New Findings Alter View of EGFR Signaling in Glioblastoma

Signaling pathway thought to be driving oncogenesis gets called into question.

The epidermal growth factor receptor (EGFR) is a key oncogene that is frequently amplified in glioblastoma, and evidence has suggested that EGFR is an important oncogenic driver in this highly invasive brain tumor. However, multiple clinical trials of EGFR targeting have failed as an effective treatment.

Surprising findings, published in Nature Cell Biology by Amyn Habib, M.D., a Professor of Neurology and member of both the Harold C. Simmons Comprehensive Cancer Center and Peter O’Donnell Jr. Brain Institute at UT Southwestern, suggest a tumor suppressive role of EGFR in a subset of EGFR-amplified glioblastomas.

“Glioblastoma’s invasive property is perhaps its most formidable barrier to treatment,” Dr. Habib says. “The prevailing paradigm was that EGFR activation was uniformly oncogenic in human cancer. In contrast, we discovered a tumor suppressive function of EGFR signaling that actually suppresses glioblastoma invasion.”

The study initially focused on analyzing data from The Cancer Genome Atlas (TCGA), a landmark cancer genomics database of over 20,000 primary cancer and matched normal samples spanning 33 cancer types. The researchers looked at the combined effect of all seven EGFR ligands, uncovering that EGFR ligands are in fact tumor suppressive in EGFR-amplified glioblastoma.

Consistent with this unexpected finding, experimental data showed that ligand-activated EGFR signaling suppresses tumor invasion. Dr. Habib and his colleagues believe this suppression of invasion mediates the tumor-suppressive effect of ligand-dependent EGFR signaling in EGFR-amplified glioblastomas.

“Previous studies have reported that EGFR stimulates invasion in glioblastoma-established cell lines that have lost EGFR amplification,” Dr. Habib says. “Our data show that the level of EGFR expression determines whether the outcome of ligand-dependent EGFR signaling is either increased or decreased invasion.”

The TCGA analysis confirmed these findings, revealing that in non-EGFR amplified glioblastomas, EGFR ligands are oncogenic while, in a striking reversal, EGFR-amplified glioblastomas’ EGFR ligands are tumor suppressive. A key reason is likely the differential upregulation of BIN3, a cytoskeletal protein, in EGFR-amplified tumors, Dr. Habib explains.

DISCOVERY OF A NEW PATHWAY

Dr. Habib and his team were the first to connect ligand-dependent EGFR signaling and suppression of invasion by upregulation of BIN3.

Using a series of siRNA and mass spectrometry experiments to identify BIN3-interacting proteins, the researchers discovered the guanine nucleotide exchange factor DOCK7, which regulates the small G proteins RAC1 and CDC42, and prioritized further investigation of the protein owing to its known role in HGF-induced invasion of glioblastoma cells.

Mechanistically, ligand-induced EGFR activation leads to upregulation of BIN3 and tyrosine phosphorylation of DOCK7, leading to a physical association of BIN3 and DOCK7. DOCK family members have a RhoGEF domain and function as guanine nucleotide exchange factors for the RhoGTPase family of signaling proteins.

Together, the experiments indicated that ligand-induced EGFR activation leads to decreased invasiveness by a BIN3-mediated inhibition of a DOCK7-RhoGTPase pathway.

“These findings show that a patient stratification based on the level of EGFR and EGFR ligands may be helpful to predict subsets of patients likely to benefit from targeting the EGFR,” Dr. Habib says. “In patients with EGFR-amplified glioblastomas, with high EGFR ligands, EGFR inhibition could actually be detrimental, since EGFR ligands and high phosphorylation of the EGFR confer a better prognosis.”

On the other hand, in EGFR-amplified tumors with a low level of ligands, a brain-penetrant drug such as tofacitinib, a selective JAK1/JAK3 inhibitor that upregulates BIN3 through upregulation of EGFR ligands and suppresses invasion, could be very useful, he adds.

REPURPOSING TOFACITINIB

From preclinical studies through regulatory approval, the average timeline for discovering, developing and bringing to market a new drug extends more than a decade and can cost hundreds of millions of dollars. With thousands of drugs now FDA-approved for use in humans, new developments have opened opportunities for potentially expanding indications of marketed drugs to other diseases.

“Tofacitinib is a drug already used to treat rheumatoid arthritis, psoriatic arthritis, and ulcerative colitis,” Dr. Habib says. “Tofacitinib could be a unique and effective treatment for glioblastoma that specifically targets invasion and upregulates BIN3 levels.”

