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Direct binding of arsenic trioxide to AMPK and generation of inhibitory effects on acute myeloid leukemia precursors.

ABSTRACT: Arsenic trioxide (As2O3) exhibits potent antineoplastic effects and is used extensively in clinical oncology for the treatment of a subset of patients with acute myeloid leukemia (AML). Although As2O3 is known to regulate activation of several signaling cascades, the key events, accounting for its antileukemic properties, remain to be defined. We provide evidence that arsenic can directly bind to cysteine 299 in AMPK? and inhibit its activity. This inhibition of AMPK by arsenic is required in part for its cytotoxic effects on primitive leukemic progenitors from patients with AML, while concomitant treatment with an AMPK activator antagonizes in vivo the arsenic-induced antileukemic effects in a xenograft AML mouse model. A consequence of AMPK inhibition is activation of the mTOR pathway as a negative regulatory feedback loop. However, when AMPK expression is lost, arsenic-dependent activation of the kinase RSK downstream of MAPK activity compensates the generation of regulatory feedback signals through phosphorylation of downstream mTOR targets. Thus, therapeutic regimens with As2O3 will need to include inhibitors of both the mTOR and RSK pathways in combination to prevent engagement of negative feedback loops and maximize antineoplastic responses.

SUBMITTER: Beauchamp EM

PROVIDER: S-EPMC4297272 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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Direct binding of arsenic trioxide to AMPK and generation of inhibitory effects on acute myeloid leukemia precursors.

Beauchamp Elspeth M EM Kosciuczuk Ewa M EM Serrano Ruth R Nanavati Dhaval D Swindell Elden P EP Viollet Benoit B O'Halloran Thomas V TV Altman Jessica K JK Platanias Leonidas C LC

Molecular cancer therapeutics 20141024 1

Arsenic trioxide (As2O3) exhibits potent antineoplastic effects and is used extensively in clinical oncology for the treatment of a subset of patients with acute myeloid leukemia (AML). Although As2O3 is known to regulate activation of several signaling cascades, the key events, accounting for its antileukemic properties, remain to be defined. We provide evidence that arsenic can directly bind to cysteine 299 in AMPKα and inhibit its activity. This inhibition of AMPK by arsenic is required in pa ...[more]

PMID: 25344585

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Project description:BackgroundThe therapeutic efficacy of arsenic trioxide (As2O3) in acute myeloid leukemia (AML) is modest, which is partly related to its limited intracellular uptake into the leukemic cells. As2O3 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 As2O3. We tested the hypothesis that azacytidine might up-regulate AQP9 and enhances As2O3-mediated cytotoxicity in AML.MethodsArsenic-induced cytotoxicity, the expression of AQP9, and the intracellular uptake of As2O3 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.ResultsAs2O3-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 As2O3 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 As2O3 entry, confirming that azacytidine enhanced As2O3-mediated cell death via up-regulation of HNF1A and hence increased AQP9 and As2O3 intracellular concentration. Azacytidine sensitization to As2O3 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.ConclusionsAzacytidine sensitizes AML cells to As2O3 treatment, and our results provide proof-of-principle evidence that pharmacological up-regulation of AQP9 potentially expands the therapeutic spectrum of As2O3. Further clinical trial should evaluate the efficacy of azacytidine in combination with As2O3 in the treatment of AML.

Project description:BACKGROUND:At present, it is generally believed that leukemia stem cells are the source of AML, so the killing of leukemia stem cells has become important. Previous studies have suggested that HHT combined with ATO can synergistically kill U937 cells, and HHT has also demonstrated the ability to kill leukemia stem cells. We evaluated whether HHT combined with ATO can systematically kill leukemia stem cells (LSCs) and explored the synergistic effect and molecular mechanism. METHODS:CCK-8 was used to detect cell viability. The changes of cell cycle (PI staining), apoptosis (Annexin V/PI) and surface markers (CD34, CD38, CD96, CD45) were detected by flow cytometry. The cells of CD34+ primary leukemia and CD38- KG-1, and TF-1 were separated by flow cytometry. High-throughput mRNA sequencing was used to analysis mRNA level changes after the application of the two drugs. Western blot was used to verify the changes of pathway protein expression. NRG mice were used as the receptor of xenograft model. Histological H&E staining assess the invaded ability of leukemia cells, and laser scanning confocal microscopy evaluated the molecule markers change. RESULTS:HHT and ATO synergistically killed KG-1 (CD34+/CD96+/CD38+/-) and Kasumi-1 (CD34+/CD38-) cells. Their combination had a stronger effect of inducing apoptosis and blocking the cell cycle than HHT or ATO administrator alone, meanwhile significantly reducing the numbers of LSCs. Further, CD34+CD38- cells in KG-1, KG-1a, TF-1, and primary leukemia cells were more sensitive to HHT and ATO. High-throughput mRNA sequencing suggested that HHT alone could significantly upregulate molecules related to the Notch, P53, and NF-?B signaling pathways. When combined with ATO, HHT further upregulated P53, whereas HHT-induced NF-?B pathway activation was significantly suppressed. Western blot analysis verified the change of protein expression in the above pathways and further demonstrated that GSI, could eliminate these effects. In vivo, HHT combined with ATO significantly reduced the LSC burden, and weakened the expression of LSC markers. CONCLUSIONS:This is the first evidence that HHT combined with arsenic can synergistically kill LSCs in vitro and in vivo, along with identification of the underlying mechanism, highlighting a potentially effective treatment strategy.

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Direct binding of arsenic trioxide to AMPK and generation of inhibitory effects on acute myeloid leukemia precursors.

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Direct binding of arsenic trioxide to AMPK and generation of inhibitory effects on acute myeloid leukemia precursors.

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