A team of scientists at the University of Toronto has successfully used CRISPR-Cas9 technology to determine likely targets for glioblastoma (GBM). This type of cancer is one of the most aggressive, having only a few existing treatment solutions. GBM is also the leading cause of cancer-related deaths in children and young adults.
Gene by gene, the strategy used genome editing to effectively reverse engineer metastatic, patient-derived GBM stem cells. In addition, it was able to mark genes that are crucial for tumor development. Apart from highlighting several possible GBM targets, the research offered new insights into the basis of glioblastoma resistance to temozolomide (TMZ) chemotherapy. It also opens pathways to potential methods for combination treatment.
One Of The First Studies Of Its Kind
The study titled “Genome-Wide CRISPR-Cas9 Screens Expose Genetic Vulnerabilities and Mechanisms of Temozolomide Sensitivity in Glioblastoma Stem Cells” was published in an open access paper in Cell Reports. Scientists at the Hospital of Sick Children, University of Calgary, and the University of Toronto, led by author Graham MacLeod, a post-doctoral fellow at University of Toronto’s Leslie Dan Faculty of Pharmacy, composed the team.
Research co-lead Stéphane Angers, Ph.D., a professor at the Leslie Dan Faculty of Pharmacy, describes the groundbreaking study as one of its kind. In fact, it’s the first time CRISPR screens are carried out directly in various newly isolated patient cells in parallel. “This study has provided a massive amount of new information that the research community can now interrogate to help design new treatment strategies,” Angers said.
GBM’s Dismal Prognosis
The authors noted that glioblastoma shows considerable heterogeneity between patients, but also in the same patient. This distinction, combined with a total lack of strong responses to therapy, means that the future where doctors can cure GBM looks grim. Even the most innovative form of therapy for this tumor type, called temozolomide, demonstrates what scientists regard as “remarkably limited” effectiveness.
What makes these GBM stem cells so difficult to cure is their resistance to treatment. Research co-lead Peter Dirks, Ph.D., senior scientist at the Hospital for Sick Children, said that they need to look for ways to disrupt malignant cells specifically if they aim to increase patients’ chance of survival. In addition, there are concerns that temozolomide chemotherapy can cause tumor cells to grow new mutations that may further propel the development of the disease.
Understanding What Fuels The Disease
Indeed, the ability to understand the factors affecting growth and drug responsiveness across this heterogeneous cancer is crucial. The team stated that this would help scientists identify new potential therapies and create strategies that could be combined with TMZ, which is now engrained in upfront GBM treatment.
GBM is believed to be driven by a small population of stem cells within the tumors. Researchers are able to look into the basis and growth of GBM in greater depth using culture systems for patient-specific GCSs. Notably, these GSC cultures safely sustain patient-derived genotypic (genes in the DNA) and phenotypic (physical traits resulting from the interaction of its genotype with the environment) characteristics. They also preserve their in vivo growth behaviors and tumorigenic capacity.
Furthermore, scientists can use CRISPR gene editing tools to carry out “cell fitness screens” that recognize both “context-specific” and “core” essential fitness genes that regulate the spread of cells. Given the heterogeneity of GBM between patients, the Canada-based team performed parallel genome-wide CRISPR screens in 10 distinct patient-specific GSC cultures. With this trial, they aim to identify the molecular basis of cell survival and growth.
Testing 20,000 Genes
The method essentially involved systematically knocking down the 20,000 genes, one after the other, from each patient sample. This allowed the researchers to see its effects on tumor survival and development. They compared information from the parallel screens with those GSC screens on two normal human fetal neural stem cell cultures. They carried out different chemogenomic screens to detect these genes involved in cancer resistance to TMZ and possible approaches for combination treatment.
The progression of the disease and the growth of tumors are driven by cancer stem cells. In order to destroy these cells effectively, having a wider understanding of the genes fueling the growth programs is crucial. If scientists find a way to pinpoint which genes are necessary for these cells to proliferate and survive, then finding a way to block or attack these genes and stop tumor growth becomes more achievable.
The findings underscored members of the SOX transcription factor family, DOT1L, USPS, and SOCS3, and protein ufmylation pathway genes as important for GSC fitness and growth. Irrespective of patient tumor genotype and despite the inherent intratumoral heterogeneity, the identification of common GSC fitness genes offers insight into the underlying biology of GBM. It also helps researchers recognize potential avenues for preclinical trial.
Inhibiting The DOT1L Gene
The authors found DOT1L to be necessary for tumor persistence in seven out of the 10 cultures. The team collaborated with Samuel Weiss, Ph.D., a professor at the University of Calgary, for additional studies. The results showed that a medication used to treat leukemia could constrain the DOT1L gene product in GSCs. It turns out that blocking this protein in this type of brain cancer minimized tumor growth and lead to longer subsistence in the preclinical model. Angers commented that this feat is promising as it uncovered a biological process, not previously known to be implicated in GBM, for which a tiny molecule drug already exists.
There’s another exciting find in this particular study. The screens were also able to detect various stress signaling pathways as critical for the development of GSCs. Together with previous trials, this supports further investigation of the success of therapeutic methods targeting JNK signaling for GBM.
In summary, the genome-wide CRISPR screens in patient-specific GSC cultures have discovered a diversity of genetic weak points. They also hold a wealth of data that can be further studied to spot drug targets for GBM. In the future, scientists seek to identify the precise mechanism by which these pathways and genes modulate the chemosensitivity and/or fitness of GSCs. They also want to know whether these factors drive the growth of the disease in vivo.
