Deferasirox-Dependent Iron Chelation Enhances Mitochondrial Dysfunction and Restores p53 Signaling by Stabilization of p53 Family Members in Leukemic Cells

Abstract

Iron is crucial to satisfy several mitochondrial functions including energy metabolism and oxidative phosphorylation. Patients affected by Myelodysplastic Syndromes (MDS) and acute myeloid leukemia (AML) are frequently characterized by iron overload (IOL), due to continuous red blood cell (RBC) transfusions. This event impacts the overall survival (OS) and it is associated with increased mortality in lower-risk MDS patients. Accordingly, the oral iron chelator Deferasirox (DFX) has been reported to improve the OS and delay leukemic transformation. However, the molecular players and the biological mechanisms laying behind remain currently mostly undefined. The aim of this study has been to investigate the potential anti-leukemic effect of DFX, by functionally and molecularly analyzing its effects in three different leukemia cell lines, harboring or not p53 mutations, and in human primary cells derived from 15 MDS/AML patients. Our findings indicated that DFX can lead to apoptosis, impairment of cell growth only in a context of IOL, and can induce a significant alteration of mitochondria network, with a sharp reduction in mitochondrial activity. Moreover, through a remarkable reduction of Murine Double Minute 2 (MDM2), known to regulate the stability of p53 and p73 proteins, we observed an enhancement of p53 transcriptional activity after DFX. Interestingly, this iron depletion-triggered signaling is enabled by p73, in the absence of p53, or in the presence of a p53 mutant form. In conclusion, we propose a mechanism by which the increased p53 family transcriptional activity and protein stability could explain the potential benefits of iron chelation therapy in terms of improving OS and delaying leukemic transformation.

Keywords: Deferasirox; MDM2; chelation; iron; leukemia; mitochondria; p21; p53; p73.

Figure 1

Iron chelation induces an altered network in acute myeloid leukemia cell lines. HL60, NB4, and MOLM-13 were treated for 48 h with 50 µM of DFX and subsequently incubated with 100 nM of MitoTracker green for mitochondrial network analysis. (A) The original green image obtained with confocal microscope is a three-dimensional (3D) image created with z-stack project (63× magnification). It has been processed using the MiNA toolset to generate an accurate skeleton. In red and black and white, the skeleton of mitochondria of the three cell lines treated with DFX is visible, compared to respective control. (B) Quantification of mitochondria footprint and branches length, which corresponds respectively to the area and connection, of mitochondria expressed in µm. Abbreviations: NT, not treated; DFX, Deferasirox. * p ≤ 0.05, ** p ≤ 0.01, and **** p ≤ 0.0001.

Figure 2

Deferasirox exerts an anti-leukemic activity on AML cell lines and on MDS patients’ cells. (A) HL60, NB4, and MOLM-13 were treated with 0, 10, 25, 50, or 100 μM DFX for 48 h and the MTT assay was performed to evaluate the proliferation index. The percentage of proliferation is expressed after normalizing with untreated cells (100%). (B) Mean fluorescence intensity (MFI) of FITC-calcein signal obtained after 48 h of DFX treatment. LIP level was inversely proportional to measured fluorescence intensity. (C) Percentage of apoptosis evaluated by flow cytometry after FITC Annexin-V assay on HL60, NB4, and MOLM-13 treated with 25 and 50 μM of DFX. (D) Western blot analysis of cleaved caspase 3 revealed that its amount increased in a DFX dose-dependent fashion, in HL60, NB4, and MOLM-13. (E) Five MDS/AML patients or 5 healthy subjects were treated with 10, 25, 50, or 100 μM of DFX for 48 h and the MTT assay was performed. (F) Representative histograms and % of apoptosis evaluated by flow cytometry after FITC Annexin-V assay on 5 MDS/AML patients or 5 healthy subjects treated with 50 μM of DFX. Abbreviations: NT, not treated; DFX, Deferasirox; Ann V, Annexin V. * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001.

Figure 3

Deferasirox activates p53 targets on acute myeloid leukemia cell lines and MDS/AML patients. (A) CDKN1A, PUMA, GADD45, and MDM2 gene expression were assayed by qRT-PCR in HL60, NB4, and MOLM-13 after 48 h treatment with DFX 50 μM. The amount is expressed as fold changes compared to untreated cells after normalizing on the ABL housekeeping gene. (B,C) Western blot analysis of target proteins p21 and MDM2 after iron chelation treatment confirmed the effects of DFX on the p53 pathway. (D,E) CDKN1A and PUMA gene expression was assayed by qRT-PCR on 15 patients’ cells after 48 h in vitro treatment with DFX 50 μM. The mRNA quantity is expressed as 2^−ΔΔCt after normalization with the ABL housekeeping gene. Abbreviations: -, not treated; + treated with DFX 50 μM; DFX, Deferasirox. * p ≤ 0.05, ** p ≤ 0.01.

Figure 4

Deferasirox regulates p53 and p73 protein stability. (A) Immunofluorescence of p53 and p73 after DFX treatment. The green signal corresponds to p53 or p73 while the red propidium is used to detect nuclei (63× magnification). (B) p73 Immunohistochemistry on MDS bone marrow samples derived from 3 different patients at diagnosis (DX) and within one year of iron chelation treatment (ICT) (20× magnification). (C) Differentially expressed genes clusters enriched according to Gene Ontology terms and ordered by ascending adjusted p-value. (D) Schematic representation of the plausible role of iron chelation on mitochondrial activity and on p53 family stability (created in biorender.com). Abbreviations: NT, not treated; DFX, Deferasirox.