Combining gemcitabine with checkpoint kinase inhibitors to sensitize pancreatic tumors

Abstract

Pancreatic tumor is one of the leading causes of cancer-related deaths in the world. Currently, the nucleoside analogue gemcitabine is the leading therapeutic drug for the treatment of pancreatic tumors. However, due to an ever-increasing number of patients developing gemcitabine resistance, there is a renewed interest in developing more efficient treatment regimes. Combination therapy that utilizes gemcitabine with other chemotherapeutic drugs or biological agents has the potential to overcome issues with traditional gemcitabine therapy. Gemcitabine acts by inducing replicative stress and consequently, cell cycle checkpoint kinases are activated. Tumor cells have more efficient checkpoint control, which could ultimately cause resistance towards gemcitabine. Therefore, inhibitors against checkpoint kinases are attractive candidates for tumor treatment in combination with gemcitabine. In this study, we have evaluated the sensitization of several pancreatic tumor cell lines (Panc1, MiaPaCa2 and BxPC3) towards gemcitabine upon inhibition of Chk1, Wee1 and ATR checkpoint kinases. We find that inhibition of these checkpoint kinases with specific chemical inhibitors sensitize pancreatic tumor cells against gemcitabine. Of these, the combination of Wee1 inhibitor, MK-1775 with gemcitabine shows high efficiency in decreasing the long-term survivability of cells and elimination of pancreatic tumor cells. Through western blot analysis, we find that Wee1 inhibition along with gemcitabine treatment causes inactivation of the ATR signaling pathway. We show that apoptosis and mitotic catastrophe do not cause the reduction in ATR-Chk1 activity. Interestingly, the attenuation of ATR-Chk1 pathway can be rescued by simultaneous inhibition of Cdks. Surprisingly, we find that simultaneous inhibition of Plk1 along with Wee1 inhibition and gemcitabine treatment can also recover the decreased ATR-Chk1 activity. We observe that activation of Plk1 upon Wee1 inhibition along with gemcitabine is dependent on Cdks. Moreover, we also show that Plk1 mediates inactivation of Chk1 through Claspin degradation. In order to reduce the toxic effects of the combined treatment of Wee1 inhibitor with gemcitabine in normal proliferating cells with wild-type p53, we tested Mdm2 antagonist, nutlin-3 pretreatment. We find that indeed nutlin-3 pretreatment can decrease the DNA damage response, apoptosis as well as the cells entering into mitosis prematurely caused by Wee1 inhibition with gemcitabine. As expected, this virtue of nutlin-3 pretreatment is dependent on p53 status of the cells. In conclusion, our study shows that the efficiency of Wee1 inhibition and gemcitabine treatment is not solely dependent on cell cycle dysregulation but also on the replicative stress. Since most of the pancreatic tumors have mutated form of p53, we propose that pretreatment with Mdm2 antagonists at sub-lethal dose can provide protection to fast proliferating cells with wild-type p53 against toxic effects of combination of Wee1 inhibition and gemcitabine treatment.