Abstract 2936: Screen for kinase inhibitors that sensitize pancreatic cancer cells to gemcitabine: When collateral damage is a good thing

Abstract

Abstract Kinase inhibitors are a growing class of therapeutically active agents that are being used in the clinic. An interesting notion is that inhibitors which show clinical efficacy likely do so by inhibiting not only their intended target but also off-target kinases. Thus, pleiotropic off-target effects of kinase inhibitors may be advantageous in preventing tumor progression. Using a panel of 160 kinase inhibitors that had been previously tested against a panel of 300 recombinant human kinases, we sought to identify kinases that sensitize PANC1 cells to a sub-lethal dose of gemcitabine. PANC1 cells were treated with gemcitabine (20nM) for 24h and then treated with increasing concentrations of kinase inhibitor before cell viability was measured 24h later. From amongst the 160 compounds, we identified 8 compounds that enhanced killing of gemcitabine-treated cells. Validation of gemcitabine sensitizers was confirmed by clonogenic assays. To assess mechanisms for sensitization, we determined the fates of cells using live cell video microscopy. We found that 4/8 compounds abrogated the DNA damage checkpoint induced by gemcitabine, and forced the cells to prematurely enter mitosis and die. Indeed, of the kinases inhibited by these compounds, Chk1 was over-represented above all others. We next queried the STRING database to elucidate pertinent protein-protein interactions of those kinases inhibited by the 8 hit compounds. We identified a signaling node centering on Src and related kinases that were selectively inhibited by Aminopurvalanol A (AMP-A) and Syk inhibitor. The importance of Src and its related kinases was confirmed when MEFs lacking Src, Yes and Fyn were found to be highly sensitive to gemcitabine compared to WT. From our in vitro data, the Syk inhibitor inhibited Src family kinases as expected, but also inhibited Chk1 (by >80%). In cells, the Syk inhibitor behaved more like a Chk1 inhibitor as it caused gemcitabine treated cells to aberrantly enter mitosis and then die. Thus, it is likely that the off-target inhibition of Chk1 is responsible for the gemcitabine sensitization. AMP-A inhibited not only cyclin dependent kinases but also the Src family kinases. By movie analysis we observed that AMP-A caused gemcitabine-treated cells to die in interphase. Which one of these family of kinases is responsible for sensitization is currently being investigated. But the results show AMP-A and the Syk inhibitor sensitize cells to killing through distinct mechanisms. Taken together, these findings demonstrate that unintended targets of kinase inhibitors may in fact play a greater role in achieving the desired biological effects of the drug. Identifying the collateral targets in vivo may reveal mechanisms of action of the kinase inhibitors that affects selection of biomarkers for drug response. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2936. doi:1538-7445.AM2012-2936