Background Relapse of acute myeloid leukemia (AML) may arise from residual chemoresistant leukemic cells. A hypoxic tumor microenvironment, such as the bone marrow, is known to enhance chemoresistance in various forms of cancer, including AML. Hypoxia inducible factor 1 alpha (HIF-1α) is an important mediator of cellular adaptation to hypoxia. HIF-1α is a constitutively expressed transcription factor that is rapidly degraded under normoxic conditions after hydroxylation by oxygen sensors, such as the HIF prolyl hydroxylases (PHDs). However, under hypoxic conditions the oxygen sensors lose the ability to induce the degradation of HIF-1α resulting in its stabilization and translocation to the nucleolus where it induces the transcription of genes associated with glucose metabolism, angiogenesis, and cell survival. This may result in proliferation of malignant cells, impaired tumor cell differentiation and chemoresistance. Reactive oxygen species (ROS) have been shown to inhibit PHDs and may thereby stabilize HIF-1α, and may thus contribute to chemoresistance. AML cells may generate ROS via the myeloid NADPH oxidase NOX2. We therefore hypothesized that NOX inhibitors would decrease chemoresistance in a hypoxic environment.

Materials and Methods The wild type (WT) AML cell line PLB-985 and its NOX2 knocked out (KO) counterpart were cultured for five days in hypoxia (1% oxygen) or normoxia (21% oxygen) in the presence or absence of the NOX inhibitors histamine dihydrochloride (HDC), diphenyleneiodonium (DPI) and GSK2795039. Thereafter cells were exposed to the chemotherapeutic agent daunorubicin for 48 hours (in hypoxia or normoxia) and cell death was determined using the XTT assay. Stabilization of HIF-1α was measured either by western blot or flow cytometry. Differentiation of cells was quantified by measuring the expression of CD14 and CD11b by flow cytometry.

Results Hypoxia reduced the sensitivity of WT PLB-985 cells to daunorubicin induced cell death (P < 0.05, n=4) whereas NOX2 KO cells were equally sensitive to daunorubicin in hypoxia and normoxia (P > 0.5, n=4). Furthermore, NOX2 KO AML cells displayed increased sensitivity to daunorubicin induced killing compared with PLB WT cells in a hypoxic environment (P < 0.05, n=4). Preliminary results show that pharmacological NOX inhibition using DPI enhanced the sensitivity of WT AML cells to daunorubicin induced killing. These results suggests that functional NOX2 contributes to chemoresistance in a hypoxic environment. As expected, hypoxia stabilized the expression of HIF-1α in AML cells. Preliminary results suggest that HIF-1α expression was reduced in the presence of NOX inhibitors.

Conclusions Genetic deletion or pharmacological inhibition of NOX2 sensitized AML cells to daunorubicin induced killing in hypoxic environments. NOX2 may thus be a target for overcoming chemoresistance in AML cells in the hypoxic bone marrow environment.

Support This work was supported by Assar Gabrielsson’s Foundation( FB19-64) and other grants used by the research group.

Disclosure Information S. Paul: None. H. Grauers Wiktorin: E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Patent. R. Kiffin: None. K. Hellstrand: E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Patent. A. Martner: E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Patent.