Potential therapeutic application of arsenic trioxide in acute myeloid leukaemia

Abstract

Acute myeloid leukaemia (AML) is characterized by the rapid proliferation of undifferentiated myeloid progenitors. Being a subtype of AML, acute promyelocytic leukaemia (APL) is currently the most curable type of leukaemia. Arsenic trioxide (As2O3) is an effective agent for the management of APL. Previous work from our laboratory has described the importance of aquaglyceroporin 9 (AQP9) in regulating arsenic trafficking and thereby governing arsenic sensitivity of leukaemic cells. Up-regulation of AQP9 expression is therefore considered as a logical means to broaden the potentiation of As2O3 in treating other malignancies. In this Ph.D. project, I hypothesized that demethylating agent, azacytidine (5′Aza), up-regulates endogenous AQP9 expression, resulting in a significant enhancement of arsenic uptake as well as As2O3-induced cytotoxicity. Pre-treatment of leukaemia cells with 5′Aza led to up-regulation of AQP9 expression and a significant increase in As2O3-induced cytotoxicity. However, such 5′Aza-induced AQP9 up-regulation was the result of direct demethylation of the gene promoter of hepatic nuclear factor 1A (HNF1A), a positive transcription regulator of AQP9 gene. Among all AML cell lines tested, HNF1A expression was markedly up-regulated upon 5′Aza treatment, and HNF1A knock-down by small-interfering RNA (siRNA) abrogated 5′Aza-induced AQP9 up-regulation and almost completely blocked intracellular As2O3 entry. In AML primary samples, HNF1A expression positively correlated with AQP9 expression, and the 5′Aza-mediated sensitization to As2O3 was also recapitulated. More importantly, 5′Aza did not enhance arsenic toxicity in MIHA liver cells, where HNF1A is largely unmethylated. These observations provide evidences that AQP9 may serve as a biomarker for predicting clinical behaviour and its up-regulation may expand the therapeutic spectrum of As2O3 to other neoplasms that are originally insensitive to As2O3. Arsenic trioxide is a highly effective salvage therapy in APL. The unique sensitivity of APL cells to As2O3 is likely to be related to the As2O3-mediated degradation of the PML-RARα. In “AML with mutated nucleophosmin (NPM1)”, the role of NPM1 cytoplasmic mutant (NPMc+) in contributing to leukaemogenesis has been previously described by a number of researches. Here, I seek to identify if As2O3 may target NPMc+ for degradation and to investigate the subsequent effects of As2O3 in AML cells harbouring NPMc+. In this study, As2O3 was found to selectively degrade NPMc+, but not the wild-type NPM1. Arsenic trioxide impaired cell colony growth, which was comparable to what has been observed in cells with NPM knock-down by siRNA. Arsenic treatment was also found to induce myeloid cell differentiation through induction of CD11b as well as p53, p21 and C/EBPα expression. Finally, proteasome inhibitor rescued the shortened half-life of NPMc+ due to As2O3 treatment. The increased ubiquitylation of NPMc+ after As2O3 treatment indicated that NPMc+ degradation mediated by As2O3 was through ubiquitin-proteasome pathway. These results suggested that As2O3 is a potential therapy for AML with NPMc+.