At St. Jude Children’s Research Hospital, John Nitiss is trying to figure out just how certain cancer drugs — those that target topoisomerases — work. In doing so, he is hoping to specifically discover where they interact on the enzyme and what exactly is going on in a cancer cell once the drug begins to act on the enzyme.
While Nitiss previously conducted this drug action research in yeast, he is now turning his attention to mammalian cells with the help of RNA interference.
Yeast has been an extremely successful model for understanding the mechanisms of cancer drugs, he told RNAi News. But “what I’ve always said is that I would start doing these sorts of experiments in mammalian cells when I could do yeast genetics in mammalian cells. That’s actually where the RNAi is starting to come in.”
Nitiss said that researchers have been using strains of yeast with particular genes deleted as a way to identify genes that are important for cell sensitivity to topoisomerase I- and topoisomerase II-targeting drugs.
“Now, we have the opportunity of taking genes that we know to be important in yeast cells for surviving these types of drugs and to demonstrate that those same genes play similar roles for mammalian cells,” he said.
Nitiss said that he and his colleagues are beginning this work by looking at Nijmegen-Breakage Syndrome. NBS is a rare genetic disease that is characterized by progressive microcephaly, short stature, and a strong predisposition to cancers, particularly lymphoma.
“Biochemical analyses show that the gene that is mutated in this disease is a component of a complex called the MRN complex,” he said, “which includes a gene called Mre-11, Rad-50, and Nbs-1.” Nitiss added that, for reasons not yet known, Nbs-1 is very poorly conserved between the yeast cells and mammalian cells, while Mre-11 and Rad-50 are very highly conserved.
Mre-11 is essential for homologous recombination and double-strand break repair, as well as damage responsiveness, he said, and the researchers are hoping to demonstrate that the individual components of Mre-11 impact sensitivity to topoisomerase-targeting drugs in mammalian cells as much as they do in yeast.
“The idea is to first have a very powerful way of identifying genes that are potentially important for drug sensitivity using yeast cells and then use RNAi techniques to verify that a loss of function of those genes actually sensitizes mammalian cells [to the drugs] as well,” Nitiss said.
First Things First
The project is already underway using Saccharomyces cerevisiae, he said. A large number of genes of interest have already been identified in yeast, he noted, but ones that have piqued the researchers’ interest are those for which there is no obvious mammalian homolog.
“So, part of our effort will require actually identifying the mammalian homologue [for various genes that may be involved in cancer drug sensitivity],” he said.
The process of doing so involves taking advantage of a previously constructed set of strains which have had their open-reading frames disrupted, Nitiss said. This provides “access to the complete set of non-essential genes in very rigorously constructed knockouts,” he said. And while this might be sufficient for many types of drugs, yeast can be resistant to many different types of drugs. In cases like these, drug-hypersensitive mutant strains are often employed, he said.
“This gives us a set of genes, some from pathways we already know are important, such a homologous recombination, but also in other pathways we didn’t suspect were important for cell survival [following the damage caused by a particular drug],” he said.
If there is an obvious mammalian homolog for the identified yeast gene, Nitiss said, “we go directly to … an RNAi-type experiment.” If no human homolog is apparent, genomes from other types of yeast are examined “to try to identify regions that are essential, [and then we] go back to query some of the mammalian genome to see if you can get things that look like homologs.”
Tool Before Treatment
In the end, Nitiss hopes that his experiments will allow him and his colleagues to figure out how the DNA damage caused by topoisomerase-targeting drugs is repaired by the cell.
“We have no possibility, at the present time, of assessing whether tumor cells have a differential capability of repairing this type of DNA damage,” he said. Additionally, many researchers are trying to develop ways of making cells more sensitive to various types of DNA damage, Nitiss added.
For example, he said, if RNAi can be used to locate those genes that control a specific repair function needed to survive the damage caused by a topoisomerase-targeting drug, those genes could be perturbed in a tumor cell in order to improve the efficacy of the therapy.
As for whether that pertur- bation can be brought about using RNAi, Nitiss is somewhat skeptical.
“Probably, [RNAi’s] strongest role is in establishing proof of principle,” he said. “Most of what I understand is that it would be very difficult to make good therapeutic molecules with RNA. So, I think its impact is going to be felt as a tool long before anyone makes a very successful RNA-based therapeutic.”
—DM