PB1124 - Effects of 5-azacytidine on zebrafish coagulation

Background: DNA hypomethylation drug, 5-azacytidine has been shown to induce fetal hemoglobin expression in adults and affect the expression of genes related to hematopoiesis. It is also used in the treatment of cancers, particularly myelodysplastic syndromes, and acute myeloid leukemia. We hypothesized that this drug may affect blood clotting by causing changes in methylation patterns of their genes.

Aims: To explore the effects of 5-azacytidine on hemostasis

Methods: Laser thrombosis assays were performed in zebrafish larvae treated with 5-azacytidine and TTO was measured. Coagulation and gill bleeding assays were performed using adult zebrafish fed with 5-azacytidine. The effects of 5-azacytidine on clotting and transcription factor mRNA levels were analyzed by qRT-PCR. Transcription factors were knocked-down using piggyback method.

Results: Prolonged TTO in arterial and venous thrombosis assays on 5-azacytidine-treated larvae suggested this drug caused bleeding. kPT and kPTT assays showed that 5-azacytidine-treated fish plasma took longer time to clot. From qRT-PCR analysis, we found that f3, f5, f8, f9b, and f9l were downregulated and f7 and f9a were upregulated after 5-azacytidine treatment; however, f9a levels were highly upregulated. Therefore, we chose f9a for studying the effects of 5-azacytidine on the expression of hepatocyte transcription factors. We found bcl6, cebpα, hnf4α, prox1a, foxa1and foxa3 were significantly upregulated and nrf2 and foxo1 were downregulated. We then tested the effects of the knockdown of bcl6, cebpα, hnf4α, prox1a, foxa1, foxa3 genes on the f9a mRNA levels. We found that bcl6, cebpα and hnf4α knockdowns affected f9a mRNA levels.

Conclusion(s): 5-azacytidine appears to increase the risk of bleeding and also causes a tremendous increase of f9a mRNA level in zebrafish by direct hypomethylation of f9a promoter and also by increasing bcl6, cebpα, and hnf4α mRNA levels by hypomethylating their promoters. Methylation sequencing analysis of these promoters may provide additional insight into these complex regulatory mechanisms.