Researchers gain insights into the deadly foes that are high-grade gliomas,
but not quickly enough for their liking

by Nick Miller

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Neurosphere cells isolated from human diffuse intrinsic pontine glioma (DIPG) tumors. Neurospheres contain neural stem and progenitor cells; researchers study the cells to learn about treatment resistance and which gene mutations might cause brain cancer.

All cancers are not created equal. So when physicians and scientists who study cancer biology talk about making strides in cure rates for many childhood cancers, there is that “other” list—the cancers that cannot be cured, or even treated with reasonable effectiveness.

Of the dozen or so different types of pediatric brain cancers, high-grade gliomas (HGG) are particularly treatment-resistant. They account for just 8 to 10 percent of central nervous system tumors in children, with an incidence rate of 0.85 cases per 100,000, according to the U.S. Central Brain Tumor Registry. Five-year survival rates are only 15 to 30 percent.

Lionel Chow, MD, PhD, is an oncologist and researcher in the Cancer and Blood Diseases Institute. He treats children with these tumors and spends long hours with research colleagues studying HGGs. The scientists are relentless in their search for better ways to treat HGG and other brain cancers. But they have yet to determine what causes them, or how to stop them.

What they do know is that they look forward to the day when they will not have to deliver the dismal prognosis that accompanies a diagnosis of an HGG to a family.

“It is extremely difficult and disheartening to deliver a diagnosis of a high-grade glioma to patients and families,” says Chow. “These are such aggressive cancers and our options for treatment are few and ineffective. We try to offer hope with new therapies and clinical trials, but we know that the patient’s outcome is determined with the diagnosis. Parents should not have to watch their child endure the suffering that this disease causes. I am a parent myself and I cannot imagine outliving my kids.”

Chow is part of a team of Cincinnati Children’s researchers who make defeating pediatric HGG their life mission. They are joined in this effort by colleagues across the globe in a variety of collaborative efforts, in particular the Pediatric Brain Tumor Consortium (PBTC), led by Maryam Fouladi, MD, who heads our Brain Tumor Program. The PBTC emphasizes a strong blend of basic cancer biology and clinical investigation.   

WHAT RESEARCHERS KNOW

Pediatric HGG tumors look similar at the microscopic level when compared to their adult counterparts, so at one time it was thought they might be driven by genetic and biologic factors similar to those seen in the adult disease. But advances in the ability to identify the precise genetic signatures of different tumors have changed this thinking.

“There is a big difference between pediatric brain tumors and adult brain tumors,” says Rachid Drissi, PhD, a scientist in the Division of Experimental Hematology and Cancer Biology. “They may look the same under the microscope, but the genetic and molecular pathways that lead to these tumors are not the same.”

Arising from glial, oligodendrocyte and ependymal brain cells, high-grade gliomas vary in their genetic drivers and molecular signatures. The precise combination of mutations that leads to HGG can depend on the type of cell being targeted or region of the brain in which the cancer originates, or even the patient’s genetic background.

Many believe these cancers have their origins from flexible progenitor cells of the central nervous system. Progenitor cells are still finalizing what cell type to become, and are easily influenced by “wrong” genetic or biologic conditions. In the context of brain cancer, they can become tumor-initiating cells (or cancer seeds), prompted by mutations in genetic pathways. During the disease process, these cancer seeds can be a source of resistance to therapy, making certain cancers harder to treat.

To thrive, glioma cells depend on what scientists call the “permissive micro-environment” of the brain, which exists in an area separated by the blood-brain barrier. Designed to protect the brain, the barrier also makes delivering therapeutic agents to diseased parts of the brain more challenging. Researchers are developing new technologies that can cross the barrier to deliver targeted treatments, such as lipid (fat)-based nanoparticles capable of toting molecular-based therapeutics.

HARD TO MAKE, HARDER TO KILL

Even in this micro-environment, it is not easy to become a brain cancer cell, says Biplab Dasgupta, PhD, whose office and laboratory are within steps of Chow’s and Drissi’s. Unlike cancers that require fewer gene mutations, cancer-initiating cells may require a larger number of mutations to form glioblastoma.

“Normal cells have a built-in mechanism to commit suicide when things go wrong,” Dasgupta says. “It is essentially a chance factor for a mutated cell to dodge the suicide mechanism. The changing environment and our changing lifestyle – including diet – likely allow mutated cells to survive long enough to acquire additional mutations and become full-blown cancer.”

As a result, brain cancer cells are smart survivors. If you block one of their mechanisms of survival, they can harness their heterogeneous nature and use genetic/molecular cross-talks to work around treatments.

“They adapt and evolve in response to therapy,” Dasgupta explains. “Glioblastoma cells are different than other cancers – they are extremely aggressive, metabolically different, and hard to grow on the petri dish.”

DIFFERENT ANGLES

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From the left: Drs. Lionel Chow, Rachid Drissi, and Biplab Dasgupta

Chow, Drissi and Dasgupta study high-grade gliomas like enthusiasts piecing together a scientific jigsaw puzzle, each working from a different angle.

Two of those angles target the abilities of brain cancer cells to use energy and to cheat nature’s rules of cell division. A third involves blocking a central molecular signaling axis that enhances the resourcefulness of cancers to adapt and work around targeted therapies.

BREAKING NATURE’S RULES

Normal cells abide by the mitotic clock and nature’s rule that cells should divide only a limited number of times. In the cell nucleus is the chromosome, which carries the genetic code and DNA that control a cell’s fate.

