An Evolutionarily Conserved Synthetic Lethal Interaction Network Identifies FEN1 as a Broad-Spectrum Target for Anticancer Therapeutic Development

摘要

Harnessing genetic differences between cancerous and noncancerous cells offers a strategy for the development of new therapies. Extrapolating from yeast genetic interaction data, we used cultured human cells and siRNA to construct and evaluate a synthetic lethal interaction network comprised of chromosome instability (CIN) genes that are frequently mutated in colorectal cancer. A small number of genes in this network were found to have synthetic lethal interactions with a large number of cancer CIN genes; these genes are thus attractive targets for anticancer therapeutic development. The protein product of one highly connected gene, the flap endonuclease FEN1 , was used as a target for small-molecule inhibitor screening using a newly developed fluorescence-based assay for enzyme activity. Thirteen initial hits identified through in vitro biochemical screening were tested in cells, and it was found that two compounds could selectively inhibit the proliferation of cultured cancer cells carrying inactivating mutations in CDC4 , a gene frequently mutated in a variety of cancers. Inhibition of flap endonuclease activity was also found to recapitulate a genetic interaction between FEN1 and MRE11A , another gene frequently mutated in colorectal cancers, and to lead to increased endogenous DNA damage. These chemical-genetic interactions in mammalian cells validate evolutionarily conserved synthetic lethal interactions and demonstrate that a cross-species candidate gene approach is successful in identifying small-molecule inhibitors that prove effective in a cell-based cancer model. Author Summary Anticancer therapeutic discovery is a major challenge in cancer research. Because cancer is a disease caused by somatic genetic mutations, the search for anticancer therapeutics is often driven by the ability to exploit genetic differences specific to tumor cells. Recently, cancer therapeutic development has sought to exploit synthetic lethality, a situation in which the combination of two independently viable mutations results in lethality. If a compound can be found to selectively kill a specific genotype via inhibition of a specific gene product, this is known as a chemical-genetic interaction, and it mimics a synthetic lethal genetic interaction. The ideal therapeutic would be broad spectrum, that is, active against multiple cancer genotypes within a tumor type and/or across a variety of cancers. We have developed an approach, taking advantage of the evolutionary conservation of synthetic lethal interactions, to identify “second-site” targets in cancer: genes whose chemical inhibition leads to selective killing of tumor cells across a broad spectrum of cancer genotypes. We identified small-molecule inhibitors of one such target, FEN1, and showed that these compounds were able to selectively kill human cells carrying cancer-relevant mutations. This approach will facilitate the development of anticancer therapeutics active against a variety of cancer genotypes.