Scientists engineer toxin-secreting stem cells to treat brain tumors

Harvard Stem Cell Institute scientists at Massachusetts General Hospital have devised a new way to use stem cells in the fight against brain cancer. A team led by neuroscientist Khalid Shah, MS, PhD, who recently demonstrated the value of stem cells loaded with cancer-killing herpes viruses, now has a way to genetically engineer stem cells so that they can produce and secrete tumor-killing toxins.

In the AlphaMed Press journal STEM CELLS, Shah’s team shows how the toxin-secreting stem cells can be used to eradicate cancer cells remaining in mouse brains after their main tumor has been removed. The stem cells are placed at the site encapsulated in a biodegradable gel. This method solves the delivery issue that probably led to the failure of recent clinical trials aimed at delivering purified cancer-killing toxins into patients’ brains. Shah and his team are currently pursuing FDA approval to bring this and other stem cell approaches developed by them to clinical trials.

“Cancer-killing toxins have been used with great success in a variety of blood cancers, but they don’t work as well in solid tumors because the cancers aren’t as accessible and the toxins have a short half-life,” said Shah, who directs the Molecular Neurotherapy and Imaging Lab at Massachusetts General Hospital and Harvard Medical School.

“A few years ago we recognized that stem cells could be used to continuously deliver these therapeutic toxins to tumors in the brain, but first we needed to genetically engineer stem cells that could resist being killed themselves by the toxins,” he said. “Now, we have toxin-resistant stem cells that can make and release cancer-killing drugs.”

Cytotoxins are deadly to all cells, but since the late 1990s, researchers have been able to tag toxins in such a way that they only enter cancer cells with specific surface molecules; making it possible to get a toxin into a cancer cell without posing a risk to normal cells. Once inside of a cell, the toxin disrupts the cell’s ability to make proteins and, within days, the cell starts to die.

Shah’s stem cells escape this fate because they are made with a mutation that doesn’t allow the toxin to act inside the cell.  The toxin-resistant stem cells also have an extra bit of genetic code that allows them to make and secrete the toxins. Any cancer cells that these toxins encounter do not have this natural defense and therefore die. Shah and his team induced toxin resistance in human neural stem cells and subsequently engineered them to produce targeted toxins.

“We tested these stem cells in a clinically relevant mouse model of brain cancer, where you resect the tumors and then implant the stem cells encapsulated in a gel into the resection cavity,” Shah said. “After doing all of the molecular analysis and imaging to track the inhibition of protein synthesis within brain tumors, we do see the toxins kill the cancer cells and eventually prolonging the survival in animal models of resected brain tumors.”

Shah next plans to rationally combine the toxin-secreting stem cells with a number of different therapeutic stem cells developed by his team to further enhance their positive results in mouse models of glioblastoma, the most common brain tumor in human adults. Shah predicts that he will bring these therapies into clinical trials within the next five years.

A discovery could prevent the development of brain tumours in children

Scientists at the IRCM discovered a mechanism that promotes the progression of medulloblastoma, the most common brain tumour found in children. The team, led by Frédéric Charron, PhD, found that a protein known as Sonic Hedgehog induces DNA damage, which causes the cancer to develop. This important breakthrough will be published in the October 13 issue of the prestigious scientific journal Developmental Cell. The editors also selected the article to be featured on the journal’s cover.

Sonic Hedgehog belongs to a family of proteins that gives cells the information needed for the embryo to develop properly. It also plays a significant role in tumorigenesis, the process that transforms normal cells into cancer cells.

“Our team studied a protein called Boc, which is a receptor located on the cell surface that detects Sonic Hedgehog,” explains Lukas Tamayo-Orrego, PhD student in Dr. Charron’s laboratory and co-first author of the study. “We had previously shown that Boc is important for the development of the cerebellum, the part of the brain where medulloblastoma arises, so we decided to further investigate its role.”

“With this study, we found that the presence of Boc is required for Sonic Hedgehog to induce DNA damage,” adds Dr. Charron, Director of the Molecular Biology of Neural Development research unit at the IRCM. “In fact, Boc causes DNA mutations in tumour cells, which promotes the progression of precancerous lesions into advanced medulloblastoma.”

