SNO 2017 in San Francisco
The Nakano Lab at the Society for Neuro-Oncology Annual Meeting, Nov. 2017, in San Francisco.
Pictured from left to right, Dr. Suojun Zhang, Dr. Ichiro Nakano, Ms. Soniya Bastola, Dr. HebaAllah Alsheikh, Dr. Kyung-Don Kang, Dr. Hai Yu, and Nakano Lab alumnus, Dr. Mayuko Nishi.
from research organizations
Anti-cancer chemotherapeutic agent inhibits glioblastoma growth and radiation resistance
This potential chemotherapeutic agent to treat glioblastoma is a novel small molecule inhibitor
Date:July 24, 2017
Source:University of Alabama at Birmingham
A unique and previously unidentified molecular mechanism has been discovered that maintains glioma stem cells, report investigators. They have tested it as a potential therapeutic target in glioblastoma, using a novel small molecule inhibitor they designed and synthesized
Glioblastoma is a primary brain tumor with dismal survival rates, even after treatment with surgery, chemotherapy and radiation. A small subpopulation of tumor cells — glioma stem cells — is responsible for glioblastoma’s tumorigenesis, treatment resistance and subsequent tumor recurrence.
A collaborative team of neuro-oncology surgeon/scientists — led by Ichiro Nakano, M.D., Ph.D., University of Alabama at Birmingham, and Maode Wang, M.D., Xi’an Jiaotong University, Xi’an, China — has discovered a unique and previously unidentified molecular mechanism that maintains glioma stem cells, and they have tested it as a potential therapeutic target in glioblastoma, using a novel small molecule inhibitor they designed and synthesized.
This international study was launched when they found that another inhibitor for advanced cancers, called OTS167, had a negative outcome in a clinical trial. The Nakano laboratory immediately began to investigate the molecular mechanism underlying glioblastoma resistance to OTS167.
They found that a different molecular target, NEK2, evolved after OTS167 treatment for glioblastoma, and they used computer-based drug design to target NEK2. The resulting NEK2 inhibitor, called CMP3a, was able to inhibit growth in pre-clinical models of glioblastoma, both in culture and in mouse brains. When combined with radiation, CMP3a has a synergistic effect to attenuate growth of glioblastoma cells in culture.
“We are currently in the process of pharmacokinetic and pharmacodynamic analyses with CMP3a to design an early-phase clinical trial for glioblastoma and other NEK2/EZH2-dependent cancers,” they said in an article published in The Journal of Clinical Investigation. “We are hopeful to add this drug candidate to our list of clinical trial protocols for glioblastoma in a year or two,” Nakano said.
Details of the research
NEK2 is a poorly characterized kinase enzyme. The researchers found that NEK2 is differentially expressed in glioma stem cells, and it is required for growth of glioma clones in culture, as well as for growth and radiation resistance of a human glioblastoma tumor in the mouse model.
Through a series of detailed experiments, the researchers revealed how NEK2 promotes tumor growth and resistance — they found that NEK2 protein binds to EZH2, an oncogenic histone H3 methyltranferase, and this binding protects EZH2 from protein degradation in the glioma stem cells. EZH2 was already known to regulate the self-renewal and survival of glioma stem cells. Thus, by stabilizing EZH2, NEK2 promotes tumor propagation.
“Disrupting the NEK2-EZH2 interaction in cancer cells has the potential to target their cancer stem cell compartment,” Nakano, Wang and colleagues wrote in their JCI report. “This strategy may serve as a new therapeutic approach for recurrent tumors and a subgroup of primary tumors.”
Recent studies had shown that elevated EZH2 expression occurs in various human cancers, including prostate cancer, breast cancer and glioblastoma, and the studies have shown that elevated EZH2 expression is associated with tumor malignancy and poor patient outcomes.
In a clinical study of 44 patient glioblastoma brain tissues, Nakano, Wang and colleagues found that NEK2 expression was closely associated with EZH2 expression. NEK2 expression also correlated with poor prognosis for the patients, and NEK2 was substantially elevated in recurrent tumors after therapeutic failure. An independent validation cohort included 56 patient samples.
NEK2, they concluded, appears to play a key role in maintaining glioma stem cells by stabilizing the EZH2 protein, and the small molecule inhibitor CMP3a is a potential novel therapeutic agent for glioblastoma.
The researchers had previously studied the MELK-EZH2-STAT3 signaling axis as a central regulator for glioma stem cells in glioblastoma. A MELK inhibitor showed short-term efficacy against human glioma xenografts in a mouse model, but the cultures soon became resistant to the MELK inhibition. Unexpectedly, those resistant glioma cultures retained their EZH2 dependence, prompting the researchers to search how the EZH2 levels were maintained.
