Author: Gianpiero Pescarmona Date: 16/03/2013
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Chelation therapy in myelodysplasticsyndromes decreases iron levels derived from red blood cells transfusions, that the majority of patients with MDS requires during the course of their disease. Iron overload causes ROS production with consequent cellular damages and organ disfunction. The administration of iron chelators in multi-trasfused MDS patients decreases the intra and extracellular free iron species, so ameliorates parameters of oxidative stress such as ROS, glutathione’s levels and membrane peroxidation and avoid DNA instability. There are different available iron chelators, in particular Deferasirox shows off target effects such as the block of NF-кB activity, the inhibition of mTOR activity, the increase of chemosensitivity and the decrease of fungal infections.
Myelodisplastic syndromes (MDS) are a heterogeneous group of disorders clinically characterized by peripheral cytopenia and an increasing risk of evolution into acute myeloid leukemia, because of acquired clonal defects of bone marrow stem cells. In bone marrow there is an expansion of a stem cell clone characterized by impaired differentiation and increased apoptosis that leads to a premature cell death and cytopenia. The natural history of MDS, ranging from indolent conditions over years to forms that rapidly progress to leukemia, complicates clinical decision-making regarding therapeutic modalities and timing of intervention. Leukemia derived from MDS, owing to a complete differentiation block, are generally drug resistant and have worse prognosis. MDS are especially typical of old people and affect above all males. The incidence estimated is 3-5/100000/year and increase to 15/50/100000 if only people older than 60-70 years old are considered. There are two different classification for this disease: FAB classification and OMS classification. The first, created in 1982, is based on blasts’ number in marrow aspirate and peripheral blood, the second, newer, addes diagnostic and prognostic criteria. Main symptoms and signs of MDS are connected to peripheral cytopenia and include: paleness, asthenia, dyspnoea, bacterial and fungal infections, bleedings, petechiae, ecchymosis. The only curative treatment in MDS patients is allogenic stem-cell transplantation (alloSCT). Long- term survival rates of between 25 and 70% were reported after transplantation. Howewer, despite advances in transplantation technology, there is still considerable morbidity and mortality associated with this approach, so an accurate selection of candidate patients is needed. The use of hypomethylating drugs such as azacitidine and decitabine may prolong survival in patients with higher-risk MDS. The majority of patients with MDS require blood transfusion during the course of their disease. Transfusion requirement can be moderate ( 2 units/month id est 24 units/year) or high (4 units/month id est 48 units/year –there are about 200/250 mg of iron in every unit, but normal body iron is 3-4 g!-). Frequent blood transfusions produce a gradual increase in transferring saturation (whose normal values are about 30%) and the appearance of nontransferring-bound iron (NTBI). Transfusions cause many complications among elderly patients, as this study shows:
Figure 1:Goldberg SL et al. Leuk Res 2009;33(1):abst P099
Of 585 cases, 231 patients (39.5%) received blood transfusions. During 3 years’ follow-up, patients receiving transfusions had higher rates of co-morbidities: lightly transfused patients had similar rates of cardiac events and diabetes as heavily transfused patients (cardiac events: 81.7% vs 79.4%, P=0.75; diabetes: 45.2% vs 41.2%, P=0.66). In conclusion, patients have a long history of transfusion, before eventually undergoing transplantation and, therefore, are at high risk of developing parenchymal iron overload with production of reactive oxygen species (ROS). In addition iron overload may persist long after transplantation. Recent data suggests that secondary iron overload is negatively associated with survival in trasfusion dependent MDS patients . This graphic shows survival of patients with MDS according to the intensity of their red cell transfusion requirement, calculated as the number of packed red cell units per month. The patients with a greater transfusion requirement have shorter median survival than those with lower transfusion requirements. These data were obtained from 426 patients diagnosed with MDS according to WHO criteria at the IRCCS Policlinico San Matteo, Pavia, Italy, between 1992 and 2004.
Figure 2: Malcovati L et al. Haematologica 2006;91(12):1588–1590.