“Our study shows that when Boc is inactivated, the number of tumours is reduced by 66 per cent,” says Frederic Mille, PhD, co-first author of the article and former postdoctoral fellow in Dr. Charron’s research unit. “The inactivation of Boc therefore reduces the development of early medulloblastoma into advanced tumours.”

Medulloblastoma ranks among the leading causes of cancer-related mortality in children. Current treatments include surgery, as well as radiation therapy and chemotherapy. Although the majority of children survive the treatment, radiation therapy damages normal brain cells in infants and toddlers and causes long-term harm.

“As a result, many children who undergo these treatments suffer serious side effects including cognitive impairment and disorders,” states Dr. Charron. “Our results indicate that Boc could potentially be targeted to develop a new therapeutic approach that would stop the growth and progression of medulloblastoma and could reduce the adverse side effects of current treatments.”

Researchers Find Promise in New Treatments for GBM

Glioblastoma multiforme (GBM) is one of the most lethal primary brain tumors, with median survival for these patients only slightly over one year. Researchers at Boston University School of Medicine (BUSM), in collaboration with researchers from the City of Hope, are looking toward novel therapeutic strategies for the treatment of GBM in the form of targeted therapies against a unique receptor, the interleukin-13 receptor α chain variant 2 (IL13Rα2).

In a review paper published in the October issue of Neuro-Oncology, the researchers discuss various targeted therapies against IL13Rα2 and early successes of clinical trials with these therapies in the treatment of GBM. The paper also highlights the need for future trials to improve efficacy and toxicity profiles of targeted therapies in this field.

Targeted therapies, which are drugs that interfere with specific molecules involved in cancer growth, have been successfully used in the treatment of many cancers, including breast and blood cancers. Successful targets for therapies are specific to tumor cells and not found on normal cells. Selectively expressed on GBM and absent on surrounding brain tissue, the interleukin-13 receptor α chain variant 2 (IL13Rα2) was identified as a potential target for therapy for GBM two decades ago. IL13Rα2 also plays an important role in the growth of tumors. In normal physiologic conditions, IL-13 binds to the receptor IL13Rα1 and helps regulate immune responses. In cancer cells, IL-13 binds to the receptor IL13Rα2 and, through a series of steps, prevents cancer cells from undergoing normal cell death. Increased expression of IL13Rα2 promotes the progression of GBM.

Since its discovery, IL13Rα2 has provided a target for therapies in GBM. These therapies have ranged from fusion proteins of IL-13 and bacterial toxins, oncolytic viruses, and immunotherapies. A phase I clinical trial and a phase III clinical trial have been completed for a T-cell based immunotherapy and IL-13/ bacterial toxin fusion protein respectively, both with promising outcomes.

“The field of targeted therapies in gliomas holds a lot of promise, and IL13Rα2 is in an optimal position to materialize these promises,” explained corresponding author Sadhak Sengupta, PhD, assistant professor of neurosurgery at BUSM and principal investigator of the Brain Tumor Lab at Roger Williams. “While early trials are encouraging, we need further research to achieve better targeting of the receptor and improved safety profiles of the treatments.”

New enzyme targets for selective cancer therapies

Thanks to important discoveries in basic and clinical research and technological advances, the fight against cancer has mobilized into a complex offensive spanning multiple fronts.

Work happening in a University of Alberta chemistry lab could help find new and more selective therapies for cancer. Researchers have developed a compound that targets a specific enzyme overexpressed in certain cancers—and they have tested its activity in cells from brain tumours.

Chemistry professor Christopher Cairo and his team synthesized a first-of-its-kind inhibitor that prevents the activity of an enzyme called neuraminidase. Although flu viruses use enzymes with the same mechanism as part of the process of infection, human cells use their own forms of the enzyme in many biological processes.

Cairo’s group collaborated with a group in Milan, Italy, that has shown that neuraminidases are found in excess amounts in glioblastoma cells, a form of brain cancer.

In a new study, a team from the University of Milan tested Cairo’s enzyme inhibitor and found that it turned glioblastoma cancer stem cells—found within a tumour and believed to drive cancer growth—into normal cells. The compound also caused the cells to stop growing, suggesting that this mechanism could be important for therapeutics. Results of their efforts were published Aug. 22 in the Nature journal Cell Death & Disease.

