PF464 DEFERASIROX DRIVES ROS‐MEDIATED DIFFERENTIATION AND INDUCES INTERFERON‐STIMULATED GENES EXPRESSION IN HEALTHY HEMATOPOIETIC STEM/PROGENITOR CELLS AND IN LEUKEMIA CELLS

Abstract

Background:The employment of the iron chelator deferasirox (DFX) in transfusion‐dependent patients affected by chronic anaemias occurring in several hematological diseases occasionally results in recovery of hematopoiesis with hematopoietic stem cells (HSCs) regaining the ability to differentiate into mature blood cells. Despite a growing body of clinical evidence, the mechanism explaining this interesting peculiarity still remains elusive.Aims:Aim of this study was to identify a general molecular mechanism underlying DFX beneficial effect on hematopoiesis both in healthy and in pathological conditions.Methods:Human healthy HSCs and two leukemia cell lines, Kasumi‐1 and K562, were treated with DFX 100 μM. N‐Acetyl Cysteine (NAC) was added as antioxidant; fludarabine was administered to inhibit STAT1 activation and interferon signaling. In vitro colony‐forming assays were assessed both in healthy and in leukemia cells to functionally test DFX ability to induce differentiation; the growth of CFU‐GM, BFU‐E and CFU‐GEMM colonies was scored and enumerated. Intracellular and mitochondrial reactive oxygen species (ROS) were assessed by cytofluorimetric analysis after DFX treatment for 24 h. The following differentiation markers were monitored after 24 h and 48 h of DFX treatment by cytofluorimetric analysis: CD14‐APC and CD36‐FITC for Kasumi‐1 cells; CD71‐FITC and CD36‐FITC for K562. Gene expression profile was performed on healthy HSCs using Illumina platform and data were analyzed by Ingenuity Pathway Analysis (IPA); differently expressed genes were validated both in healthy and leukemia cells by Realtime PCR. The expression and phosphorylation state of STAT1 were assessed by western blotting.Results:DFX, at clinically relevant concentrations, increased the clonogenic capacity of healthy human CD34+ HSC to form erythroid colonies in a ROS dependent fashion and the whole gene expression analysis of DFX‐treated HSCs unveiled up‐regulation of genes linked to interferon (IFN) signaling. The study, extended on human‐derived leukemia cell lines, revealed a DFX‐mediated overproduction of mitochondria‐generated reactive oxygen species (ROS), accompanied by a ROS‐dependent up‐regulated expression of specific markers of hematopoietic commitment. Similarly to HSCs, DFX treatment of Kasumi‐1 and K562 resulted in a significant up‐regulation of IFN‐related genes expression as well as in a marked hyper‐phosphorylation of the signal transducer and activator of transcription 1 (STAT1), known to be involved, in turn, in the IFN signaling. Most notably, treatment of Kasumi‐1 and K562 cell lines with the antioxidant NAC completely prevented the DFX‐mediated phosphorylation of STAT1 as well as the expression of the IFN‐stimulated genes. Conversely, the inhibition of STAT‐1 alone, by Fludarabine, was not sufficient to affect differentiation processes in both cell lines.Summary/Conclusion:The novel findings here reported suggest a hitherto strong impact of ROS signaling and strengthen its role as master regulator of multiple orchestrated events triggered by DFX, like IFN pathway up‐regulation and differentiation induction. Interestingly, DFX property seems to be independent on the physiological or pathological state of the cells. The identification of key factors earlier modulated by DFX and able to activate downstream events involved in the regulation of cell survival, proliferation and differentiation, provides new insights that can be developed to anticipate its administration or to extend the use of the drug also to other categories of patients.image