NEWS

Running Interference: Pace Picks Up on Synthetic Lethality Research

Ken Garber

Visionary ideas sometimes languish for years. Back in 1995, cell biologist Lee Hartwell, Ph.D., and pediatric oncologist Stephen Friend, M.D., Ph.D., backed by the National Cancer Institute, launched the Seattle Project. The goal: identify novel anticancer drug targets by performing full-genome genetic screens in mutant strains of yeast. However, Hartwell soon departed to take charge of the Fred Hutchinson Cancer Research Center, and Friend left to join the biotechnology company Rosetta Inpharmatics. (Hartwell won the 2001 Nobel Prize for Physiology or Medicine for his work on the cell cycle.) The Seattle Project lost momentum and did not achieve its goals. But one of its main concepts lay dormant, waiting for technology to catch up.

That moment has now arrived. The new technology is RNA interference (RNAi), unknown back in 1997. Thanks to RNAi, Hartwell and Friend's idea, synthetic lethality, is now enjoying a resurrection. It is being used not only in yeast, but in human cells, and on a scale unimaginable 7 years ago. "RNAi is exquisitely suitable to find genes that are synthetically lethal," said René Bernards, Ph.D., head of the division of molecular carcinogenesis at The Netherlands Cancer Institute in Amsterdam.

Hartwell and Friend borrowed the term "synthetic lethality" from classical genetics to describe situations in which a mutation and a drug together cause a cell's death. ("Synthetic" is used in the sense of synthesis, or coming together.) The presence of the mutation alone, or the drug alone, cannot kill. Put them together, and the cell dies. Several experimental anticancer drugs, in retrospect, appear to work through synthetic lethality. (See News, Nov. 20, 2002, Vol. 94, No. 22, p. 1666.)



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Dr. Lee Hartwell

 

Synthetic lethality is a new way to find cancer drug targets. Mainstream cancer drug discovery aims to block oncogenic signaling or, more rarely, to restore tumor suppressor gene function. Synthetic lethality, on the other hand, seeks not to change these defining features of cancer cells but to instead use them to cause the cells' downfall. "The synthetic lethal approach you might compare to judo, or jujitsu," said Kim Quon, Ph.D., a group leader at the Genomics Institute of the Novartis Foundation (GNF) in San Diego. "Rather than trying to stop what is giving the cancer cell strength, you're using the cancer cell strength as its weakness."

TRAIL of Death

Quon's group recently demonstrated the power of synthetic lethality in work reported in May in Cancer Cell. Antibodies activating the DR5 "death receptor" caused apoptosis, or programmed cell death, in cells overexpressing the myc oncogene, whereas normal cells survived, proving that activated myc and DR5 are synthetically lethal. The group confirmed this result using transformed cells transplanted into immune-deficient mice—possibly the first in vivo demonstration of synthetic lethality in mammals. Myc-overexpressing tumors disappeared after antibody therapy, while non-myc tumors proved resistant.

DR5, also known as TRAIL receptor 2, is a promising anticancer drug target, with at least two companies currently testing TRAIL receptor antibodies in the clinic. The Quon work partly explains why these antibodies have shown selectivity for cancer cells in animal models. More importantly, "it's the first demonstration that the synthetic lethal approach can work as a cancer therapy," said Quon.

Many such synthetic lethal interactions exist, just waiting to be discovered and mined as anticancer drug targets, Hartwell believes. In yeast, about 70% of genes are nonessential. "If you take one of those nonessential genes and knock it out, the cell grows fine," Hartwell said at the annual meeting of the American Association of Cancer Research in March. "[But] you can find 30 other targets for every gene which, when knocked out at the same time, will kill the cell. So there's a huge redundancy, and there's a huge number of secondary targets for each primary target."

Synthetic lethal interactions have been thoroughly documented in model organisms, agreed Bernards, but have been demonstrated only rarely in mammalian cells. "The reason is not that they don't exist—of course they exist—but we never had the tools to find them," he said.

Bernards himself has now developed one such tool. In work published in the March 25 issue of Nature, Bernards' group in Amsterdam and Greg Hannon's group at Cold Spring Harbor Laboratory in New York both demonstrated, for the first time, the use of large-scale RNA interference libraries to screen human cells. In RNA interference, small double-stranded RNAs efficiently and specifically silence gene expression by chopping up messenger RNA. The phenomenon was discovered in 1998 and shown to work in human cells 3 years later. (See News, April 2, 2003, Vol. 95, No. 7, p. 500.)