Cairo said these findings establish that an inhibitor of this enzyme could work therapeutically and should open the door for future research.

“This is the first proof-of-concept showing a selective neuraminidase inhibitor can have a real effect in human cancer cells,” he said. “It isn’t a drug yet, but it establishes a new target that we think can be used for creating new, more selective drugs.”

Long road from proof of concept to drug

Proving the compound can successfully inhibit the neuraminidase enzyme in cancer cells is just the first step in determining its potential as a therapy.

In its current form, the compound could not be used as a drug, Cairo explained, largely because it wasn’t designed to breach the blood-brain barrier making it difficult to reach the target cells. The team in Milan had to use the compound in very high concentrations, he added.

The research advances our understanding of how important carbohydrates are to the function of cells. Although most of us think of glucose (blood sugar) as the only important sugar in biology, there is an entire area of research known as glycobiology that seeks to understand the function of complex carbohydrate structures in cells. Carbohydrate structures cover the surface of cells, and affect how cells interact with each other and with pathogens.

Scientists have known for decades that the carbohydrates found on cancer cells are very different from those on normal cells. For example, many cancers have different amounts of specific residues like sialic acid, or may have different arrangements of the same residues.

“The carbohydrates on the cell surface determine how it interacts with other cells, which makes them important in cancer and other diseases. So, if we can design compounds that change these structures in a defined way, we can affect those interactions,” Cairo explained. “Finding new enzyme targets is essential to that process, and our work shows that we can selectively target this neuraminidase enzyme.”

Although there has been a lot of work on targeting viral neuraminidase enzymes, Cairo’s team has found inhibitors of the human enzymes. “The challenge in human cells is that there are four different isoenzymes. While we might want to target one for its role in cancer, hitting the wrong one could have harmful side-effects,” he said.

The U of A team reached out to their colleagues in Milan who were studying the role of a specific neuraminidase isoenzyme in cancer cells isolated from patients. Cairo approached them about testing a compound his team identified last year, which was selective for the same isoenzyme.

“I expected it would do something, but I didn’t know it would be that striking. It came out beautifully,” Cairo said.

The U of A team is already working on improving the compound, and developing and testing new and existing inhibitors using a panel of in vitro assays they developed.

“We’ve been working on these enzymes for about five years. Validation of our strategy­­­—design of a selective neuraminidase inhibitor and application in a cell that overexpresses that enzyme—is an achievement for us.”

Researchers reveal weakness in defenses of deadly brain tumor

Glioblastoma is a complex, deadly, and hard-to-treat brain cancer, but Yale School of Medicine researchers may have found the tumor’s Achilles heel.

The researchers report in the Aug. 12 issue of the journal Science Signaling that targeting a protein crucial in the early development of the brain can block multiple signaling pathways implicated in glioblastoma growth. The approach also reduced human tumors in mouse models of the disease.

“In neurodevelopment, this protein (atyptical protein kinase or aPKC) helps regulate proliferation and migration of cells but when active in adults, can cause formation and spread of cancer,” said Sourav Ghosh, assistant professor of neurology and co-senior author of the paper.

About 13,000 people die of primary malignant brain tumors annually in the United States. Glioblastomas are particularly hard to treat because these tumors grow rapidly, spread quickly, and respond poorly to current anti-tumor therapies.

The new study shows that targeting this protein works in several ways. Inhibiting aPKC blocks a signal pathway that is the target of existing glioblastoma therapy. But it also blocks the action of some immune system cells called macrophages, which instead of attacking tumors, actively promote their growth.

“This is exciting because it ends up targeting multiple pathways involved in cancer,” said Carla Rothlin, assistant professor of immunobiology and co-senior author of the paper.

Herpes-loaded stem cells used to kill brain tumors

Harvard Stem Cell Institute (HSCI) scientists at Massachusetts General Hospital have a potential solution for how to more effectively kill tumor cells using cancer-killing viruses. The investigators report that trapping virus-loaded stem cells in a gel and applying them to tumors significantly improved survival in mice with glioblastoma multiforme, the most common brain tumor in human adults and also the most difficult to treat.

