Abstract 862: Sensitization of pancreatic cancer to chemoradiation by the Wee1 inhibitor AZD1775
Authors: Kausar Tasneem , Parsels Leslie A. , Parsels Joshua D. , Karnak David , Davis Mary A. , Maybaum Jonathan , Lawrence Theodore S. , Morgan Meredith A. 
Published: May 2015

Abstract
Gemcitabine-based chemoradiation is standard therapy for locally advanced pancreatic cancer and is associated with an approximately one year median survival. In order to improve the efficacy of this therapy, integration of agents which target the DNA damage response, such as inhibitors of Wee1, is a promising strategy. The goals of this study were to determine the efficacy of the Wee1 inhibitor AZD1775 in sensitizing pancreatic cancer cells to gemcitabine-radiation and to begin to identify predictive biomarkers of response. Sensitization was assessed by clonogenic survival in MiaPaCa-2 cells. Potential biomarkers, γH2AX and pChk1 S345, were analyzed by FACS, immunofluorescence and immunoblotting. In addition, we tested a novel biomarker platform which utilizes ex vivo-treated 200µm tumor slices to query biomarkers of DNA damage with the ultimate goal of being able to predict patient response from a single biopsy. We found that AZD1775 in combination with gemcitabine further increased radiosensitization in MiaPaCa-2 cells with an enhancement ratio (ER) of 2.1±0.2 compared to radiosensitization by gemcitabine alone (100nM; ER 1.3±0.2; P&lt;0.05). In order to begin to identify predictive biomarkers of chemoradiosensitization by AZD1775, we assessed γH2AX by FACS in MiaPaCa-2 cells and found that while gemcitabine/radiation resulted in γH2AX positivity, the addition of AZD1775 caused the appearance of a more intensely stained γH2AX population of cells. Upon immunoflourescent analysis of γH2AX we found that this more intensely stained γH2AX population was associated with pan-nuclear γH2AX versus the focal γH2AX observed in response to gemcitabine/radiation. In addition, we examined pChk1 S345 as a predictive biomarker and found that sensitization of MiaPaCa-2 cells to gemcitabine-radiation was associated with a concentration-dependent increase in pChk1 S345 protein levels. Thus, we went on to assess pChk1 S345 in ex vivo-treated slices of two independent patient-derived tumor xenografts. Following ex vivo treatment of tumor slices with AZD1775 and gemcitabine-radiation, we observed that AZD1775 induced pChk1 S345 in response to gemcitabine-radiation in one xenograft but not the other suggesting the possibility of differential sensitivity to AZD1775 treatment across patient-derived xenografts. Together, our results suggest that AZD1775 sensitizes pancreatic cancers to gemcitabine-radiation and in association with induction of pharmacodynamic biomarkers such as pChk1 S345 and γH2AX. These results support our approved clinical trial assessing the efficacy of AZD1775 with gemcitabine-radiation in locally advanced pancreatic cancer. Future studies will establish correlations between these biomarkers and therapeutic responses in vivo as well as investigate these biomarkers in primary pancreatic tumor specimens.
Citation Format: Tasneem Kausar, Leslie A. Parsels, Joshua D. Parsels, David Karnak, Mary A. Davis, Jonathan Maybaum, Theodore S. Lawrence, Meredith A. Morgan. Sensitization of pancreatic cancer to chemoradiation by the Wee1 inhibitor AZD1775. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 862. doi:10.1158/1538-7445.AM2014-862