A research team led by investigators at McMaster University has identified an axonal guidance signaling pathway that cancer cells use to infiltrate the brain. The combined results of preclinical in vitro and in vivo tests evaluating either a small molecule inhibitor of the protein PTP4A2, or a newly developed CAR-T cell-based therapeutic approach against the PTP4A-ROBO1 signalling axis demonstrated increased cell survival in brain cancer models. ROBO1 CAR T cells eradicated tumors in 50–100% of cancer-bearing mice. The team says their research offers new hope and potential treatment strategies for glioblastoma (GBM), the most aggressive form of brain cancer, and potentially other brain malignancies.

“In glioblastoma, we believe that the tumor hijacks this signalling pathway and uses it to invade and infiltrate the brain,” said Sheila Singh, MD, PhD, professor with McMaster’s department of surgery and director of the Centre for Discovery in Cancer Research. “If we can block this pathway, the hope is that we can block the invasive spread of glioblastoma and kill tumor cells that cannot be removed surgically.” Singh, and Jason Moffat, PhD, head of the Genetics and Genome Biology program at The Hospital for Sick Children (SickKids) are co-senior authors of the team’s published paper in Nature Medicine, titled “Targeting axonal guidance dependencies in glioblastoma with ROBO1 CAR T cells.” In their paper they stated “Our study identifies a promising multi-targetable PTP4A–ROBO1 signaling axis that drives tumorigenicity in recurrent GBM, with potential in other malignant brain tumors.”

GBM remains the most aggressive and prevalent malignant primary brain tumor in adults, the authors wrote. Treatment for the cancer has not advanced beyond surgery, chemotherapy and radiotherapy for two decades. “Unchanged since 2005, standard of care (SoC) consists of surgical resection followed by radiation therapy plus concurrent and adjuvant chemotherapy with temozolomide (TMZ)” they stated. However, tumors often return, and patient survival is limited to only a few months.

To discover the pathway that cancer cells use to infiltrate the brain the researchers used large-scale gene editing technology to compare gene dependencies in glioblastoma when it was initially diagnosed and after it returned following standard treatments. “… we conducted a genome-scale comparison between patient-matched pre-treatment and post-treatment GBM models at the mutational, functional genetic, transcriptomic and proteomic levels,” they explained. Their analyses identified the PTP4A2-ROBO1 pathway which is involved in axonal guidance—a signalling axis that helps establish normal brain architecture —that can become overrun by cancer cells. Their combined results, they suggested “…  indicate that modulation of the PTP4A–ROBO1 axis could be a viable strategy to target rGBM and requires further mechanistic investigation and optimization of therapeutic interventions.”

The team evaluated different methods to prevent cancer cells hijacking this pathway, including use of a pan-PTP4A chemical inhibitor. The team also developed a CAR T cell therapy to target the signaling pathway in the brain through inhibition of ROBO1, the protein that helps guide certain cells, similar to a GPS. “ROBO1 is an axonal guidance gene associated with migration and invasion of GBM cells,” they explained.

“We created a type of cell therapy where cells are taken from a patient, edited and then put back in with a new function,” said lead author Chirayu Chokshi, PhD, who worked alongside Singh at McMaster University. “In this case, the CAR T cells were genetically edited to have the knowledge and ability to go and find ROBO1 on tumor cells in animal models.”

Singh and Chokshi say the CAR T cell approach could also be relevant to other invasive brain cancers. In their reported study the researchers examined models for three different types of cancer including adult glioblastoma, adult lung-to-brain metastasis, and pediatric medulloblastoma. In all three models, treatment led to a doubling of survival time. In two of the three diseases therapy led to tumor eradication in at least 50% of the mice. “Our study establishes ROBO1 CAR T cells as a potent immunotherapy for GBM and other highly invasive brain cancers,” the investigators stated.

“In this study, we present a new CAR T therapy that is showing very promising preclinical results in multiple malignant brain cancer models, including recurrent glioblastoma,” Singh added. “We believe our new CAR T therapy is poised for further development and clinical trials.”