Identification and Mechanistic Analysis of Synergistic Anti-Cancer Drug Interactions by Combinatorial Screening and Analytical Approaches.

摘要

Resistance to single agent targeted therapies may be due to the activity of alternate signaling pathways that functionally compensate for the inhibited pathway. We hypothesized that the simultaneous inhibition of compensatory pathways would result in synergistic growth inhibition. In order to identify compensatory signaling pathways, we designed and executed a small molecule inhibitor combination screen and looked for drug combinations that caused synergistic cytotoxicity. Using this method, we identified 192 such combinations. We then performed a series of follow-up analyses designed to confirm the drug targets that were necessary and sufficient for synergy, determine the biological outcomes of combination treatment, and elucidate the functional relationship between the signaling pathways. The combination of an epidermal growth factor receptor (EGFR) inhibitor and a phosphoinositol-3-kinase (PI3K) inhibitor was studied as a proof of principle. We found that these two signaling pathways converge on the p70 ribosomal protein S6 kinase (p70S6K), and that this protein regulates survival and proliferative signaling coming from these pathways in the UMUC-6 bladder cancer cell line. We also investigated the combination of an EGFR inhibitor and a p70S6K inhibitor in the KU-7 bladder cancer cell line. The synergistic cytotoxicity caused by this combination may be due to the activation of the pro-apoptotic protein BAD in response to the combination treatment. Lastly, we attempted to elucidate the mechanism underlying the synergistic cytotoxicity caused by the combination of nordihydroguareacetic acid (NDGA) and Trichostatin A (TSA). Although this combination produced robust synergy in a number of cell lines, we were unable to identify the relevant targets of these drugs using a drug substitution method, and were therefore unable to discern the mechanism of synergy. We concluded that using combinatorial drug screening is a valid method for identifying compensatory pathways, but that drug specificity limits the generality of this approach. In addition, we found that the role of p70S6K in the cell signaling network can vary in different cellular contexts. It is likely that many other proteins will likewise have context-dependent roles in cell signaling networks, underscoring the need to develop a thorough knowledge of the signaling network in order to rationally design effective treatment strategies.