Abstract 2622: Fluorocyclopentenylcytosine (RX-3117) is activated by uridine-cytidine kinase 2, a potential biomarker
Authors: Peters Godefridus J. , Julsing Joris R. , Smid Kees , De Klerk Daniel , Sarkisjan Dzjemma , Yang Mi Y. , Lee Young B. , Kim Deog J. 
Published: August 2015

Fluorocyclopentenylcytosine (RX-3117) is an orally bioavailable novel cytidine analog, currently being tested in a Phase I clinical trial. RX-3117 shows promising antitumor activity in various human tumor xenografts including patient derived xenografts resistant to gemcitabine. Initial characterization of RX-3117 indicates that this compound is incorporated into both RNA and DNA, and downregulates DNA methyltransferase I (DNMT1). RX-3117 is not deaminated by cytidine deaminase, an enzyme that limits the efficacy of most cytidine analogs due to extensive deamination. Our studies also demonstrate that RX-3117 is taken up by the equilibrative nucleoside transporter and is activated by uridine-cytidine kinase (UCK), since co-administration of uridine or cytidine protects cells against cytotoxicity of RX-3117, and inhibits RX-3117 phosphorylation in enzyme activity assays. Since UCK exists in two forms, UCK1 and UCK2, we investigated which form is responsible for RX-3117 phosphorylation by transfecting two non-small cell lung cancer cell lines, A549 and SW1573 with siRNA directed against either UCK1 or UCK2 to determine whether downregulation would protect cells against RX-3117. UCK1-siRNA and UCK2-siRNA efficiently downregulated the expression of UCK1 and UCK2, respectively, in both cell lines, up to at least 72 hr after transfection. Exposure of UCK1-siRNA transfected cells to IC50 concentrations of RX-3117 did not affect the sensitivity of either A549 or SW1573 cells; however, transfection of UCK2-siRNA completely protected both A549 and SW1573 from the cytotoxic effects of RX-3117. In order to further investigate the role of UCK2, we tested the sensitivity of transfected cells to ethynylcytidine (ETC), a known substrate for UCK2. UCK2-siRNA transfection protected cells against ETC, but transfection with UCK1-siRNA did not. In order to explore whether we can use UCK2 expression in tumors as a predictive biomarker, we measured UCK enzyme activity, using radioactively labelled RX-3117 and the natural substrate uridine in 6 tumor cell lines to determine whether the enzyme activity correlates with UCK1 or UCK2 mRNA expression. UCK2 mRNA expression was significantly (p&lt;0.001) correlated with enzyme activity measured with both uridine (r = 0.828) and RX-3117 (r = 0.803) but not for UCK1 (r = -0.721 and -0.585, respectively). Phosphorylation of RX-3117 in intact cells was significantly correlated with UCK2 expression (r = 0.936), but not with UCK1 (R = -0.620). In conclusion, RX-3117 is activated by UCK2 which may be used to select patients potentially sensitive to RX-3117.

Citation Format: Godefridus J. Peters, Joris R. Julsing, Kees Smid, Daniel De Klerk, Dzjemma Sarkisjan, Mi Y. Yang, Young B. Lee, Deog J. Kim. Fluorocyclopentenylcytosine (RX-3117) is activated by uridine-cytidine kinase 2, a potential biomarker. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2622. doi:10.1158/1538-7445.AM2015-2622