Session Item

Pancreatic ductal adenocarcinoma (PDAC) is radioresistant histology, and inhibition of cyclin-dependent kinase 1 (CDK1) in combination with radiation therapy (RT) is a potentially attractive strategy to overcome radioresistance.

Standard clonogenic assays, γH2aX foci staining, HR-GFP reporter assay, and western blot analysis of DNA damage response proteins were performed in MIA-PaCa2 (MP2) and PANC-1 cells following drug targeting of CDK1 through the use of a selective CDK1 inhibitor, RO-3306. Knockdown of CDK1 was achieved in PDAC cells using a doxycycline Tet-On 3G promoter of the CDK1 shRNA gene. One million PDAC cells with the inducible sh-CDK1 were implanted orthotopically in the pancreas of athymic nude mice along with 50 uL BioXmark liquid fiducial adjacent to the injection. After one week of tumor formation, tumors with Luciferase expression were treated with imaged-guided radiation therapy (IGRT) on a Xstrahl small animal radiation research platform (SARRP) to a dose of 20 Gy in 5 daily fractions. Tumor growth was measured via bioluminescence on IVIS Lumina every 2 weeks.

We found CDK1 inhibition therapy combined with RT lead to greater cell death than either treatment alone.  Our results show that treatment of PDAC cells in vitro with a selective CDK1 inhibitor, RO-3306, reduced activated CDK1 and results in a 2-fold increase of DNA damage following 4 Gy of radiation via measurement of gamma H2AX nuclear foci. Furthermore, we found that RO-3306 (10 μM) treatment concurrent with radiation resulted in decreased clonogenic survival with increasing radiation dose. DNA repair markers and HR repair efficacy were both reduced following CDK1 inhibition. Mice with orthotopic inducible shCDK1 PDAC tumors treated with doxycycline 48 hours prior to IGRT showed significantly longer tumor growth delay following in vivo IGRT compared to the CDK1 control tumors (2.5-fold increase, p<0.001). After the last dose of RT, expression of the DNA damage marker yH2aX in tumors was significantly higher in the CDK1 knockdown tumors compared to controls indicating reduced DNA repair in tumors deficient in CDK1.

Our analysis indicates a novel role of CDK1 targeting to overcome PDAC radioresistance. Targeting CDK1 prior to RT is a promising strategy to overcome the radioresistance of PDAC.