5-Azacytidine, a possible new inhibitor of ribonucleotide reductase (R2), and its combined biological effect with GTI 2040 in chronic and acute myeloid leukemia cells
Authors: Josephine Aimiuwu, Ping Chen, Ming Chiu, Zhiliang Xie, Zhongfa Liu, Shujun Liu, Guido Marcucci, Kenneth Chan
Journal: Cancer Research (2008)

AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA

2614 

Purpose: 5-Azacytidine appears to exert its anticancer activity not only through DNA incorporation after conversion to its deoxynucleotide, but also by direct incorporation into RNA, thereby leading to disruption of RNA enlongation. In order to mechanistically enhance the incorporation of 5-azacytidine into RNA, we combined 5-azacytidine with GTI 2040, an antisense to the R2 subunit of ribonucleotide reductase (RNR), which catalyzes conversion of ribonucleotides into deoxyribonucleotides. Methods: MTS cytotoxicity assay was used to determine the synergistic/additive cytotoxic effects of GTI 2040 and 5-azacytidine. GTI 2040 was introduced into cells by electroporation. Quantitative RT-PCR was used to measure the transcriptional level of the R2 subunit of RNR and DNA methyltransferase I (DNMT1); Western blot assay was used to evaluate the corresponding protein levels. In vivo studies were conducted by inoculating Nu/Nu mice with MV4-11 acute myeloid leukemia cells. Tumor size and weight of the xenografted implants were measured following treatments with the combination and compared with those of animals treated with the single drugs or vehicle alone. GTI 2040 levels were measured by a hybridization ELISA method; 5-azacytidine levels were quantified by a LC-MS/MS method. A non-radioactive, sensitive and specific LC-MS/MS method was used to quantify nucleotides (NTP) and deoxynucleotides (dNTP) pools before and after drug treatments. Results: 5-Azacytidine showed significant cytotoxicity with an IC50 of 7.5µM in K562 cells and GTI-2040 transfection displayed slight cytotoxicity at higher (>5μM) than achievable concentration in human studies. Combination of the two compounds, 0.5 μM GTI plus variable 5-azacytidine, showed a synergistic effect based on the combination index. Treatment with GTI 2040 induced down-regulates R2 RNR mRNA and RNR protein after 24 hrs exposure. At 24 hrs, 5-azacytidine depleted DMNT1 levels, but most surprisingly also down-regulated R2 RNR mRNA expression, which was further down-regulates at both mRNA and protein levels in cell treated with the combination. GTI 2040 also depleted the dNTP pool particularly dCTP with no change in the NTP pools, supporting functional inhibition of RNR. All drug treatment but control decreased tumor size and weight after 32 days with suggestion of higher effect for the combination. Conclusion: These studies demonstrate that GTI 2040 induced imbalance of endogenous NTP and dNTP pools as results of antisense activity on RNR. Unexpectedly, we showed that 5-azacytidine down-regulates R2 RNR mRNA but not the protein and this may relate to its RNA effect. The combination of low doses of GTI 2040 and 5-azacytidine seems to potentiate the antileukemic activity of the two compounds and will be explored in the clinic. Supported by NIH-UO1CA076576.