Abstract 3413: Dissecting the combination of gemcitabine and the Chk1 inhibitor MK-8776 in vitro and in vivo: cell cycle perturbation and the impact of administration schedule.
Authors: Eastman Alan , Montano Ryan , Thompson Ruth , Khan Nadeem , Hou Huagang , Lewis Lionel D. , Seigne John D. , Ernstoff Marc S. 
Published: August 2014

Abstract
Gemcitabine inhibits ribonucleotide reductase, prevents synthesis of deoxyribonucleotides, and induces arrest of cells in S phase. Subsequent inhibition of Chk1 causes the stalled replication forks to collapse, thereby increasing cell death. We have observed up to 10-fold decrease in the growth-inhibitory concentration (IC50) of gemcitabine upon incubation with the Chk1 inhibitor MK-8776 across multiple cell lines. In contrast to most in vitro studies, we have briefly incubated cells with gemcitabine to more closely reflect the exposure to drug that occurs in cancer patients. This permits analysis of the recovery from S phase arrest as would occur clinically. Following treatment, the rate of recovery depends on the concentration of gemcitabine, with S phase arrest lasting for 24 h at low concentrations and 48 - 72 h at higher concentrations. MK-8776 (2 μM) was then added for 6 h periods reflecting the approximate exposure that occurs in human plasma at its maximum tolerated dose. The maximum decrease in IC50 occurred when MK-8776 was added 18-24 h after gemcitabine. In addition to there being more cells in S phase at this time, we observed that replication forks also evolve over time to become more Chk1 dependent. Homologous recombination of DNA as assessed by the formation of RAD51 foci does not occur at the time cells initially arrest, but begins to occur 8 - 16 h later. Inhibition of Chk1 prevents RAD51 foci formation leading to replication fork collapse. Hence there are two interacting mechanisms that contribute to the optimum schedule of this drug combination: gemcitabine-mediated accumulation of cells in S phase, and Chk1-mediated stabilization of replication forks.
To assess cell cycle perturbation in vivo, tumors were stained for Ki67 which detects cells at all phases of the cell cycle except Go, and geminin which stains cells in S and G2. Results were expressed as the ratio of geminin:Ki67; i.e., the ratio of cells in cycle that are in S/G2. In untreated AsPc-1 human pancreas tumor xenografts, about 25% of the cells were in S/G2, while administration of 150 mg/kg gemcitabine (equates to 450 mg/m2 in humans) showed 80-90% of the cells in S/G2 24 h after drug administration. When 50 mg/kg MK-8776 was administered 18 h after 150 mg/kg gemcitabine, there was greater tumor growth delay than concurrent administration of the drugs.
We are currently conducting a clinical trial in patients with bladder cancer to assess the cell cycle perturbation that occurs following administration of standard dose of 1000 mg/m2 gemcitabine. Initial results show a significant S/G2 arrest 24 h after drug administration. These results emphasize the importance of assessing cell cycle perturbation in patient tumors, and provide justification for a clinical trial of gemcitabine followed 1 day later by MK-8776.
Citation Format: Alan Eastman, Ryan Montano, Ruth Thompson, Nadeem Khan, Huagang Hou, Lionel D. Lewis, John D. Seigne, Marc S. Ernstoff. Dissecting the combination of gemcitabine and the Chk1 inhibitor MK-8776 in vitro and in vivo: cell cycle perturbation and the impact of administration schedule. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3413. doi:10.1158/1538-7445.AM2013-3413