Pancreatic cancer has only a 6% 5-year survival rate. Gemcitabine is the standard treatment in conjunction with surgery to remove cancerous pancreatic tissue in the minority of the cases where the disease is caught soon enough. However, the gemcitabine response rate is less than 20%, and combinations with other targeted drugs have not improved this significantly so far.
To address this treatment problem, Dr. Michael Boettcher’s group at DKFZ used DECIPHER short-hairpin RNA (shRNA) libraries to run loss-of-function genetic screens on pancreatic cancer cells to identify genes that enable cells to resist gemcitabine-induced lethality. They screened the cells with both DECIPHER Library Modules 1 and 2 which target approximately 10,000 human genes in total. Each lentiviral-based library module consists of 27,500 shRNA expression constructs targeting approximately 5,000 human genes—each gene targeted by 5-6 hairpins.
To run the screens, cells were transduced with the libraries then treated with gemcitabine with the goal to identify genes that, when knocked down, made the cell more susceptible to gemcitabine. Interference with the function of these genes, then, would appear to be synthetically lethal with gemcitabine. Thus, these genes might be good potential targets for a combination treatment with the drug.
In the screening, the researchers identified about 70 genes with synthetic lethal effects in combination with gemcitabine. Highly represented in the hits were genes involved in DNA damage response and repair, which was expected since gemcitabine is a DNA damaging agent. They focused specifically on genes identified in the screen that were upstream of the checkpoint kinase 1 (CHK1) of the ATR/CHK1 pathway. In particular, RAD17, HUS1, WEE1, and RFC3 all turned up in the screen, and all but RCF3 were subsequently confirmed with 3 independent shRNA constructs.
The rest of the study focused on the RAD17 gene. The researchers demonstrated that knocking down this gene increased lethality of gemcitabine by forcing cells with damaged DNA to enter mitosis. A similar effect was shown previously for WEE1 by Aarts, et al., which was also identified in the screen. In fact, a potent inhibitor of WEE1 kinase (MK-1775) is scheduled in combination with gemcitabine and platinum-based drugs for phase I clinical studies.
The study is published in the Journal of Cell Science.