Suicide molecules kill any cancer cell

January 05, 2018

CHICAGO – Small RNA molecules originally developed as a tool to study gene function trigger a mechanism hidden in every cell that forces the cell to commit suicide, reports a new Northwestern Medicine study, the first to identify molecules to trigger a fail-safe mechanism that may protect us from cancer.

The mechanism — RNA suicide molecules — can potentially be developed into a novel form of cancer therapy, the study authors said.

Cancer cells treated with the RNA molecules never become resistant to them because they simultaneously eliminate multiple genes that cancer cells need for survival.

“It’s like committing suicide by stabbing yourself, shooting yourself and jumping off a building all at the same time,” said Northwestern scientist and lead study author Marcus Peter. “You cannot survive.”

The inability of cancer cells to develop resistance to the molecules is a first, Peter said.

“This could be a major breakthrough,” noted Peter, the Tom D. Spies Professor of Cancer Metabolism at Northwestern University Feinberg School of Medicine and a member of the Robert H. Lurie Comprehensive Cancer Center of Northwestern University.  

Peter and his team discovered sequences in the human genome that when converted into small double-stranded RNA molecules trigger what they believe to be an ancient kill switch in cells to prevent cancer. He has been searching for the phantom molecules with this activity for eight years.

“We think this is how multicellular organisms eliminated cancer before the development of the adaptive immune system, which is about 500 million years old,” he said. “It could be a fail safe that forces rogue cells to commit suicide. We believe it is active in every cell protecting us from cancer.”

This study, which will be published Oct. 24 in eLife, and two other new Northwestern studies in Oncotarget and Cell Cycle by the Peter group, describe the discovery of the assassin molecules present in multiple human genes and their powerful effect on cancer in mice.

Looking back hundreds of millions of years

Why are these molecules so powerful?

“Ever since life became multicellular, which could be more than 2 billion years ago, it had to deal with preventing or fighting cancer,” Peter said. “So nature must have developed a fail safe mechanism to prevent cancer or fight it the moment it forms. Otherwise, we wouldn’t still be here.”

Thus began his search for natural molecules coded in the genome that kill cancer.

“We knew they would be very hard to find,” Peter said. “The kill mechanism would only be active in a single cell the moment it becomes cancerous. It was a needle in a haystack.”

But he found them by testing a class of small RNAs, called small interfering (si)RNAs, scientists use to suppress gene activity. siRNAs are designed by taking short sequences of the gene to be targeted and converting them into double- stranded RNA. These siRNAs when introduced into cells suppress the expression of the gene they are derived from.Peter found that a large number of these small RNAs derived from certain genes did not, as expected, only suppress the gene they were designed against. They also killed all cancer cells. His team discovered these special sequences are distributed throughout the human genome, embedded in multiple genes as shown in the study in Cell Cycle.

When converted to siRNAs, these sequences all act as highly trained super assassins. They kill the cells by simultaneously eliminating the genes required for cell survival. By taking out these survivor genes, the assassin molecule activates multiple death cell pathways in parallel.

The small RNA assassin molecules trigger a mechanism Peter calls DISE, for Death Induced by Survival gene Elimination.

Activating DISE in organisms with cancer might allow cancer cells to be eliminated. Peter’s group has evidence this form of cell death preferentially affects cancer cells with little effect on normal cells.

To test this in a treatment situation, Peter collaborated with Dr. Shad Thaxton, associate professor of urology at Feinberg, to deliver the assassin molecules via nanoparticles to mice bearing human ovarian cancer. In the treated mice, the treatment strongly reduced the tumor growth with no toxicity to the mice, reports the study in Oncotarget. Importantly, the tumors did not develop resistance to this form of cancer treatment. Peter and Thaxton are now refining the treatment to increase its efficacy.

Peter has long been frustrated with the lack of progress in solid cancer treatment.

“The problem is cancer cells are so diverse that even though the drugs, designed to target single cancer driving genes, often initially are effective, they eventually stop working and patients succumb to the disease,” Peter said. He thinks a number of cancer cell subsets are never really affected by most targeted anticancer drugs currently used.

Most of the advanced solid cancers such as brain, lung, pancreatic or ovarian cancer have not seen an improvement in survival, Peter said.

“If you had an aggressive, metastasizing form of the disease 50 years ago, you were busted back then and you are still busted today,” he said. “Improvements are often due to better detection methods and not to better treatments.”

Cancer scientists need to listen to nature more, Peter said. Immune therapy has been a success, he noted, because it is aimed at activating an anticancer mechanism that evolution developed. Unfortunately, few cancers respond to immune therapy and only a few patients with these cancers benefit, he said.

“Our research may be tapping into one of nature’s original kill switches, and we hope the impact will affect many cancers,” he said. “Our findings could be disruptive.”

