NEW YORK (GenomeWeb) – Researchers from Vanderbilt University have developed a multi-omics platform for large-scale investigations into the mechanisms of action of drugs and other agents.
In a study published last week in the Journal of Proteome Research, they demonstrated the platform's capabilities in an analysis looking at the effects of the chemotherapeutic cisplatin on the lung cancer cell line A549.
Using a combination of RNA sequencing; label free, SILAC, and phospho-enriched SILAC LC-MS/MS; and mass spec-based metabolomics, the researchers over the course of 30 days measured and interpreted more than 10,000 molecular changes in the cisplatin-treated cells, identifying nearly all the known cellular responses to cisplatin as well as a host of other mechanisms with potential links to phenomena that include drug resistance and off-target effects.
The results suggest that omics tools have advanced to the point where they can deliver large-scale mechanistic data in a rapid, high-throughput manner suitable for applications such as drug development work, said Jeremy Norris, a research associate professor of biochemistry at Vanderbilt and the first author of the paper.
"I think there has been an attitude [within pharma] towards omics technologies that, yes, they can play a role, but they are too slow and not responsive enough [to be used] for every compound brought through the pipeline," he said. "And I think what we've shown here with this experiment is that those technologies have matured a great deal, especially over the last five years. This idea that you can get high-throughput mechanistic data in a span as short as 30 days, I think, will be eye-opening for a lot of people."
While the platform has obvious applications to drug development, it was in fact developed as part of a project funded by the US Defense Advanced Research Projects Agency (DARPA) and the Army Research Office (ARO). In 2014, Vanderbilt researchers led by Richard Caprioli, senior author of the JPR paper, received a grant from these agencies worth up to $16.5 million over five year to develop an omics-based platform for rapidly identifying how toxic agents and drugs affect human cells.
The grant was one of three awarded through DARPA's Rapid Threat Assessment program for such work, with the other two awards going to the University of Colorado, Boulder, and George Washington University.
The JPR study consisted of two phase. In the first, the researchers used MALDI-based proteomic analysis to determine what the relevant doses would be for the study.
This effort, Norris said, stemmed from the realization that if they were, in fact, presented with an unknown compound, as envisioned in the scenario the DARPA project is designed to tackle, the researchers would first need to establish at what levels of exposure the mechanisms of action could best be measured.
"If we get a true unknown, we're going to have to do experiments on it just to figure out how much to put on a cell," he said.
In the second phase, the researchers applied the aforementioned sequencing, proteomic, phosphoproteomic, and metabolomic techniques to track molecular changes in A549 cells treated with 50 μM of cisplatin at time points of one, six, 24, and 48 hours, collecting a total of 254,470 measurements on 53,500 molecules, 13,483 (or 24 percent) of which showed significant changes.
To test their findings, Norris and his colleagues developed a canonical mechanism of action for cisplatin, based on published literature investigating the drug. That MOA consisted of changes to 33 different molecules via which cisplatin causes DNA damage, which launches a cellular response ending in apoptosis.
The dataset the researchers generated in the JPR study observed changes in 32 of the 33 molecules comprising the canonical MOA. Regarding the one molecule they missed, the protein Mdm2, Norris said this was a function not of their assays but of the cell type they used for the study.
"We have some evidence that that miss was justified," he said.
The vast quantity of data collected also allowed the researchers to expand their analyses to a wide variety of pathways and mechanisms outside the canonical MOA. For instance, they were able to elucidate multiple modes of cisplatin resistance, including resistance mediated by the protein ATP1A1 and by estrogen.
"We were able to extend into some areas beyond what was known as the canonical mechanism and into some areas and pathways that have never been implicated with cisplatin biology," Norris said.
"I think the broader, more interesting thing, not specific to cisplatin, is this ability to, perhaps, at a very early stage in development, use a systems approach to understand or anticipate unintended consequences of giving a drug to a human cell," he added. "I think that's a very exciting possibility that comes out of this."
"I don't think any of us would be so bold as to say that data of this type is the end point," Norris said. "But what I think it can do is generate new hypotheses that can be tested that potentially could head off what might be a very problematic situation if you proceeded with a drug and didn't know about it."
He noted that while the methods used in the study were not for the most part novel, coordinating the various elements of the platform — from cell culturing to the omics analyses through to data interpretation — and completing the study within the 30-day window was a significant challenge.
"Our approach was not to completely reinvent how one does proteomics and RNA-seq and metabolomics," Norris said, “but instead to take what we knew to be reliable, and integrate all of it in such a way that we could seamlessly collect the data and then wrap [that data] in a computational capability that allowed us to sort through it quickly."
The DARPA initiative consists of periodic tests, one of which was the cisplatin study published in JPR. The requirements for various metrics go up with each test, Norris said, adding that because of this, his colleagues would need to further improve the platform as the effort continued.
He declined to say specifically what sort of hardware or informatics improvements the Vanderbilt team was currently pursuing, but noted that they were "challenging [the platform] on every level.”
"Hardware, software, how we prepare samples in order to multiplex them — every step of the process gets broken down and rebuilt after every challenge to try to make it better," he said. "We still have a couple years left in the program and now we find ourselves at the point where we're trying to push the technology beyond its current limits and develop ways to get more time resolution and more sensitivity and better computational tools to integrate the data.”