The researchers confirmed the effect of tofacitinib in vivo in several orthotopic PDX models. In these experiments, they engineered tumor cells to express the EGFR ligand TGF?, so that tumor cells secreted this ligand in an autocrine manner.

Dr. Habib further explains that this type of treatment could be used to suppress invasion prior to surgical resection and shrink the main tumor mass. Since invasion is thought to be a key determinant of glioblastoma recurrence, tofacitinib could also be used later in the course of the disease to prevent or treat recurrence.

More generally, for EGFR non-amplified tumors with a high level of ligands, or possibly for EGFR-amplified tumors with a low level of ligands, EGFR inhibition combined with blunting of the accompanying adaptive response could be a useful therapeutic strategy, Dr. Habib says.

A NEW CLINICAL TRIAL

In collaboration with Pfizer, Dr. Habib and Michael Youssef, M.D. – an Assistant Professor of Neurology and Hematology-Oncology and member of both the Simmons Cancer Center and the O’Donnell Brain Institute at UT Southwestern – are leading a new clinical trial examining the effects of tofacitinib in patients with recurrent glioblastoma. The study (NCT05326464) is currently accruing patients at Simmons Cancer Center.

The co-investigators anticipate the trial has the potential to advance the standard of care and improve the quality of life of patients with this debilitating malignancy. Moreover, if the results are positive, it could prompt the FDA to approve the drug for this indication.

“We have already validated the feasibility of tofacitinib in preclinical models of amplified EGFR glioblastoma tumors,” Dr. Youssef says. “This trial provides us with the opportunity to confirm these observations with evidence from a Phase 3 study.”

In the trial, eligible patients with recurrent glioblastoma are being treated with 10 milligrams of tofacitinib twice daily until evidence of progression, intolerance of treatment, withdrawal of consent, or death.

The primary outcome is progression-free survival as defined by RANO criteria. Secondary outcomes include overall survival, safety, and tolerability, and tumor response by RANO criteria up to two years after study treatment.

“We expect this trial to also provide further insight into the mechanisms underlying recurrent disease, which has not been well characterized,” Dr. Youssef adds.

As the trial continues to accrue, the researchers eagerly await the forthcoming results, which they expect by mid to late 2024.

FUTURE DIRECTIONS

A next direction for the research could be to determine whether distinct ligands differentially affect EGFR signaling downstream from EGFR, Dr. Habib says.

“Further studies may also test how distinct ligands differentially affect downstream signaling, and the ability to block EGFR signaling at multiple nodes both upstream and downstream of this receptor tyrosine kinase,” Dr. Habib notes. “These findings could inform future clinical approaches in patients with EGFR-amplified glioblastoma.”

More generally, additional research is needed to understand how the current findings affect our treatment regimens using EGFR inhibitors, Dr. Habib explains.

Dr. Habib acknowledges that in theory, adapting the current precision-medicine workflow by stratifying patients by their levels of EGFR ligands represents an opportunity to improve outcomes but could present practical challenges.

“If we were to stratify patients, we’d have to determine how and when a patient’s level of EGFR ligands would be assessed,” he adds.

CONCLUDING REMARKS

While a clinical paradigm that incorporates EGFR amplification status may benefit some patients, curative treatment regimens will probably continue to rely on combination therapy.

According to Dr. Habib, glioblastoma has historically proved intractable to treatment, likely due to incomplete understanding of the complex signaling networks required for tumor cells to proliferate, invade, and develop resistance to treatment.

Future studies, both in the clinical and basic science realms, will be needed to fully elucidate whether outcomes in patients with EGFR-amplified glioblastoma can be improved by targeting oncogenic functions of EGFR while sparing its potential tumor suppressive roles.

“The O’Donnell Brain Institute is truly a unique environment for neuroscience and clinical care,” Dr. Habib says. “Abundant grant and funding opportunities, such as the Investigator Program and the Sprouts Grant Program, encourage creative, high-quality research that continues to make a difference in our patients’ lives.”

This study was funded by grants from the Department of Veterans Affairs (VA) (2I01BX002559-08) and the National Institutes of Health (1R01CA244212-01A1 and 1R01NS119225-01A1). The VA has filed a patent on the use of tofacitinib in glioblastoma, listing Dr. Habib as the inventor. The clinical trial will be conducted at the Simmons Cancer Center.