Researchers at the Stanford University School of Medicine, led by Dr. Michelle Monje, have discovered that DIPG tumors stop growing when the tumor cells do not have access to a protein known as neuroligin-3. The researchers also showed in a mouse model of DIPG that a class of drugs known as ADAM10 inhibitors can block neurologin-3 and stop the growth of DIPG tumors. These results were recently published in Nature, and have generated significant media coverage because it provides hope of possible treatment for DIPG.
We recently discussed the paper with Dr. Monje. Here are her views on the potential impact of this study.
Defeat DIPG: This paper seems to build on your prior work by showing that a molecule known as neuroligin-3 plays an important role in driving DIPG tumor growth. Can you explain what neuroligin-3 is? What role it plays in normal cells? And how it drives tumor growth?
Dr. Monje: We had previously discovered that the activity of normal brain cells called “neurons” drives DIPG growth. We found two factors that are released as a result of neural activity that promote the proliferation of DIPG cells, a known growth factor called brain-derived neurotophic factor (BDNF) and, unexpectedly, a protein called neuroligin-3. Neuroligin-3 is a molecule present at the connection between neural cells called a “synapse”. Synapses are the place where communication between brain cells takes place, and neuroligin-3 helps to hold this connection together. It is a long molecule that sticks out of the cell and binds to another similar molecule on the other cell, sort of like holding hands. As a result of brain activity, neuroligin-3 is cleaved and released from the cell. We don’t yet know why this happens, but it is likely part of the activity and experience-dependent plasticity of the brain. When neuroligin-3 is released, it can interact with DIPG cells infiltrating the brain, and it acts as a powerful growth signal for DIPG cells. We were startled to realize that DIPG and other high-grade gliomas depend on this molecule to a dramatic extent. The tumors don’t grow without its presence in the brain.
We have determined that the enzyme that cleaves neuroligin-3 a protease called ADAM10. If we block neuroligin-3 release by inhibiting ADAM10 function, this strongly reduces DIPG growth in the brains of mice.
Here is a more technical explanation:
Neuroligin-3 is a post-synaptic adhesion molecule, meaning that it is present on the membrane of post-synaptic cells (most of the protein sticks out of the cell) and normally adheres to a binding partner on the presynaptic membrane to stabilize synapses between cells. Neuroligin-3 is thus important for synaptic function as well as synapse formation and remodeling in the normal brain. We discovered it is released as a result of neuronal activity and that, quite unexpectedly, is a potent mitogen for high-grade glioma cells such as DIPG. Neuroligin-3 stimulates numerous oncogenic signaling pathways in DIPG and other glioma cells, including Focal Adhesion Kinase (FAK) and downstream PI3K-mTOR, SRC and RAS pathways.
We had shown back in 2015 that neuroligin-3 is a potent growth factor released in the glioma microenvironment, and expression levels of neuroligin-3 correlate inversely with survival in a large database of glioblastoma patients. But at that time we did not know the relative importance of neuroligin-3 to glioma growth. Even with respect to the influences of neuronal activity on DIPG growth, we know that BDNF is also important, and there are numerous other factors in the microenvironment or intrinsic to DIPG cells that can promote growth. So to test the role of neuroligin-3, we combined a genetic mouse model that lacks neuroligin-3 with a mouse model that can accept human cell xenografts and then tested how patient-derived xenografts of DIPG and other high-grade gliomas grow when neuoligin-3 is lacking in the brain microenvironment. We were startled to find that that the xenografts simply did not grow. The profound growth stagnation of DIPG and other high-grade gliomas indicates that neuroligin-3 is necessary for something fundamental to glioma progression. While we have shown that it stimulates numerous important pathways for cell proliferation, I suspect there is substantially more we need to understand about its role in glioma and the reasons for its necessity to DIPG growth. Some hints about this were gleaned from the gene expression changes observed after neuroligin-3 exposure, but we still have a great deal of work to do.
Defeat DIPG: Should we be concerned about blocking neuroligin-3? Does normal brain cells need this protein to function properly?
Dr. Monje: Neuroligin-3 knock out mice exhibit largely normal brain function, because other neuroligin family members compensate for neuroligin-3 loss in normal cells. Interestingly, only neuroligin-3, but not neuroligin-1, 2 or 4 influences glioma growth.
Defeat DIPG: The paper shows that a class of drugs known as ADAM10 inhibitors are effective in decreasing neuroligin-3, which then slows tumor growth. Can you explain what ADAM10 inhibitors are, and how they affect tumor growth?
Dr. Monje: Because neuroligin-3 has such a strong effect on DIPG growth, it represents an important target for therapy. One way to target neuroligin-3 is to block its cleavage and release into the glioma microenvironment, so we set about determining the protease responsible for cleavage of neuroligin-3 at the membrane. Using a combination of computational prediction tools, pharmacological protease inhibitors and genetic mouse modeling, we found that an enzyme called ADAM10 is responsible for neuroligin-3 secretion. When we treated mice bearing xenografts of patient-derived DIPG or pediatric glioblastoma, ADAM10 inhibition blocked tumor growth.
Defeat DIPG: As we understand it, ADAM10 inhibitors are not yet FDA approved for any use, but that there are several ongoing clinical trials testing these drugs for use in other cancers. What have we learned about these inhibitors from those trials? Do we have any idea whether these inhibitors effectively cross the blood-brain barrier?
Dr. Monje: There have been two ADAM10 inhibitors in clinical trials for breast cancer and lymphoma. These were well-tolerated in adults, but development stopped when ADAM10 inhibition was found to not be more effective than existing treatments for those diseases. We tested blood-brain-barrier penetration of both ADAM10 inhibitors and found one to be superior to the other. We then tested the ADAM10 inhibitor shown to be safe in adults and found to get into the brain at reasonable concentration for a reasonable amount of time in our study. This ADAM10 inhibitor substantially reduced glioma xenograft growth.
Defeat DIPG: What are the next steps in your research? Do you plan to test the effectiveness of ADAM10 inhibitors in combination with other drugs, such as panobinostat? Is any more lab work necessary before DIPG patients can be treated with these inhibitors?
Dr. Monje: We are presently in discussions with the company who owns this ADAM10 inhibitor compound. We would like to extend the preclinical studies to generate enough evidence to support a clinical trial for DIPG. It is difficult to predict the timeline, but we hope to translate this as quickly as we possible can for children with DIPG and other forms of high-grade glioma.
It should be noted that loss of neuroligin-3 signaling in DIPG and other gliomas causes stagnation of growth, but not glioma cell death. Targeting neuroligin-3 with ADAM10 inhibition could be complementary to cytotoxic therapies. A theory that we need to test experimentally is the idea that growth stagnation caused by blockade of neuroligin-3 secretion could help to prevent the evolution of therapy resistance that we have observed with agents such as panobinostat.
Defeat DIPG: Thank you Dr. Monje. We appreciate your efforts to find effective treatments for DIPG. We hope that DIPG children will soon benefit from your research and can be treated with ADAM10 inhibitors.