Abstract 1770: Metabolism and mechanism of action of fluorocyclopentenylcytosine (RX-3117)
Authors: Peters Godefridus J. , Smid Kees , Vecchi Leonardo , Kathmann Ietje , Loosekoot Nienke , Lee Young B. , Kim Deog J. , Ahn Chang-Ho , Jeong Lak S. , Orbach Aric , Ohne Osnat , Blaugrund Eran 
Published: August 2012

Abstract
Cytidine analogs play an important role in the treatment of various types of cancer, both solid tumors and leukemia. A novel cytidine analog fluorocyclopentenylcytosine (RX-3117) was characterized for its cytotoxic effects, its metabolism and its mechanism of action in a panel of 9 solid tumor and leukemic cell lines, as well as 6 variants resistant to gemcitabine, cytarabine and other pyrimidine analogs. Sensitivity in the parent cell lines after 72 hr exposure varied 75 fold with IC50 values from 0.4 to 30 µM RX-3117. The human equilibrative nucleoside transporter mediates transport of RX-3117, since its inhibition protected cells. Uridine and cytidine also protected cells against RX-3117, indicating that activation of RX-3117 is dependent on phosphorylation catalyzed by uridine-cytidine kinase (UCK), which was abundant in all tested cell lines, including the gemcitabine resistant variants. Deoxycytidine did not protect cells against RX-3117. RX-3117 was a very poor substrate for cytidine deaminase (66,000-fold less than gemcitabine). After its uptake in cells, RX-3117 was rapidly metabolised to its nucleotides with the triphosphate being the most prominent form (90% of all nucleotides), while synthesis of the nucleotides was highest in the most sensitive cell lines (U937 and A2780 cells) and lowest in the least sensitive cells (CCRF CEM cells). No difference in nucleotide formation was observed between the SW1573 and its gemcitabine resistant variant SW1573/G. In the AG6000 cells, the dCK- variant of A2780 and resistant to gemcitabine and RX-3117, a normal monophosphate level was found, but no di-and triphosphates were formed, explaining its resistance. Similarly incorporation of RX-3117 into RNA and DNA was higher in the sensitive A2780 and low in the insensitive SW1573 cells, with no difference between the gemcitabine sensitive and resistant variants. The effect of RX-3117 on synthesis of RNA and DNA was quite different; in the sensitive U937 cells 10 µM RX-3117 inhibited RNA synthesis 90%, while in A2780 and CCRF-CEM cells 100 µM RX-3117 was required for 90% inhibition of RNA synthesis. The effect on DNA synthesis was quite different. 1 µM RX-3117 completely inhibited DNA synthesis in the sensitive U937 cells, 80-90% inhibition was achieved with 10 µM in both CCRF CEM variants and with 100 µM in SW1573/G and AG6000, but in A2780 and SW1573 cells 100 µM only resulted in a partial or no inhibition, respectively. In conclusion, RX-3117 showed a completely different sensitivity profile compared to other cytidine analogs. Its uptake is transporter dependent; it is not activated by dCK, but by UCK. RX-3117 is incorporated into RNA and DNA; RX-3117 hardly affected RNA synthesis at IC50 values, but inhibited DNA synthesis. Its metabolism to nucleotides is related with its sensitivity, possibly because they directly inhibit the target presumably DNA methyltransferase.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1770. doi:1538-7445.AM2012-1770