They found that EZH2 was protected post-translationally. They also found that NEK2 was the most up-regulated kinase gene in glioma spheres and that NEK2 expression showed a strong association with EZH2 expression in protein profiles for human cancers in the Human Protein Atlas. So, they then looked to see if NEK2 played an essential role in post-translational regulation of EZH2.
Experiments with short hairpin RNAs to knock down NEK2 expression showed that NEK2 is required for glioma stem cell propagation in culture and for glioma tumor growth in the animal model. The researchers found that NEK2 formed a protein complex with EZH2 to phosphorylate and protect EZH2 from proteasome-dependent degradation in glioma stem cells. They also showed that NEK2 promoted radiation resistance in glioma stem cells.
The researchers next designed their novel, clinically applicable, small molecule inhibitor, CMP3a. CMP3a selectively inhibits NEK2 kinase activity in glioma stem cells. Glioma sphere cells showed high sensitivity to this lead candidate for glioblastoma therapy, and normal human astrocytes were markedly resistant to CMP3a. When CMP3a was tested against a screen of 97 kinases, representing all kinase clusters, only three kinases showed more than 65 percent inhibition, indicating that CMP3a is relatively selective for NEK2 inhibition.
Nakano is an academic neurosurgeon at UAB who conducts both brain tumor translational research and clinical brain tumor surgery. He is professor of neurosurgery in the UAB School of Medicine and a senior scientist for the UAB Comprehensive Cancer Center.
- Jia Wang, Peng Cheng, Marat S. Pavlyukov, Hai Yu, Zhuo Zhang, Sung-Hak Kim, Mutsuko Minata, Ahmed Mohyeldin, Wanfu Xie, Dongquan Chen, Violaine Goidts, Brendan Frett, Wenhao Hu, Hongyu Li, Yong Jae Shin, Yeri Lee, Do-Hyun Nam, Harley I. Kornblum, Maode Wang, Ichiro Nakano. Targeting NEK2 attenuates glioblastoma growth and radioresistance by destabilizing histone methyltransferase EZH2. Journal of Clinical Investigation, 2017; DOI: 1172/JCI89092
UAB researchers identify protein that plays key role in brain cancer stem cell growth and survival
by Beena Thannickal
February 10, 2016
A team of physicians and scientists at the University of Alabama at Birmingham discovered that a kinase protein, mixed lineage kinase 4, also known as MLK4, plays a crucial role in survival of patient-derived brain cancer stem cells in pre-clinical animal models. The findings suggest that MLK4 could potentially be a useful target for cancer treatment.
Protein kinases are key regulators of cell function that constitute one of the largest and most functionally diverse gene families. Until recently, MLK4 was considered a poorly characterized kinase. The UAB team, however, identified this gene from a stepwise screening of molecules that are elevated in cancer stem cells isolated from brain cancer patients.
The findings, published this week online in Cancer Cell, nailed down the novel molecular mechanisms for which MLK4 is essential in cancer stem cells and not in normal cells in the human body. Most importantly, brain cancer patients with higher MLK4 expression have shorter survival despite the current intensive therapies including surgery, chemotherapy and radiotherapy. Nonetheless, there are no MLK4-targeting therapies or clinical trials currently available for patients.
“There is no doubt that society desperately needs new and effective therapies for this life-threatening brain disease. Improvement of patient survival for the past 50 years has been counted by months and not years,” said Ichiro Nakano, M.D., Ph.D., professor in the UAB Department of Neurosurgery and principal investigator of the study. “We, as an international collaborative team centered at UAB, focus on cancer stem cells as a therapeutic target in brain cancers.”
“There is no doubt that society desperately needs new and effective therapies for this life-threatening brain disease. Improvement of patient survival for the past 50 years has been counted by months and not years. We, as an international collaborative team centered at UAB, focus on cancer stem cells as a therapeutic target in brain cancers.”
In early 2000, Nakano was involved in a team that isolated cancer stem cells from brain cancers at the University of California at Los Angeles. This discovery gained attention from physicians and scientists because accumulating evidence suggested that cancer stem cells are relatively therapy-resistant and appear to contribute to re-generation of recurrent tumors that subsequently kill affected patients.
“Cancer stem cells share many of the properties of normal stem cells but have also gained transformed cancerous phenotypes,” said Sunghak Kim, Ph.D., an instructor in the UAB Department of Neurosurgery who has led much of the research. “We have been trying to identify the cancer stem cell-specific Achilles heel that could make all the difference.”
