Synergistic Anti-tumor Interactions Between The Novel Atr Inhibitor AZ20 And Gemcitabine In Pancreatic Cancer Cells

Pancreatic cancer is a highly malignant disease with poor prognosis. Surgery remains the only chance of cure. However, less than 20% of diagnosed patients are suitable for surgery. Chemotherapy is the main treatment for patients with advanced disease. Gemcitabine has been the first-line chemotherapeutic drug for pancreatic cancer since 1997 thoughits clinical activity is very limited. Therefore, there is an urgent need for new drugs to enhance the anti-tumor activity of gemcitabine against this deadly disease.To maintain genomic integrity, eukaryotic cells evolve a crucial cellular network named the DNA damage response(DDR), which includes DNA replication,DNA repair, cell cycle checkpoints and other signaling pathways. The DDR plays critical roles in responding to exogenous and endogenous DNA damage stress. ATR(ataxia telangiectasia and Rad 3 related) plays a critical role in sensing DNA damage and is a key mediator of the DDR. To respond to DNA damage, ATR phosphorylates numerous downstream proteins including CHK1 to stop the cell cycle providing cells time to repair damaged DNA, and up-regulate the expression of ribonucleotide reductase(RNR) to supply sufficient d NTP for DNA repair. Notably, tumor cells harbor defects in the DDR more frequently promoting carcinogenesis and rendering them more sensitivity to ATR inhibition compared normal cells. Thus, ATR is an attractive therapeutic target for treating cancer. AZ20 is a potent ATR-selective inhibitor. However, the anti-tumor activity of AZ20 against pancreatic cancer has not been tested.In this study, we first determined the cytotoxicity of AZ20 in a panel of pancreatic cancer cell lines. The IC50 of AZ20 determined by MTT assays ranged from 0.8 to 4.1 μM in the pancreatic cancer cell lines. AZ20 decreased thephosphorylation of CHK1 at Ser345, phosphorylation of CDC25 C at Ser216, and phosphorylation of CDK1 at Tyr15, demonstrating that inhibition of ATR resulted in inactivation of CHK1 and CDC25 C and activation of CDK1. Interestingly, AZ20 treatment of pancreatic cancer cells caused arrest of the cell cycle at S and G2/M phases, rather than progression of the cell cycle. AZ20 treatment induced a small degree of cell death suggesting that the anti-tumor activity of AZ20 single drug treatment was majorly due to cell cycle arrest.Then, we tested the combined anti-tumor activity of AZ20 and gemcitabine in the pancreatic cancer cell lines. As expected, combination of AZ20 with gemcitabine indeed resulted in synergistic inhibition of cell growth in all of the pancreatic cancer cell lines. Further, AZ20 also enhanced gemciatbine-induced DNA damage and cell death. AZ20 decreased gemciatbine-induced cell cycle arrest and RNR expression which would impair the repair of damaged DNA. These results indicate that AZ20 enhances the anti-tumor activity of gemciatbine by inhibiting DNA damage repair,pushing cell cycle progression, and reducing cellular d NTP pools.Taken together, our data show that targeting ATR by using AZ20 can enhance the anti-tumor activity of gemcitabine through induction of DNA damage in pancreatic cancer cells. Accordingly, combination of AZ20 with gemciatbine might represent a potential chemotherapeutic regimen for treating pancreatic cancer.