At the end of every chromosome is the telomere, a series of DNA sequences that, like the plastic caps on the ends of shoelaces, keep the whole works from unraveling. These “caps” help preserve the genetic stability of cells. Each time a cell divides, the telomere gets shorter. After so many divisions, the telomere gets short enough it tells the cells to stop dividing.

Not so with cancer cells, explains Drissi. In brain cancer cells, there are two molecular processes that let the cells ignore nature’s stop signs. One is a protein complex called telomerase. The second is a process called ALT (Alternative Lengthening of Telomeres). Normal cells do not produce telomerase or have ALT, but brain cancer cells do.

When telomerase or ALT kicks in, prompted by genetic mutation, telomeres preserve their length and brain cancer cells grow, spread and kill.

Drissi and his team are testing ways to block the activity of telomerase, to prevent cancer cell telomeres from preserving length. Lab data show that blocking telomerase kills brain cancer cells. The real plus is that it also makes brain cancer cells more sensitive to radiation.

“Radiation treatment causes devastating side effects for children, so being able to make cancer cells more sensitive to radiation and lowering radiation doses would be very beneficial,” he explains.

Drissi had been testing a molecular inhibitor that successfully blocked telomerase activity in cancer cells. It led to a multi-institutional Phase II clinical trial, although the death of a patient already very sick with brain cancer ended the study and the testing of that drug. He is now working on a new inhibitor to stop telomerase, and testing a molecular inhibitor that appears to block the establishment of ALT.

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The panel on the left shows a confocal microscopic image of undifferentiated brain cells. The panel on the right shows differentiated DIPG cells. Both differentiated and undifferentiated cells have the potential to form tumors. Color coding in the images helps researchers compare the genetic makeup of normal brain cells to those with cancer. Their goal is to isolate, characterize and ultimately target these cells to eradicate brain cancer.

FOSTERING NEGATIVE ENERGY

Dasgupta takes aim at glioblastoma cells by messing with their energy. One tactic involves a study he led that helped answer a controversy over how the popular diabetes drug metformin - and its analog phenformin – slow the growth of glioblastoma cells. Another tactic focuses on the ultimate goal of being able to use lipid-based nanoparticles to deliver a molecular inhibitor of AMPK, an enzyme that helps control glioblastoma cells’ energy.

The metformin controversy centered on the widely accepted notion that it slowed glioblastoma by activating AMPK, then blocked the protein mTOR. Gene mutations in the mTOR molecular pathway are a key driver of many cancers. Clinical trials testing metformin for cancer were built on this premise. Dasgupta and his colleagues proved the theory wrong in a study published in PNAS: Proceedings of the National Academy of Sciences. Their study showed that metformin directly inhibited mTOR to cause tumor regression without involvement from AMPK.

Another observation in the study, Dasgupta says, is that while metformin slowed glioblastoma growth, the tumors managed to survive in a diminished state. Metformin shut down the cells’ ability to use oxygen as energy, but they immediately switched to a different energy source – sugar – through a process called glycolysis. So although tumors regressed, they survived. And the longer they survived, the less effective metformin became – helping illustrate the importance of targeted and combined treatments for glioblastoma.

Dasgupta and colleagues recommended that clinical trials of metformin consider these newly discovered mechanisms.

Although AMPK is not part of metformin’s anti-cancer properties, Dasgupta and Chow have found that the enzyme remains important to the survival of cancer cells under severe metabolic stress, like glioblastoma cells.  In other less-stressed cancer cells, AMPK works in reverse as part of the Lkb1-AMPK tumor suppressor pathway.

Using glioblastoma cells taken directly from human surgical biopsies, Dasgupta is now testing the use of “silencing RNA” that turns off AMPK. The silencing RNA is delivered directly to the glioblastoma cells via an engineered virus delivered in vitro. If the model is successful, scientists will test the inhibitor in mouse models.

CHOKING OFF CANCER’S ROOT SYSTEM

Chow studies a core molecular signaling axis for adult and pediatric glioblastoma known as the PI3K/AKT/mTOR pathway. His laboratory developed a mouse model that revs up this pathway to mimic human glioblastoma.

He compares PI3K/AKT/mTOR in glioblastoma to plants that appear to be freestanding, but have an underground network of complex roots. Although PI3K/AKT/mTOR may be at the core, its components can activate a number of downstream molecules and pathways. This gives glioblastoma plenty of escape routes from therapeutic agents.

Using the mouse model, Chow’s team tests combinations of molecular inhibitors of PI3K/AKT/mTOR’s components to see how effectively they slow tumor growth and block escape routes. Hitting critical bottlenecks or junctions for molecular signaling could inhibit tumor growth enough to be a key component of combination treatments. One tactic involves using two agents: rapamycin, which blocks the well-established mTOR pathway, and an inhibitor of PI3K, an enzyme that triggers the disease process. The combined agents have had a dramatic effect on cell death in the mouse model.

These results are important to keep in mind, says Chow, as the PBTC is preparing to initiate a Phase 1 clinical trial to test a PI3K inhibitor.

He emphasizes that success with a drug or combination of treatments does not mean the search ends – it’s just a promising beginning. Given the genetic diversity and complexity of glioblastomas, and how the cancers differ in each patient, the hunt for additional targeting inhibitors to block brain cancer pathways must continue.

“We have to take an unbiased approach and say ‘it could be anything, so let’s look at everything,’” he says.