While conducting this study, the investigators also found that MLK4-high tumors appear to have Mesenchymal signature, considered to be one process cancers use to become aggressive and therapy-resistant.
“Approximately 35 to 40 percent of glioblastoma patients appear to have Mesenchymal signature. It is also interesting that some non-Mesenchymal cancers seem to shift their phenotype to a Mesenchymal one after therapeutic failure,” Kim said. “We are still collecting more data on this additional piece of information to prove that this is a universal event in brain cancers.”
It is important to note that MLK4 is not expressed in all brain cancers. But now that research indicates that MLK4 is elevated in a subset of brain cancer patients and plays a key role in brain cancer stem cell growth, the next step is to identify targeted therapies that affect the MLK4 in the cancer stem cells.
“We have begun to collaborate with Southern Research Institute to screen drug candidates that selectively target MLK4 in brain cancers,” said Nakano, also a senior scientist at the UAB Comprehensive Cancer Center. “Targeting strategies for MLK4 may work for other cancer types, as we already know that MLK4 is highly expressed in some other malignant types of cancers.”
Nakano added, “Ultimately, we want better outcomes for patients with brain cancer. There’s no question that this is not an easy battle. But by further understanding the molecular mechanisms and applying new targeted therapeutic strategies including MLK4, we are hoping to provide brain cancer patients with more promising and tailored therapeutic approaches.”
Collaborative participants on this project include M.D. Anderson, Ohio State University, University of Texas, Northwestern University, Cincinnati Hospital Medical Center, and a variety of German and Japanese research departments and institutes.
The work was supported by the American Cancer Society, the Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science and Takeda Science Foundation.
Attractive drug candidate identified to target glioma brain tumors
by Jeff Hansen
December 15, 2016
In a paper published today in Cancer Research, researchers: 1) identify a biomarker enzyme associated with aggressive glioma brain tumors, 2) reveal the regulatory mechanism for that enzyme, and 3) demonstrate potent efficacy, using a mouse model of glioma, for a small molecule inhibitor they have developed.
The inhibitor, GA11, retains a core structure that resembles natural inhibitors of the biomarker enzyme; but the inhibitor has been modified to help it pass through the blood-brain barrier.
“In principle, both these features make GA11 an attractive drug candidate to target glioma stem-like cells in glioblastoma multiforme tumors,” said Ichiro Nakano, M.D., Ph.D., and colleagues in the paper.
Nakano, a professor of neurosurgery and academic neurosurgeon at the University of Alabama at Birmingham, and Vito Coviello and Concettina La Motta, University of Pisa, Italy, are doing further preclinical evaluation of the GA11 and its analogs.
Glioblastoma multiforme, or GBM, is a formidable cancer foe. Only two therapeutic improvements have appeared in the past 30 years, increasing the average survival of patients from five months to 15 or 16 months, Nakano says.
A GBM tumor is a mix of different cells that respond differently to therapies. Small numbers of glioma stem-like cells, or GSCs, drive the tumorigenicity of GBM and thus are prime targets for possible treatments. One GSC subtype called the mesenchymal GSC is more malignant and the most therapy-resistant, so Nakano and fellow researchers reasoned that identifying the regulatory molecules active in mesenchymal GSCs might lead to novel and effective therapeutics.
Nakano and colleagues found that one form of the enzyme aldehyde dehydrogenase — ALDH1A3 — is a specific marker for mesenchymal GSCs, and his group is the first to show that, among the heterogeneous mix of cells in a GBM tumor, cells with high levels of ALDH1A3 expression were more tumorigenic in vivo than are cells that are low in ALDH1A3.
The researchers also found that the FOXD1 transcription factor regulates the production of ALDH1A3 in mesenchymal GSCs. In clinical samples of high-grade gliomas from patients, the expression levels of both FOXD1 and ALDH1A3 were inversely correlated with disease progression — gliomas with high levels were more rapidly fatal than were gliomas with low levels.
Astonishingly, the same mechanism that drives the mesenchymal GSC tumorigenicity in humans acts in an evolutionarily distant organism, the fruit fly. Knocking down the expression of either the fruit fly version of the FOXD1 gene or the fruit fly version of ALDH1A3 blocks the formation of brain tumors in a brain cancer model of the fruit fly species Drosophila melanogaster, the researchers found. Thus, this signaling has been highly conserved in evolution.
The FOXD1 transcription factor is normally active during development from a fertilized egg and embryo to a fetus, and it is silent after birth. The role of FOXD1 in GBM, Nakano and colleagues say, suggests that the mesenchymal GSCs have hijacked the molecular mechanism of normal embryonic development to promote tumor growth.
