Abstract 5627: Involvement of pyrimidine metabolism pathway in 5-azacytidine resistance.
Authors: Imanishi Satoshi , Umezu Tomohiro , Kobayashi Chiaki , Ohyashiki Kazuma , Ohyashiki Junko 
Published: August 2014

Background and aim

5-azacytidine (AZA) is a DNA demethylating agent that is clinically used for patients with myelodysplastic syndrome and acute myeloid leukemia. However, the mechanism of AZA-resistance remains unclear although many patients become resistant to AZA after short-term use. To investigate the mechanism of AZA resistance, we established an AZA-resistant cell line and analyzed its cellular and molecular biological characteristics.

Experimental procedures

To establish the AZA-resistant cell line, U937 cells were incubated in a gradually increasing dose of AZA from 0.1 μM to 3 μM. Surviving cells were named R-U937. The profiles of mRNA expression and DNA methylation in U937 and R-U937 cells were obtained and pathway analysis was performed to determine differences between U937 and R-U937. Experiments using specific inhibitors were also performed.

Results

Proliferation and drug resistance of R-U937

R-U937 cells showed a lower proliferation rate than their parent cells, but their viability was not affected by AZA or 2’-deoxy-AZA (DAC).

Role of pyrimidine metabolism pathway in AZA resistance

The pathway analysis of mRNA expression profiles revealed differences between U937 and R-U937 in terms of the mRNA expression level of 4 genes involved in the ‘Pyrimidine Metabolism Pathway’. The observation was confirmed by quantitative RT-PCR. R-U937 cells re-acquired sensitivity to AZA when they were incubated with an inhibitor of CTP synthase. These results indicated that adaptation in pyrimidine metabolism, particularly the accelerated synthesis of CTP from UTP, played important roles in the development of an AZA-resistant phenotype.

Role of DNA hypomethylation in AZA resistance

The DNA demethylation process induced by AZA involves inhibition of DNA methyltransferases (DNMTs) and DNA damage. It is not clear which process is the major mechanism of the anti-leukemia activity. To clarify this, we compared the effects of AZA, DAC and RG108, a DNMT inhibitor with no DNA damaging activity, on viability and DNA damage. AZA and DAC significantly decreased U937 cell viability, but not RG108. U937 incubated with AZA or DAC showed an increased ratio of p-H2AX-positive cells, indicating that these agents caused DNA damage. However, the ratio of p-H2AX-positive cells in U937 incubated with RG108 was equal to that of cells incubated without the agents. In R-U937, all agents did not increase the ratio of p-H2AX-positive cells. The DNA methylation profiles revealed global hypomethylation in R-U937. These results indicated that DNA damage but not DNMT inhibition played a major role in the anti-leukemia activity of AZA and DAC.

Conclusion

The present results showed that the development of an AZA-resistant phenotype of R-U937 depended on the adaptation in pyrimidine metabolism and global DNA hypomethylation. To overcome AZA resistance, pyrimidine metabolism, particularly CTP synthase, can be a good target.

Citation Format: Satoshi Imanishi, Tomohiro Umezu, Chiaki Kobayashi, Kazuma Ohyashiki, Junko Ohyashiki. Involvement of pyrimidine metabolism pathway in 5-azacytidine resistance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5627. doi:10.1158/1538-7445.AM2013-5627