However, until the Bernards and Hannon innovations, it was not practical (at least outside of industry) to conduct large-scale screens using RNAi in human cells because of the expense and labor involved. To perform even a simple genome-wide knockout screen, an RNAi construct or vector representing each gene would have had to be separately introduced into dishes containing cells and then assayed for effect. For a single screen "you would have to do 25,000 experiments," said Bernards. "And now we can basically do it in one experiment."

Bernards and Hannon accomplished this feat by inventing "barcode" systems for identifying RNAi vectors in cell culture. First, both groups created large human RNAi "libraries" by generating viral vectors containing DNA constructs coding for double-stranded RNAs, each capable of silencing a gene. (Hannon's library encompassed 9,610 human genes; Bernards' had 7,914.) Following introduction into one cell culture pool, all DNA sequences are pulled out in a single polymerase chain reaction using primers specific to the vector sequence and labeled with fluorescent dye. Then, hybridized to a microarray containing the complementary sequence for each gene, the amplified DNA is combined with the DNA from a control set that is labeled with a different color. Any variation from the intermediate (mixed) color signals an effect—either inhibitory or enhancing—of RNAi on cell proliferation. In a synthetic lethality assay, "basically you see all the possible genetic interactions with your stress signal in one experiment," said Bernards.

"A very nice technical advance," commented Bill Kaelin, M.D., associate professor of medicine at Harvard Medical School, Boston. "It's what everyone was hoping for."

Lending Libraries

Academic groups are already taking advantage of this work. Kaelin, for example, is planning to undertake a genome-wide screen in human cells, using the Hannon RNAi library, to test for genes synthetically lethal with the mutated VHL (von Hippel-Lindau) tumor suppressor gene. "We've also had discussions with some pharmaceutical companies about doing a synthetic siRNA [small interfering RNA] screen," Kaelin said. That approach, using double-stranded RNAs themselves instead of DNA vectors encoding RNA, is more laborious and expensive but is within the means of most large drug companies.

The pharmaceutical industry, in fact, is enthusiastically adopting synthetic lethality screening to identify novel drug targets. "I know of at least three or four major pharmaceutical companies that are talking about doing these screens," Kaelin said. Bernards said that is an understatement. "All the pharma companies are doing this," he said. The appeal is obvious—quick target identification with built-in cancer specificity. "If we find a gene that is synthetically lethal with myc overexpression, now we inhibit that gene with a small molecule that should be preferentially toxic to cells that have overexpression of myc, which is the cancer cells," said Bernards. "You can play the same game with p53 loss." Bernards' group is looking for genes synthetically lethal with p53, myc, and ras, among other oncogenes and tumor suppressor genes, as well as genes synthetically lethal with known anticancer drugs.



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© 2004 Nature Publishing Group. Reprinted with permission.

Library privileges: The Netherlands Cancer Institute built a retrovirus-based RNA interference library targeting about one-third of all human genes. It is now publicly available. (Source: Berns K, et al. A large-scale RNAi screen in human cells identifies new components of the p53 pathway. Nature 2004; 428:431–7.)

 

Quon's group at GNF has already performed a screen to find siRNAs that kill cancer cell lines that overexpress myc. "We did find agents that would synergize with the DR5 antibody," Quon said. Some of the synthetically lethal interactions seemed able to kill cell lines normally resistant to TRAIL therapy, he added, suggesting possible future drug combinations for tumors known to be resistant to TRAIL receptor antibody treatment. Although this screen involved only 400 siRNAs, GNF is gearing up for a much larger screen involving roughly 5,000 genes.

RNAi libraries have made such screening feasible for academic laboratories as well. "The libraries are publicly available at a reasonable price, both our library and Greg Hannon's library," said Bernards. Meanwhile, the Broad Institute at MIT is coordinating construction of yet another genome-scale RNAi library. "Our goal is to make large-scale RNAi screens efficient and widely accessible for the entire scientific community," wrote David Root, Ph.D., project leader of the Broad Institute's RNAi consortium, in a conference program brochure. (Root and institute communications director Scott Turner, however, declined requests for information, since the consortium is still "being organized," in Turner's words.)

Almost a decade after Hartwell and Friend first tried using synthetic lethality against cancer, the concept has come of age. "What's unique about cancer cells is their genetic instability," Hartwell said. "And we have not, despite the fact that we've known this for decades, really taken advantage of that fact." With the maturing of RNA interference, that long period of neglect seems to be ending.



             
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