samus Therapeutics, a spinout dedicated to developing heat shock protein inhibitors such as PU-H71, which inhibits heat shock protein 90 (Hsp90).

Hsp90 has long been associated with cancer and has been known to complex with other proteins, but the new study establishes chaperone complexes as essential to the cancers that feature them.

"Before, people thought chaperones are important to all the tumors," Chiosis said, namely the over-expression of particular proteins. "We're saying it's not the total level of expression of Hsp90 or Hsp70 or any of the 300 to 500 chaperones in the cells, it's how they network with each other that matters," she said.

Chaperones can regulate key cancer-driving proteins, like HER2 in breast cancer. One experiment in the new study shows that while heat shock protein expression from two separate HER2-overexpressing primary tumor samples may look the same at the aggregate level, their propensity to form complexes can be totally different. Some were enriched for the chaperone complexes, while others were not. Other types of cancer, including pancreatic cancers and lymphomas, also showed this delineation.

The scientists came up with three assays for different tumor analysis styles: a positron emission tomography imaging scan for lymphomas, a flow cytometry-based assay for liquid tumors, and a native isoelectric focusing assay for biopsies — kind of like a Western blot, except the protein complex is preserved and the proteins aren't denatured, Chiosis said, adding that the first two are already being used with patients at MSKCC in exploratory trials.

The assays showed that the researchers could delineate tumors into those with and without chaperone complexes. Moreover, the chaperone proteins don't form complexes in non-cancer cells.

The researchers then used mass spectrometry to identify the proteins involved in the complexes, as well as the proteins that they regulate.

Different "teams" of proteins form up in different contexts, Chiosis said. "Each tumor has different proteins that go out of whack and no two tumors are the same. But the core team is the same," and that's what the assays can pick up on, she said.

"The mass spec analysis really gave us an idea of what's at the core of this change. It also told us what drives this change," she said. The researchers performed liquid chromatography tandem mass spec analysis using a 6410 system from Agilent Technologies in multiple reaction-monitoring mode and positive-ion electrospray ionization. By looking at the proteome being regulated by the chaperone proteins in the complexes, she was able to zero in on the oncogene MYC as a possible driver for complex formation.

"When you turn up MYC, you all of a sudden create a large destabilization of the proteome," Chiosis said. "A lot of things go awry in the cell. Hsp70 or Hsp90 alone cannot deal with that, so they reach to each other for help and team up to control this big mess."

The researchers were able to validate that by turning on MYC, enabling them to create chaperone complexes in cancer cells that previously didn't have them.

The chaperone complex helps keep the cell alive and even proliferate, but the complex also retains a key vulnerability. It's an all-hands-on-deck situation, and if even one core protein fails to do its part, the whole operation falls apart.

"If you pluck at any point, you destabilize the whole network," Chiosis said. Using siRNA knockdown of core heat shock proteins, the scientists were able to kill the cancer cells featuring the complexes (measured by a lactate dehydrogenase assay), but not in cells without complexes.

Those results suggested that if a cell dependent on such a chaperone complex were to receive a dose of an Hsp90 inhibitor, say PU-H71, it will die. In ex vivo experiments, the researchers were able to use PU-H71 to increase the number of cells undergoing apoptosis in complex-forming primary tumor specimens — over 50 percent compared to the control at doses of more than 0.5 micro molar.

"In other cells, you don't need all these proteins to team up, they don't need to create this network," Chiosis explained. "Normal cells need chaperones, too, for protein folding and transporting. But when you inhibit any of them, the inhibition is local, because they need not team up for these functions."

That means heat shock protein inhibitors may only be valuable to patients whose cancers are dependent on these complexes. The assays developed by Chiosis' team could be vital to finding patients likely to respond to heat shock protein inhibitors.

And the inhibitors have the potential to be broadly applicable, regardless of cancer type. In the study, the researchers looked at lymphoma, leukemia, pancreatic, gastric, lung, and breast cancer cell lines, finding that more than half "presented medium to high levels of epichaperome complexes," they wrote, adding that follow-up experiments suggested that response to an inhibitor could be dependent on the abundance of chaperone complexes.

"If patients were to be selected for epichaperome therapy, not only the existence of this species but also its abundance should be measured," the authors wrote.

Chiosis said her pharmaceutical spinout, Samus Therapeutics, has already licensed all three of the assays described in the paper. Details of the licensing agreement were not disclosed and as of press time, MSKCC had not responded to GenomeWeb's request for information about the deal.