The work, led by Khalid Shah, MS, PhD, an HSCI Principal Faculty member, is published in the Journal of the National Cancer Institute. Shah heads the Molecular Neurotherapy and Imaging Laboratory at Massachusetts General Hospital.

Cancer-killing or oncolytic viruses have been used in numerous phase 1 and 2 clinical trials for brain tumors but with limited success. In preclinical studies, oncolytic herpes simplex viruses seemed especially promising, as they naturally infect dividing brain cells. However, the therapy hasn’t translated as well for human patients. The problem previous researchers couldn’t overcome was how to keep the herpes viruses at the tumor site long enough to work.

Shah and his team turned to mesenchymal stem cells (MSCs)—a type of stem cell that gives rise to bone marrow tissue—which have been very attractive drug delivery vehicles because they trigger a minimal immune response and can be utilized to carry oncolytic viruses. Shah and his team loaded the herpes virus into human MSCs and injected the cells into glioblastoma tumors developed in mice. Using multiple imaging markers, it was possible to watch the virus as it passed from the stem cells to the first layer of brain tumor cells and subsequently into all of the tumor cells.

“So, how do you translate this into the clinic?” asked Shah, who also is an Associate Professor at Harvard Medical School.

“We know that 70-75 percent of glioblastoma patients undergo surgery for tumor debulking, and we have previously shown that MSCs encapsulated in biocompatible gels can be used as therapeutic agents in a mouse model that mimics this debulking,” he continued. “So, we loaded MSCs with oncolytic herpes virus and encapsulated these cells in biocompatible gels and applied the gels directly onto the adjacent tissue after debulking. We then compared the efficacy of virus-loaded, encapsulated MSCs versus direct injection of the virus into the cavity of the debulked tumors.”

Using imaging proteins to watch in real time how the virus combated the cancer, Shah’s team noticed that the gel kept the stem cells alive longer, which allowed the virus to replicate and kill any residual cancer cells that were not cut out during the debulking surgery. This translated into a higher survival rate for mice that received the gel-encapsulated stem cells.

“They survived because the virus doesn’t get washed out by the cerebrospinal fluid that fills the cavity,” Shah said. “Previous studies that have injected the virus directly into the resection cavity did not follow the fate of the virus in the cavity. However, our imaging and side-by-side comparison studies showed that the naked virus rarely infects the residual tumor cells. This could give us insight into why the results from clinical trials with oncolytic viruses alone were modest.”

The study also addressed another weakness of cancer-killing viruses, which is that not all brain tumors are susceptible to the therapy. The researchers’ solution was to engineer oncolytic herpes viruses to express an additional tumor-killing agent, called TRAIL. Again, using mouse models of glioblastoma—this time created from brain tumor cells that were resistant to the herpes virus—the therapy led to increased animal survival.

“Our approach can overcome problems associated with current clinical procedures,” Shah said. “The work will have direct implications for designing clinical trials using oncolytic viruses, not only for brain tumors, but for other solid tumors.”

Further preclinical work will be needed to use the herpes-loaded stem cells for breast, lung and skin cancer tumors that metastasize to the brain. Shah predicts the approach will enter clinical trials within the next two to three years.

Blocking DNA repair mechanisms could improve radiation therapy for brain cancer

UT Southwestern Medical Center researchers have demonstrated in both cancer cell lines and in mice that blocking critical DNA repair mechanisms could improve the effectiveness of radiation therapy for highly fatal brain tumors called glioblastomas.

Radiation therapy causes double-strand breaks in DNA that must be repaired for tumors to keep growing. Scientists have long theorized that if they could find a way to block repairs from being made, they could prevent tumors from growing or at least slow down the growth, thereby extending patients’ survival. Blocking DNA repair is a particularly attractive strategy for treating glioblastomas, as these tumors are highly resistant to radiation therapy. In a study, UT Southwestern researchers demonstrated that the theory actually works in the context of glioblastomas.

“This work is informative because the findings show that blocking the repair of DNA double-strand breaks could be a viable option for improving radiation therapy of glioblastomas,” said Dr. Sandeep Burma, Associate Professor of Radiation Oncology in the division of Molecular Radiation Biology at UT Southwestern.

