Therapeutic Toxins

by Cheryl SooHoo | photography by Teresa Crawford

Northwestern investigators explore the transformative potential of bacterial toxins.

 

 

 

 

The human body has a love-hate relationship with bacteria. While trillions of bacteria found on earth — and on and in our bodies — do no harm, the toxic effects of a very small percentage of these microscopic organisms can be downright lethal. But even as bacterial pathogens cause serious health problems such as tuberculosis and meningitis, the toxins they secrete can be turned into therapeutic agents to effectively ward off disease.

Karla Satchell, PhD, professor of Microbiology-Immunology, has spent her career — including nearly two decades at Northwestern — breaking ground in the niche area of toxin biology. In her basic science research, she delves into the mechanisms of bacterial proteins to understand how they result in deadly infections. Focusing on MARTX (multifunctional-autoprocessing repeats-in-toxin) proteins, Satchell discovered a novel toxin of the bacterium that causes cholera, Vibrio cholerae, when she was a postdoctoral fellow at Harvard in the late 1990s. Her research resume also includes the identification of a hypervirulent variant of V. cholerae responsible for the 2010 cholera epidemic in Haiti.

 

(above) A 3D computer illustration depicting the activation process of a Ras protein. Mutations in Ras genes have been found in many types of cancer, including 95 percent of pancreatic cancer, but effectively targeting Ras proteins has been one of the hardest challenges in cancer research and drug discovery for decades.

Expanding her interest in the MARTX family, Satchell also studies the virulence factors that make the bacterium Vibrio vulnificus so dangerous. Every year in the United States, this particular organism and several other Vibrio species infect some 52,000 individuals who eat raw or undercooked shellfish or develop a wound infection after wading or swimming in coastal waters. About one in four people with vibriosis die due to severe sepsis — sometimes within days. Survivors may require limb amputation due to necrotizing fasciitis or what has been dubbed “flesh-eating bacteria.” Teasing out the pathogenesis of this complicated toxin protein, little did Satchell and her team expect to find a promising avenue for inhibiting tumor cell growth.

Vibrio vulnificus MARTX is an exceptionally large and complex single protein that cuts itself into little bits. We have been methodically looking at the biochemical actions of each of those pieces,” she explains. “One of them turns out to cleave or cut the protein Ras and, by doing so, stops its activity. Because mutations of Ras drive 30 percent of cancers, we immediately recognized the importance of this toxin as a potential cancer treatment.”

In 2015, Satchell’s laboratory revealed this discovery to the scientific community with a publication in Nature Communications. Many papers later, Satchell and her team continue to collaborate with Northwestern colleagues and others at leading institutions to find ways to turn this novel finding into a viable anti-cancer drug.

“We are taking a slow and steady basic science approach to understand the underlying biochemistry … to ensure that by the time we can safely test it in humans it will truly work to stop cancer.”

Karla Satchell, PhD

Professor of Microbiology-Immunology

INFECT AND DESTROY

Ras oncogenes have been implicated in a variety of cancers, with pancreatic, lung and colorectal malignancies high on the list. Usually Ras proteins contribute to normal cell development, and they cycle between being active and inactive to control cell growth and migration. When mutated, though, a breakdown in signaling causes Ras to stay switched on all the time, leading to the proliferation of tumor cells that is the hallmark of cancer. Even cancers with no mutations in Ras have been found to have other alterations that impair the “off” switch in the oncogene.

Additionally, Ras has another task: recognizing pathogens and helping to mount an appropriate immune response.

Satchell’s team found that a specific component of the MARTX toxin of Vibrio vulnificus, an effector domain called DUF5, zeros in on and cleaves Ras without modifying it — a novel mechanism for inactivating the protein. A naturally occurring toxic effector domain, DUF5 also inactivates Ras’s co-conspirator in promoting cancer growth, a protein called Rap1.

Now dubbed “RRSP” for Ras/Rap1 specific peptidase, this new class of endopeptidase uncovered by the Satchell group has been shown to disable signaling through the ERK1/2 pathway essential for cancer cell survival. Ultimately, the hope is that by inactivating Ras and Rap1 signaling, RRSP can paralyze the normal immune response to a pathogen, increase its own virulence and spread throughout the host. Basically, do what harmful bacterial toxins do best: infect, overwhelm and destroy.

Since the discovery of this novel protein activity, Satchell’s laboratory has been fully exploring its anti-cancer capabilities. “We are very interested in breast cancer,” says Satchell. “While not typically a Ras-driven disease, signals do flow through the Ras pathway, possibly due to the overexpression of growth factor receptors. This activity provides opportunities for using RRSP to interrupt the cancer process downstream.”

