Deferasirox Is the Only Iron Chelator Acting as a Potent NF-KB Inhibitor in Myelodysplastic Syndromes
Authors: Messa Emanuela , Defilippi Ilaria , Roetto Antonella , Messa Francesca , Arruga Francesca , Carturan Sonia , Rosso Valentina , Bracco Enrico , Cilloni Daniela , Saglio Giuseppe 
Published: April 2020

Abstract
Iron overload is a critical issue for low risk myelodisplastic syndrome (MDS) patients with a long tranfusional history and often requires chelation therapy. Iron chelation is as an independent prognostic factor for survival in MDS but can also improve haemoglobin level in some cases with different drugs and modalities. Recently a once daily oral chelator Deferasirox became available for the treatment of secondary hemosiderosis also in MDS patients and it has been described an haemoglobin improvement in a many patients within a few months of treatment. Moreover it was demonstrated that the transcriptional factor NF-kB is abnormally activated in MDS blast cells. Its pathway can be mediated by a broad variety of stimuli, sometimes dependent by reactive oxygen species generated by iron overload but it is activated even in the absence of iron overload. Aim of our study was to compare the effects of the 3 commercially available iron chelators on NF-kB activity in MDS and to identify a possible mechanism responsible for the observed reduced transfusion requirement during iron chelation therapy. We collected 40 PB samples from MDS patients: 18 RA, 14 RAEB, and 8 s-AML. Thirty of them presented hepatic iron overload (measured by SQUID biomagnetic liver susceptometry) and serum ferritin levels over 5000 ng/ml. Ten samples were collected before starting transfusion therapy. MNC cells were incubated with 50 microM Deferasirox for 3 hrs. K562 and HL60 cells were analyzed as controls and were incubated with Deferasirox 50 mM, Deferiprone 0,3 mM and Deferioxamine 0,3 mM for 18 hours and with the DL-Dithiothreitol (DTT) 100 mM for 18 hours as control. NF-kB activity was evaluated using both EMSA and ELISA methods. Apoptosis was evaluated by FACS for the detection of annexin V. Deferasirox incubation induced a significant decrease of NF-kB activity both in HL60 and K562 cells lines (EMSA method). We detected an increased activation of NF-kB as compared to healthy subjects in 6 RA, 12 RAEB, in all the s-AML PB samples and in cell lines. No significant difference was detected in NF-kB activity by comparing patients with or without iron overload (p=0,5). The percentage of samples presenting NF-kB activity increases during disease progression being higher in RAEB and s-AML as compared to RA (p=0,003). Among patients with increased NF-kB (n=14) the incubation with Deferasirox induced a significant reduction of NF-kB activity (p=0,0002). The degree of NF-kB inhibition was not significantly different according to the level of iron overload. Apoptosis was not significantly triggered by incubation with any of the different chelators. HL60 and K562 cells incubation with the Deferiprone and Deferioxamine failed to induce a significant reduction of NF-kB activity. NF-kB is abnormally activated in MDS patients and this activity is not strictly related to iron overload being present in patients with normal serum ferritin levels and in cell lines. Deferasirox is the only commercially available iron chelator acting as a potent NF-kB inhibitor and this effect is not shared by Deferioxamine, Deferiprone or DTT. This behaviour suggests that it is independent from ROS scavenging that is a common feature of all the mentioned drugs. Our in vitro observation could be an explanation for the early improvement of hemoglobin levels observed in some patients under deferasirox chelation that seems not related to a sharp decrease of iron overload.