Abstract 4725: Inhibition of DNA methyltransferase by RX-3117 (fluorocyclopentenylcytosine) leads to upregulation of hypomethylated targets
Authors: Peters Godefridus J. , Sarkisjan Dzjemma , Julsing Joris J. , Hassan Ahmed , Smid Kees , Kathmann Ietje , Lee Young , Kim Deog J. 
Published: July 2016

RX-3117 (Fluorocyclopentenylcytosine; FCPEC) is an orally bioavailable novel cytidine analog which is currently being evaluated in a Phase I clinical study. Downregulation of DNA methyltransferase 1 (DNMT1) by RX-3117 was shown in various cell lines with different histological backgrounds. In the present study we determined the effect of RX-3117 on DNMT1 at the DNA, RNA, protein and enzyme activity, and reactivation of suppressed target genes, including p16INK4A, methylguanine methyltransferase (MGMT) and the proton coupled folate transporter (PCFT).

In the moderately sensitive non-small cell lung cancer (NSCLC) cell lines such as A549 and SW1573, 5-50 μM RX-3117 downregulated DNMT1 protein expression by 5-20% after 24 hr exposure and &gt;90% after 48 hr. DNMT1 mRNA was not affected after 24 hr exposure but was affected moderately after 48 hr. In the sensitive ovarian cancer cell line A2780, protein down regulation was already observed after 24 hr at 1 μM RX-3117. DNMT1 activity was inhibited by 1 μM RX-3117 by 32% which was similar to the percent inhibition with 5 μM of the reference compound 5-aza-2’-deoxycytidine (DAC, 31%). In A549 cells 5 μM RX-3117 decreased overall methylation of DNA (detected by an antibody against 5-methylcytosine) by 25% after 48 hr exposure, while 5 μM DAC only inhibited 9%. For several genes known to be affected by methylation, we evaluated their protein expression and activity. In A549 and SW1573 cells a 24 hr exposure to 5 μM RX-3117 increased the expression of the cell cycle protein p16INK4A and of the DNA repair enzyme MGMT.

For PCFT we evaluated its functional activity in CCRF-CEM leukemic cells which have a highly methylated PCFT promoter and in CEM-MTX cells which are deficient for the reduced folate carrier (RFC). PCFT is a specific folate transporter responsible for uptake of folic acid and the folate analogs methotrexate (MTX) and pemetrexed. Incubation of both CEM and CEM-MTX cells with either 29.6 μM RX-3117 or DAC as a positive control markedly increased PCFT mediated transport of MTX. This was more pronounced in CEM-MTX cells, 10-11-fold increase for both RX-3117 and DAC, compared to a 4-fold increase in CEM cells.

In conclusion, the down-regulation of DNMT1 by RX-3117 was accompanied by a decrease in DNMT1 protein expression and enzyme activity, resulting in up-regulation of proteins of MGMT, PCFT, and the tumor suppressor gene p16INK4A. These data underline DNMT1 inhibition as a novel mechanism of RX-3117.

Citation Format: Godefridus J. Peters, Dzjemma Sarkisjan, Joris J. Julsing, Ahmed Hassan, Kees Smid, Ietje Kathmann, Young Lee, Deog J. Kim. Inhibition of DNA methyltransferase by RX-3117 (fluorocyclopentenylcytosine) leads to upregulation of hypomethylated targets. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4725.