Arsenic Trioxide Potentiates Extracellular Traps-Formation Cell Death in Acute Promyelocytic Leukemia through mTOR-Regulated Autophagy

Background:Emerging clinical data shows that arsenic trioxide (ATO) exerts selective cytotoxicity against acute promyelocytic leukemia (APL) without severe side effects that mainly ascribed to nonspecific induction of apoptosis. It is attractive to speculate whether other uncovered APL cell death exists which can be specifically sparked by ATO administration. We have recently demonstrated that APL cells can undergo a previously unrecognized pathway for death by releasing extracellular DNA traps (ETs), termed ETosis (Ma R et al, Cell Death Dis 2016). However, besides apoptosis, whether ATO induces this APL-specific ETotic cell death remains to be explored. We wereto investigated the effects of a wide range of concentrations of ATO on ETotic death in APL cells and elucidate the underlying molecular mechanisms.

Methods: Bone marrow samples were obtained from sixteen APL patients before and after the continuous administration of ATO for two weeks. APL cells were isolated and cultured in the presence and absence of ATRA for 3 days. We used confocal microscopy to image ET formation by APL cells and the percentage of ETotic cells was simultaneously quantified. ELISA was used to measure the concentration of myeloperoxidase (MPO)-DNA complexes in the supernant. We also assessed the effects of a wide range of concentrations (0.1, 0.25, 0.5, 0.75, 1.0, 2.0 μM) of ATO treatment for 24, 48 and 72 hours on ETosis in APL-cell line NB4 cells in vitro. Autophagy activation and leukemia-initiating cell (LIC) activity were evaluated by immunoblotting and imaged by immunostaining. LICs were analyzed using colony-forming unit (CFU) assays, and identified and quantified by flow cytometry.

Results: APL cells isolated from ATO-treated APL patients underwent significantly increased spontaneous (P = 0.005) and ATRA-stimulated (P = 0.002) ETosis compared to those from newly diagnosed patients (n = 16). In vitro ATO treatment showed that the percentage of ETotic NB4 cells dramatically increased at 0.5 μM (8 ± 1.6%), peaked at 0.75 μM (15 ± 2.4%) and was gradually suppressed at higher concentrations. The concentration of MPO-DNA complexes, an indirect marker of ETosis, parallelled the dose-dependent change in the percentage of ETotic cells. Interestingly, inhibition experiments indicated that ATO caused concentration-dependent APL cell death: ATO primarily triggered ETosis at moderate concentrations (0.5 to 0.75 μM) and switched to apoptosis at relatively high doses (1.0 to 2.0 μM). Furthermore, We found that ATO induced ETosis through mammalian target of rapamycin (mTOR)-regulated autophagy. Surprisingly, inhibition of ETosis spared LIC activity from ATO reduction, while combined treatment with ATO and rapamycin further increased ETosis-mediated LIC loss (~3.5-fold).

Conclusions: This is the first study to show that ATO potentiates ETotic death in APL cells through mTOR-regulated autophagy. Importantly, further investigation suggests that ATO specifically targets the APL LICs to ETosis. This implies that, in combination with ATO, therapy-triggered ETosis by targeting the corresponding signaling pathways could be a novel and effective strategy to improve a long relapse-free survival through LIC clearance, avoid lethal apoptosis-related complications and overcome apoptosis resistance.

Conflict of interest statement: None.