BACKGROUND. The redox metabolism is a key actor in the pathophysiology of acute myeloblastic leukemia (AML). High level of reactive oxygen species (ROS) is observed in the leukemic bulk, whereas leukemic stem cells (LSCs) have a low level of ROS, and we reported that glutathione peroxydase 3 (GPx-3) promotes competitiveness of LSCs in which Gpx3 overexpression is associated with a decreased level of ROS and inactivation of p38 MAPK (Herault O et al, J Exp Med 2012, 209:895).
NF-E2-related factor 2 (Nrf2) transcription factor, the master regulator of antioxidant responses, is highly expressed in acute myeloblastic leukemia (AML) and is involved in its chemoresistance (Rushworth SA et al, Blood 2012, 120,5188). Since leukemic cells interact with mesenchymal stem/stromal cells (MSCs) in the bone marrow (BM) niche, the aim of our study was to evaluate the effects of MSC-contact on the Nrf2 sub-cellular localization in acute myeloid leukemia (AML) cells and to concomitantly assess the the GPx-3/ROS/p38 MAPK axis and cell cycle status.
METHODS. BM MSCs (from consenting patients undergoing orthopaedic surgery) were used at P2. The mesenchymal differentiation capacity was systematically checked. The KG1a (FAB M0 AML) cell line and primary AML blasts were cultured for 72h in presence of MSCs and compared to their counterpart cultured in presence of supernatant from MSCs alone or medium alone. ROS level measurements (DCFDA), cell cycle analyses (Vignon C et al, PLOS One 2013, 8: e68425) and proliferation assays (CFSE) of the leukaemic cells were performed by flow cytometry. Expression levels of 24 key antioxidant genes, CDKN1A (p21, Cip1) and 22 Nrf2-regulated genes were quantified by qRT-PCR. Western-blot analyses were also realized to determine the expression levels of GPx-3 and p21(Cip1), the activation of p38 MAPK (T180/Y182) and the localization of Nrf2 after subcellular fractionation.
RESULTS. Adherent KG1a cells were characterized by major decrease in nuclear translocation of Nrf2 and expression of Nrf2-regulated genes. We concomitantly observed overexpression of GPX3 mRNA and protein, major decrease in ROS levels and inactivation of p38 MAPK. Similar results were obtained with adherent primary leukemic cells. These effects were concomitantly observed with major decrease in nuclear translocation of Nrf2 and expression of Nrf2-regulated genes. Interestingly, the increased G0 phase, together with the decreased S and M phases, and overexpression of CDKN1A mRNA and protein demonstrated cell cycle inhibition and reduced mitotic activity. The supernatant of MSCs did not impact on Nrf2-subcellular localization, GPx-3 expression and ROS levels in leukemic cells.
CONCLUSION. Altogether these findings suggest that the niche (direct contact to MSCs) plays a key role in the regulation of the oxidative metabolism in leukemic cells by modifying the subcellular localization of Nrf2 and by promoting the inhibition of the ROS/p38 MAPK axis through the induction of GPx-3, resulting in a reduced proliferation. Targeting the Nrf2 pathway in leukemic cells by a modulation of microenvironmental interactions may be a clinically relevant chemosensitizing approach.
Citation Format: Olivier Herault, Christine Vignon, Christelle Debeissat, Florence Rouleux-Bonnin, Emmanuel Gyan, Frederic Mazurier, Fabrice Gouilleux, Jorge Domenech. The Nrf2-subcellular localization in human acute myeloblastic leukemic cells is regulated by the bone marrow mesenchymal stem/stromal cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-334.