In preclinical testing, GA11 was validated several ways. The researchers showed that it inhibited ALDH in yeast, reduced ALDH1 activity in cell-culture spheres of mesenchymal GSCs, inhibited proliferation of glioma spheres in cell culture, and inhibited xenograft growth of GBM in mouse brains.
“In conclusion,” Nakano and fellow researchers wrote, “the FOXD1-ALDH1A3 axis is critical for tumor initiation in mesenchymal GSCs, therefore providing possible new molecular targets for the treatment of GBM and other ALDH1-activated cancers.”
Nakano says his study of the role of GSCs in GBM is just one approach to treat glioma tumors. Other labs are pursuing immunotherapy, the use of check-point inhibitors, vaccination and efforts to increase sensitivity to radiotherapy.
It will take combined therapies to treat glioblastoma, says Nakano, a senior scientist of the UAB Comprehensive Cancer Center. “We don’t believe that one therapy will be effective.”
Nakano expects to launch a new clinical trial for glioblastoma in 2017, in conjunction with Burt Nabors, M.D., professor of neurology at UAB. Nakano says UAB will be the only site in the Deep South for this unique trial aimed at a molecular target in glioma stem cells, a target that is different from the ones described in the Cancer Research paper. The referral contact to Nakano’s service will be Lydia P. Harrell.
The Nakano lab is also working on brain metastases, tumors that spread into the brain from other parts of the body. Similar to high-grade gliomas, which originate in the brain, these metastatic brain tumors are lethal, and there are very few therapeutic options. Nakano believes the core stem cell genes and signaling pathways are shared between gliomas and brain metastases.
“If so,” he said, “the molecular targets identified for gliomas are most likely essential in brain metastases. Studies are underway, and similar to the glioma therapy development, I am working to develop clinical trials for brain metastasis, together with medical oncologists Mansoor Saleh, M.D., Andres Forero, M.D., and others at UAB.”
Besides Nakano, Coviello and La Motta, authors of the Cancer Research paper, “FOXD1-ALDH1A3 signaling is a determinant for the self-renewal and tumorigenicity of mesenchymal glioma stem cells,” are Peng Cheng, Jia Wang, Zhuo Zhang, Sung-Hak Kim, Marat S. Pavlyukov and Mutsuko Minata, UAB Department of Neurosurgery and Comprehensive Cancer Center; Indrayani Waghmare and Madhuri Kango-Singh, Department of Biology, University of Dayton, Ohio; Stefania Sartini, Department of Pharmacy, University of Pisa, Italy; Ahmed Mohyeldin, Department of Neurological Surgery and the James Comprehensive Cancer Center, The Ohio State University; Claudia L.L. Valentim, Department of Stem Cell Biology and Regenerative Medicine, Lerner Research Institute, Cleveland Clinic; Rishi Raj Chhipa and Biplab Dasgupta, Department of Oncology, Cincinnati Children’s Hospital Medical Center; and Krishna P.L. Bhat, Department of Translational Molecular Pathology, The University of Texas, M.D. Anderson Cancer Center, Houston.
Funding came from National Institutes of Health grants P01CA163205, R01NS083767, R01NS087913 and R01CA183991. Support also was provided by The First Hospital of China Medical University, and by the Graduate Program and startup funds at the University of Dayton.
My name is Shinji Kawamura and I am a 5th year medical student of Osaka University. I have decided to become a neurosurgeon in the future, so I am interested in malignant brain tumors. Needless to say, surgery is very important for treatment. However, non-invasive treatment such as chemotherapy is very important as well. So, I think we have to research diseases and develop better treatment for patients. I have come here to learn how to research because I will have to research malignant brain tumor in the future.
Currently, I learn various research techniques and discuss research which the lab members have been doing. Discussing with them can broaden my horizons. I am convinced that the experience here is helpful for my future.
AUGUST 11, 2016
“My name is Zahab Aleezada and I am a senior at the Alabama School of Fine Arts in its Math and Science Department. I enjoy studying Science, especially Biology, and would like to pursue a career in Medicine. I decided to volunteer in Dr. Nakano’s lab this summer because I am interested in neuroscience research. I want to major in neuroscience in college as well.
Currently I am learning how to do a Western Blot. Also I have successfully learned how to make gels for gel electrophoresis. Dr. Hirokazu Sadahiro and I have been working on seeing if BGB324, an experimental drug, is successful in lowering the concentration of AXL, a protein found in the glioblastoma (GBM). We are also seeing what the safety of BGB324 is for mice with induced tumors. In addition, we are seeing the effect BGB324 has on human GBM stem cell lines.
Understanding how my body works and how different organisms function fascinates me. Learning about the brain is one way to truly understand those things.”