Azacytidine sensitizes acute myeloid leukemia cells to arsenic trioxide by up-regulating the arsenic transporter aquaglyceroporin 9

Abstract

Abstract Background: The therapeutic efficacy of arsenic trioxide (As 2 O 3) in acute myeloid leukemia (AML) is modest, which is partly related to its limited intracellular uptake into the leukemic cells. As 2 O 3 enters cells via the transmembrane protein aquaglyceroporin 9 (AQP9). Azacytidine, a demethylating agent that is approved for the treatment of AML, has been shown to have synergistic effect with As 2 O 3. We tested the hypothesis that azacytidine might up-regulate AQP9 and enhances As 2 O 3 -mediated cytotoxicity in AML. Methods: Arsenic-induced cytotoxicity, the expression of AQP9, and the intracellular uptake of As 2 O 3 were determined in AML cell lines and primary AML cells with or without azacytidine pre-treatment. The mechanism of AQP9 up-regulation was then investigated by examining the expression of transcription factors for AQP9 gene and the methylation status of their gene promoters. Results: As 2 O 3 -induced cytotoxicity in AML cell lines was significantly enhanced after azacytidine pre-treatment as a result of AQP9 up-regulation, leading to increased arsenic uptake and hence intracellular concentration. Blocking AQP9-mediated As 2 O 3 uptake with mercury chloride abrogated the sensitization effect of azacytidine. AQP9 promoter does not contain CpG islands. Instead, azacytidine pre-treatment led to increased expression of HNF1A, a transcription activator of AQP9, through demethylation of HNF1A promoter. HNF1 knockdown abrogated azacytidine-induced AQP9 up-regulation and almost completely blocked intracellular As 2 O 3 entry, confirming that azacytidine enhanced As 2 O 3 -mediated cell death via up-regulation of HNF1A and hence increased AQP9 and As 2 O 3 intracellular concentration. Azacytidine sensitization to As 2 O 3 treatment was re-capitulated also in primary AML samples. Finally, azacytidine did not enhance arsenic toxicity in a liver cell line, where HNF1A was largely unmethylated. Conclusions: Azacytidine sensitizes AML cells to As 2 O 3 treatment, and our results provide proof-of-principle evidence that pharmacological up-regulation of AQP9 potentially expands the therapeutic spectrum of As 2 O 3. Further clinical trial should evaluate the efficacy of azacytidine in combination with As 2 O 3 in the treatment of AML.