Northwestern co-authors include first authors William Putzbach, Quan Q. Gao, and Monal Patel, and coauthors Ashley Haluck-Kangas, Elizabeth T. Bartom, Kwang-Youn A. Kim, Denise M. Scholtens, Jonathan C. Zhao and Andrea E. Murmann.

The research is funded by grants T32CA070085, T32CA009560, R50CA211271 and R35CA197450 from the National Cancer Institute of the National Institutes of Health.

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High-speed drug screening

October 11, 2014


MIT engineers have devised a way to rapidly test hundreds of different drug-delivery vehicles in living animals, making it easier to discover promising new ways to deliver a class of drugs called biologics, which includes antibodies, peptides, RNA, and DNA, to human patients.

In a study appearing in the journal Integrative Biology, the researchers used this technology to identify materials that can efficiently deliver RNA to zebrafish and also to rodents.

This type of high-speed screen could help overcome one of the major bottlenecks in developing disease treatments based on biologics: how to find safe and effective ways to deliver them.

“Biologics is the fastest growing field in biotech, because it gives you the ability to do highly predictive designs with unique targeting capabilities,” says senior author Mehmet Fatih Yanik, an associate professor of electrical engineering and computer science and biological engineering. “However, delivery of biologics to diseased tissues is challenging, because they are significantly larger and more complex than conventional drugs.

Automating large-scale studies 

Zebrafish are commonly used to model human diseases, in part because their larvae are transparent, making it easy to see the effects of genetic mutations or drugs.

In 2010, Yanik’s team developed a technology for rapidly moving zebrafish larvae to an imaging platform, orienting them correctly, and imaging them. This kind of automated system makes it possible to do large-scale studies because analyzing each larva takes less than 20 seconds, compared with the several minutes it would take for a scientist to evaluate the larvae by hand.

For this new study, Yanik’s team developed a new technology to inject RNA carried by nanoparticles called lipidoids. These fatty molecules have shown promise as delivery vehicles for RNA interference, a process that allows disease-causing genes to be turned off with small strands of RNA.

Yanik’s group tested about 100 lipidoids that had not performed well in tests of RNA delivery in cells grown in a lab dish. They designed each lipidoid to carry RNA expressing a fluorescent protein, allowing them to easily track RNA delivery, and injected the lipidoids into the spinal fluid of the zebrafish.

To automate that process, the zebrafish were oriented either laterally or dorsally once they arrived on the viewing platform. Once the larvae were properly aligned, they were immobilized by a hydrogel. Then, the lipidoid-RNA complex was automatically injected, guided by a computer vision algorithm. The system can be adapted to target any organ, and the process takes about 14 seconds per fish.

A few hours after injection, the researchers imaged the zebrafish to see if they displayed any fluorescent protein in the brain, indicating whether the RNA successfully entered the brain tissue, was taken up by the cells, and expressed the desired protein.

The researchers found that several lipidoids that had not performed well in cultured cells did deliver RNA efficiently in the zebrafish model. They next tested six randomly selected best- and worst-performing lipidoids in rats and found that the correlation between performance in rats and in zebrafish was 97 percent, suggesting that zebrafish are a good model for predicting drug-delivery success in mammals.

The idea is to identify useful drug delivery nanoparticles using this miniaturized system.

New leads

The researchers are now using what they learned about the most successful lipidoids identified in this study to try to design even better possibilities. “If we can pick up certain design features from the screens, it can guide us to design larger combinatorial libraries based on these leads,” Yanik says.

Yanik’s lab is currently using this technology to find delivery vehicles that can carry biologics across the blood-brain barrier — a very selective barrier that makes it difficult for drugs or other large molecules to enter the brain through the bloodstream.

The research was funded by the National Institutes of Health, the Packard Award in Science and Engineering, Sanofi Pharmaceuticals, Foxconn Technology Group, and the Hertz Foundation.

Abstract of Organ-targeted high-throughput in vivo biologics screen identifies materials for RNA delivery

Therapies based on biologics involving delivery of proteins, DNA, and RNA are currently among the most promising approaches. However, although large combinatorial libraries of biologics and delivery vehicles can be readily synthesized, there are currently no means to rapidly characterize them in vivo using animal models. Here, we demonstrate high-throughput in vivo screening of biologics and delivery vehicles by automated delivery into target tissues of small vertebrates with developed organs. Individual zebrafish larvae are automatically oriented and immobilized within hydrogel droplets in an array format using a microfluidic system, and delivery vehicles are automatically microinjected to target organs with high repeatability and precision. We screened a library of lipid-like delivery vehicles for their ability to facilitate the expression of protein-encoding RNAs in the central nervous system. We discovered delivery vehicles that are effective in both larval zebrafish and rats. Our results showed that the in vivo zebrafish model can be significantly more predictive of both false positives and false negatives in mammals than in vitro mammalian cell culture assays. Our screening results also suggest certain structure–activity relationships, which can potentially be applied to design novel delivery vehicles.