His lab works on understanding basic mechanisms by which DNA breaks are repaired, with the translational objective of improving cancer therapy with DNA damaging agents. Recent research from his lab has demonstrated how a cell makes the choice between two major pathways that are used to repair DNA breaks – non-homologous end joining (NHEJ) and homologous recombination (HR). His lab found that enzymes involved in cell division called cyclin-dependent kinases (CDKs) activate HR by phosphorylating a key protein, EXO1. In this manner, the use of HR is coupled to the cell division cycle, and this has important implications for cancer therapeutics. These findings were published April 7 in Nature Communications.

While the above basic study describes how the cell chooses between NHEJ and HR, a translational study from the Burma lab demonstrates how blocking both repair pathways can improve radiotherapy of glioblastomas. Researchers in the lab first were able to show in glioblastoma cell lines that a drug called NVP-BEZ235, which is in clinical trials for other solid tumors, can also inhibit two key DNA repair enzymes, DNA-PKcs and ATM, which are crucial for NHEJ and HR, respectively. While the drug alone had limited effect, when combined with radiation therapy, the tumor cells could not quickly repair their DNA, stalling their growth.

While excited by the initial findings in cell lines, researchers remained cautious because previous efforts to identify DNA repair inhibitors had not succeded when used in living models – mice with glioblastomas. Drugs developed to treat brain tumors also must cross what’s known as the blood-brain-barrier in living models.

But the NVP-BEZ235 drug could successfully cross the blood-brain-barrier, and when administered to mice with glioblastomas and combined with radiation, the tumor growth in mice was slowed and the mice survived far longer – up to 60 days compared to approximately 10 days with the drug or radiation therapy alone. These findings were published in the March 1 issue of Clinical Cancer Research.

“The consequence is striking,” said Dr. Burma. “If you irradiate the tumors, nothing much happens because they grow right through radiation. Give the drug alone, and again, nothing much happens. But when you give the two together, tumor growth is delayed significantly. The drug has a very striking synergistic effect when given with radiation.”

The combination effect is important because the standard therapy for glioblastomas in humans is radiation therapy, so finding a drug that improves the effectiveness of radiation therapy could have profound clinical importance eventually. For example, such drugs may permit lower doses of X-rays and gamma rays to be used for traditional therapies, thereby causing fewer side effects.

“Radiation is still the mainstay of therapy, so we have to have something that will work with the mainstay of therapy,” Dr. Burma said.

While the findings provide proof that the concept of “radiosensitizing” glioblastomas works in mouse models, additional research and clinical trials will be needed to demonstrate whether the combination of radiation with DNA repair inhibitors would be effective in humans, Dr. Burma cautioned.

“Double-strand DNA breaks are a double-edged sword,” he said. “On one hand, they cause cancer. On the other, we use ionizing radiation and chemotherapy to cause double-strand breaks to treat the disease.”

Another recent publication from his lab highlights this apparent paradox by demonstrating how radiation can actually trigger glioblastomas in mouse models. This research, supported by NASA, is focused on understanding cancer risks from particle radiation, the type faced by astronauts on deep-space missions and now being used in cutting-edge cancer therapies such as proton and carbon ion therapy.

Dr. Burma’s lab uses the high-tech facilities and large particle accelerator of the NASA Space Radiation Laboratory at the Brookhaven National Laboratory in New York to generate heavy ions, which can be used to irradiate glioblastoma-prone mice to test both the cancer-inducing potential of particle radiation as well as its potential therapeutic use.

“Heavy particles cause dense tracks of damage, which are very hard to repair,” Dr. Burma noted. “With gamma or X-rays, which are used in medical therapy, the damage is diffuse and is repaired within a day. If you examine a mouse brain irradiated with heavy particles, the damage is repaired slowly and can last for months.”

These findings, published March 17 in Oncogene, suggest that glioblastoma risk from heavier particles is much higher compared to that from gamma or X-rays. This study is relevant to the medical field, since ionizing radiation, even low doses from CT scans, have been reported to increase the risk of brain tumors, Dr. Burma said.