Abstract
BackgroundThe histone methyltransferase G9a has recently been identified as a potential target for epigenetic therapy of acute myeloid leukemia (AML). However, the effect of G9a inhibition on leukemia stem cells (LSCs), which are responsible for AML drug resistance and recurrence, is unclear. In this study, we investigated the underlying mechanisms of the LSC resistance to G9a inhibition.MethodsWe evaluated the effects of G9a inhibition on the unfolded protein response and autophagy in AML and LSC-like cell lines and in primary CD34+CD38− leukemic blasts from patients with AML and investigated the underlying mechanisms. The effects of treatment on cells were evaluated by flow cytometry, western blotting, confocal microscopy, reactive oxygen species (ROS) production assay.ResultsThe G9a inhibitor BIX-01294 effectively induced apoptosis in AML cell lines; however, the effect was limited in KG1 LSC-like cells. BIX-01294 treatment or siRNA-mediated G9a knockdown led to the activation of the PERK/NRF2 pathway and HO-1 upregulation in KG1 cells. Phosphorylation of p38 and intracellular generation of reactive oxygen species (ROS) were suppressed. Pharmacological or siRNA-mediated inhibition of the PERK/NRF2 pathway synergistically enhanced BIX-01294-induced apoptosis, with suppressed HO-1 expression, increased p38 phosphorylation, and elevated ROS generation, indicating that activated PERK/NRF2 signaling suppressed ROS-induced apoptosis in KG1 cells. By contrast, cotreatment of normal hematopoietic stem cells with BIX-01294 and a PERK inhibitor had no significant proapoptotic effect. Additionally, G9a inhibition induced autophagy flux in KG1 cells, while autophagy inhibitors significantly increased the BIX-01294-induced apoptosis. This prosurvival autophagy was not abrogated by PERK/NRF2 inhibition.ConclusionsPERK/NRF2 signaling plays a key role in protecting LSCs against ROS-induced apoptosis, thus conferring resistance to G9a inhibitors. Treatment with PERK/NRF2 or autophagy inhibitors could overcome resistance to G9a inhibition and eliminate LSCs, suggesting the potential clinical utility of these unique targeted therapies against AML.
Highlights
The histone methyltransferase G9a has recently been identified as a potential target for epigenetic therapy of acute myeloid leukemia (AML)
Among the Leukemia stem cell (LSC)-like cell lines, KG1 cells were very insensitive to BIX-01294 treatment; we used these cells as representative BIX01294-resistant cells in further experiments
G9a inhibition-induced PERK signaling conferred resistance to LSCs via NRF2/HO-1 activation We found that BIX-01294 upregulated NRF2, which is a direct substrate of the PERK kinase, resulting in increased HO-1 expression in KG1 cells (Fig. 5a and b). Small interfering RNA (siRNA)-mediated PERK knockdown effectively abolished the BIX-01294-mediated upregulation of NRF2 and HO1 in KG1 cells (Fig. 5c)
Summary
The histone methyltransferase G9a has recently been identified as a potential target for epigenetic therapy of acute myeloid leukemia (AML). The effect of G9a inhibition on leukemia stem cells (LSCs), which are responsible for AML drug resistance and recurrence, is unclear. Leukemia stem cells (LSCs) play important roles in the relapse and drug resistance, which hamper complete cure of the disease [2]. Studies have indicated that mutations related to DNA methylation could mediate abnormal self-renewal and differentiation of LSCs, suggesting that drugs targeting epigenetic enzymes may provide a new AML treatment option [5,6,7]. High levels of G9 expression are associated with unfavorable clinical outcomes, including disease progression, metastasis, development of stem cell-like characteristics, resistance to treatment, and poor survival [17, 18]. The tolerance of LSCs to epigenetic therapies causes treatment failure, hampering the clinical applicability of these therapies, while inhibition of LSC tolerance is considered a promising therapeutic strategy
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