The investigators are also looking at colon cancer, of which 45 percent of diagnoses have some Ras mutation involvement. Once the scientists have achieved proof of concept with the other malignancies, they intend to turn their attention to pancreatic cancer — a 95 percent Ras-driven disease. “This is one of the most challenging cancers to treat, with the lowest survival rates,” says Satchell, who is also a member of the Robert H. Lurie Comprehensive Cancer Center of Northwestern University. “It is also hard to work with in the laboratory.”

At the moment, her group is conducting early pancreatic cancer studies. One member of her team, Marco Biancucci, PhD, a postdoctoral fellow, recently published findings from this research in the October issue of Science Signaling.

Usually, Ras proteins switch on (by binding to GTP) and off (by binding to GDP) as they control cell growth and migration. When mutated, they stay on, leading to the proliferation of cancer cells. Satchell’s laboratory discovered that a component of the bacterial toxin MARTX, dubbed RRSP, can specifically inactivate Ras and disable a signaling pathway that perpetuates cancer cell survival.  Illustration by Christina Wheeler.

A BIG DEAL

For the past 30 years, knowledge of Ras and its role in cancer development has confounded the cancer research community. To date, attempts to effectively block Ras gene function have gained little traction, compelling the National Cancer Institute (NCI) to launch the Ras Initiative in 2013 to mobilize scientists around the world to find a solution. (In fact, the Northwestern study recently published in Science Signaling was conducted in collaboration with the NCI’s Ras Initiative.) But despite the expenditure of millions of dollars and the efforts of many in cancer research and drug discovery, a targeted anti-cancer agent that effectively inhibits Ras proteins remains elusive.

“Everyone is searching for anything that targets Ras,” says David Gius, MD, PhD, coleader of the Women’s Cancer Research Program at the Lurie Cancer Center and professor of Radiation Oncology and Pharmacology. “Dr. Satchell has a bacterial protein that binds to Ras. If she can demonstrate proof of principle that this action reverses tumor growth, then that will be a very big deal.”

A cancer biologist, Gius and his laboratory provide tumor tissue and mutant Ras tumor models essential for carrying out Satchell’s Ras studies. Their collaboration has resulted in the characterization of one of the MARTX toxin proteins as a Ras protease — it breaks down the malicious protein.

Beyond cancer, Ras has been found to have a role in cardiac arrhythmias and other diseases of the heart and skin. This summer a new Northwestern Medicine study revealed its link between desmoplakin and connexin-43 — the former, a protein that helps cells stick together, controls expression of the latter, a protein that facilitates communication between cells. When mutated, desmoplakin causes cell signaling malfunctions that can lead to conditions such as arrhythmogenic cardiomyopathy, an inherited heart disorder that can result in sudden death in young adults. The use of RRSP as a Ras inhibitor opens the door to another therapeutic use of the bacterial toxin. Satchell was a co-author on the study, which appeared in the June issue of the Journal of Cell Biology.

OUT(SIDE) FOR DELIVERY

Therapeutic agents are only as good as their drug delivery systems. Satchell believes she has “good but still untested cargo” in her Ras-cleaving MARTX toxin. She has been “test driving” vehicles for transporting the RRSP into living beings where it would go after intended cancer targets.

A problem generating drugs from the MARTX toxins is that they are about 200 times larger than most proteins. “The toxins I study are enormous,” she says. “They typically can’t be druggable because of their sheer size.” One tactic is to fuse the activity domain of the RRSP to another smaller toxin because “toxins are really good at getting into cells,” Satchell explains.

Partnering with external collaborators, Satchell has been exploring a variety of delivery strategies. For example, her group has been working with scientists from the Massachusetts Institute of Technology (MIT) who have discovered a unique use for the Bacillus anthracis bacterium. Leading to the development of lethal anthrax, the bacterial toxin is particularly adept at transporting large enzymes into cells. In 2014, MIT announced that a team of chemists had disarmed the anthrax toxin to deliver small protein molecules, or cargo proteins, into cells to administer cancer drugs.

Satchell and her team’s novel approach to solving the mutant Ras cancer conundrum offers much potential. However, she cautions taking too big a leap just yet. Much more work on the activity, efficacy and immunotoxicity of RRSP and its cancer-fighting abilities is still to come.

“Everyone wants to jump to the end when it comes to developing therapeutics and that’s why many attempts disastrously fail,” she says. “We are taking a slow and steady basic science approach to understand the underlying biochemistry of what we are doing to ensure that by the time we can safely test it in humans it will truly work to stop cancer.”