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

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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13045-015-0143-3) contains supplementary material, which is available to authorized users.