Abstract 672: Targeting metabolic pathways to radiosensitize pancreatic cancer
Authors: Seshacharyulu Parthasarathy , Nimmakayala Ramakrishna , Rachagani Satyanarayana , Wang Shuo , Li Sicong , Talmon Geoffrey , Kaur Sukhwinder , Ponnusamy Moorthy P. , Jain Maneesh , Lin Chi , Batra Surinder K. 
Published: August 2018

Abstract
Background: Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer related mortality killing 117 PC patients every day in the United States. The efficacy of systemic chemotherapy and radiotherapy (RT) is tempered by the acquired and innate therapeutic resistance resulting in limited survival benefits to PDAC patients. Our bioinformatics and high throughput database analysis revealed that cholesterol biosynthesis (CBS) pathway is involved in radioresistance (RR) of PC patients. In addition, our previous studies suggested the involvement of FDPS, a CBS pathway enzyme, in imparting radioresistance to PC cells. Here, we tested the utility of Zoledronic acid (ZOL), a specific inhibitor of FDPS, in radiosensitization (RS) of PC cells in vitro and in vivo and investigated the underlying RS mechanisms. Material and Methods: RR PC cell lines (CD18/HPAF and Capan-1) were developed using fractionated irradiation (a total of 20 Gray) and validated using functional assays. RT-PCR and western blotting analysis were performed to determine the expression of CBS pathway associated transcripts and proteins. Stable CRISPR-Cas9 mediated knockout of FDPS in PC cells was developed to examine its role in PC radio sensitivity. Feasibility of pharmacologically inhibiting FDPS in in vitro and in vivo was tested using zoledronic acid (ZOL). Effect of ZOL on FDPS activity was studied by measuring intracellular cholesterol and by observing its inhibition on protein prenylation. Radiosenstization effects of ZOL on PC cells was analyzed using in vitro (FACS, cell cycle and clonogenic survival assay) and in vivo (orthotopic model) assays. Histological and immunohistochemistry analysis were performed to determine the levels of DNA damage markers on PC cells and pancreatic xenograft tissues. Results: Persistent exposure of PC cells to RT (20 Gy) resulted in altered cellular morphology, G2/M arrest and enriched side population accompanied by increased levels of FDPS and cell cycle regulatory proteins. Knockout of FDPS in PC cells enhanced radio-sensitivity. Pharmacological inhibition of FDPS in conjunction with radiation resulted in reduced number of colonies, increased apoptosis and abrogated radiation induced G2/M arrest. ZOL pre-treatment resulted in inhibition of prenylation of CDC42 and RAC1 GTPases and decreased accumulation of intracellular cholesterol. The combined therapy of ZOL and radiation resulted in significant reduction in tumor growth and metastasis as compared to ZOL and radiation alone treatment. Finally, we observed marked reduction in DNA repair proteins such as RAD50 and phosphorylation of P95 in cell lines knockout for FDPS/treated with ZOL and/or RT and xenograft tissues excised from single and combination treatment regimens. Conclusion: FDPS inhibition radiosensitizes PC cells by inducing G2/M cell cycle arrest and by abrogating radiation induced DNA repair mechanism thereby resulting in enhanced apoptosis.
Citation Format: Parthasarathy Seshacharyulu, Ramakrishna Nimmakayala, Satyanarayana Rachagani, Shuo Wang, Sicong Li, Geoffrey Talmon, Sukhwinder Kaur, Moorthy P. Ponnusamy, Maneesh Jain, Chi Lin, Surinder K. Batra. Targeting metabolic pathways to radiosensitize pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 672.