NEW YORK (GenomeWeb) – CRISPR/Cas9 screens can offer a systematic high-throughput approach to finding novel cancer drug targets, according to a new study from scientists at Cold Spring Harbor Laboratory.
Led by Junwei Shi and Christopher Vakoc, the researchers designed CRISPR/Cas9 systems to knock out 192 specific protein domains in five chromatin regulator genes (rather than knock out the entire gene) to find those essential to the survival of acute myeloid leukemia cells. They found 25 domains without which cancer cells could not survive, including six known drug targets. They published their study this week in Nature Biotechnology.
"We can now take a very complicated protein and scan across it and try to visualize which surfaces matter most for that protein to work. We can draw a bulls-eye around the domains critical for a certain disease," Vakoc told GenomeWeb. "We now feel like we have technology to see every weakness that a cancer cell possesses along its cell proteins. It collapses the druggable genome."
The idea for comprehensive screening came about by interrogating the domains of the BRD4 gene, Vakoc said. Perturbing an exon in the gene that corresponds to a protein domain can have the same effect as a small molecule blocking that binding site, removing the function of that domain. Several of the BRD4 protein's domains are known to be small molecule binding sites; perturbating the section of the genome coding for those domains with CRISPR/Cas9 confirmed that without them the cancer cells die. "We kind of rediscovered BRD4 as a drug target," Vakoc said.
Vakoc had previously led a group that discovered BRD4 as a drug target with RNA interference (RNAi) screens, so they were intimately familiar with the protein and had a point of comparison. The researchers began to suspect that CRISPR/Cas9 could systematically point out discrete domains essential to AML and set about designing a way to see them.
The new screen consisted of over 1,000 CRISPR/Cas9 systems programmed to target sequences coding for almost 200 domains essential for chromatin regulation. Each CRISPR/Cas9 system targeted only one domain, but there were several systems targeting each domain.
It revealed 19 brand new domains essential to AML survival along with six domains already being targeted in clinical trials. "Our CRISPR strategy easily identified all of those targets in one experiment. We can rediscover everything we already knew in the course of two weeks," Vakoc said, noting that finding those six known targets took decades of research.
The method could help find ways to disable large proteins with many domains that are known to have an effect on cancer cells. "There may be only one [domain] that you might consider druggable," he said, but the cancer cell's survival might not depend on it. "Pharma can burn a lot of energy on those complicated targets. Our method would immediately home in on that target being crucial."
Vakoc suggested that CRISPR screens could find new targets for well established classes of drugs, like kinase inhibitors. "You could take the druggable domains, survey them, and find which kinase domains do ovarian cancers need to survive," he said. "The low-hanging fruit is to do deep dive on druggable domains we know can be targeted with small molecules. If you put out a compelling case for a kinase domain being important to cancer, there are a lot of pharma groups that would be willing to swoop in," he added.
Vakoc also believes that CRISPR screens will allow researchers to dismiss drug targets, something that couldn't be done with RNAi screens.
"It's clear that RNAi missed a lot of things," Vakoc said. "CRISPR feels like it doesn't miss anything. It's because we're targeting the gene rather than going for the mRNA.
"With RNAi we couldn't be confident in a negative result, maybe we just didn't knock down the protein enough," he said. "I'm starting to believe in a negative result [with CRISPR]. If you don't score a phenotype, I'm starting to believe that domain doesn't matter."
Vakoc said his group envisions a comprehensive map of all the protein domains essential to many types of cancers, which could serve as a resource for future drug development.
"Maybe we don't know how to drug certain domains now, but if we discover a compelling domain, that might motivate someone to figure out how to drug it," he said.