The iron overload and its consequences are typical of all trasfusional dependent diseases. In an autopsy study of 135 subjects with chronic acquired anemia, 60% of patients who had received more than 75 units of red blood cells had cardiac iron deposits Non-specific serum iron in thalassaemia: An abnormal serum iron fraction of potential toxicity.. In 1981, Schafer et al. Clinical consequences of acquired transfusional iron overload in adults. reported the clinical consequences of acquired transfusional iron-overload in adult patients with refractory anemia or aplastic anemia who had received a mean of 120 units of red blood cells. In this study, 10 out of 15 liver biopsy specimens contained between 7 and 26 times the normal levels of iron and typically showed portal fibrosis. Cardiac left ventricular function was impaired in only the most heavily transfused patients, or in those with coexisting coronary artery disease; all 15 patients had glucose intolerance associated with a significantly reduced insulin output. Another problem is iron accumulation in pineal gland with secondary hypogonadotrophic hypogonadism. In fact, in rare MDS young patients is important additional evaluation as assessment of growth rate and sexual development. Iron Body Status in MDS patients should be determined with different methods described in the table below:
Table 1: Iron overload and iron chelation therapy in patients with myelodysplastic syndrome treated by allogenic stem-cell trasplantation: Report from the working conference on iron chelation of the Gruppo Italiano Trapianto di Midollo Osseo..
Iron is an important metal with a wide range of physiological roles within the body, as the following table summarize:
Table 2
Iron is also a potentially toxic metal, because it can catalyze the production of toxic radicals:
FENTON REACTION:
Fe2+ + H2O2 → Fe3+ + OH• + OH− Fe3+ + H2O2 → Fe2+ + OOH• + H+ Ferrous iron (II) is oxidized by hydrogen peroxide to ferric iron(III), a hydroxyl radical and a hydroxyl anion. Iron(III) is then reduced back to iron(II), a peroxide radical and a proton by the same hydrogen peroxide (disproportionation).The hydroxyl free radical generated by Fenton’s reagent is a powerful, non-selective oxidant. Oxidation of an organic compound by Fenton’s reagent is rapid and exothermic (heat-producing) and results in the oxidation of contaminants to primarily carbon dioxide and water. ROS are toxic for cells:
• Lead to pro-apoptotic pathways through the activation of the caspasis cascade: caspasi 9 causes apoptosis and consequent ineffective erythropoiesis. • Induce DNA damage → mutations with major risk of progression to AML. ROS induce genomic instability in MDS mouse model and can promote progression to AML in mice. Reactive oxygen species, DNA damage, and error-prone repair: a model for genomic instability with progression in myeloid leukemia?. • Induce mtDNA damage (Damage to mtDNA accumulates in vivo is a function of age. mtDNA is much more sensitive to oxidative damage. The mutation rate in mtDNA has is 5–10 times greater than in nuclear DNA. Mitochrondria generate reactive oxygen species and are intrinsically rich in iron. Repair of damage to mtDNA is slower and less effective). Mitochondrial DNA Damage in Iron Overload.
• Damage proteins, enzymes → inactivation, denaturation, structural and metabolical alterations. • Arouse lipid peroxidation with damage of cell membrane. Living organisms have evolved different molecules (anti-oxidants) that speed up termination by catching free radicals and, therefore protecting the cell membrane. • Alteration of lisosomial mechanism.
Figure 3
Iron overload increases degree of ineffective erythropoiesis (charateristic of the MDS) through ROS production and consequent excessive apoptosis in the bone marrow precursors. Iron overload derives from trasfusions and increased intestinal assorbtion. In a normal balanced state, 1–2 mg of iron enters and leaves the body every day. Dietary iron is absorbed by duodenal enterocytes and circulates in the plasma bound to transferrin, the main iron transport protein. Most of the circulating iron is used by the bone marrow to generate hemoglobin for red blood cells, while around 10–15% is utilized by muscle fibers to generate myoglobin. Iron released by tissue breakdown is absorbed and recycled. Excess iron is stored by parenchymal cells in the liver and reticuloendothelial macrophages. Traces of iron are lost each day by sloughing of mucosal cells, loss of epithelial cells and any blood loss. However the human body has not evolved a mechanism to clear excess iron. Bowel assorbtion is regulated by hepcidin, a protein placed on basolateral membrane of enterocytes. Hepcidin responds to several physiological states including inflammation, iron, hypoxia and anemia. Decreased production of hepcidin leads to increased absorption of iron which magnifies the effect of secondary iron overload. Hepcidicin reduction in MDS is caused by the increased of GDF15, a marker of ineffective erythropoiesis. GDF15 shows increased expression and secretion during erythroblast maturation. This marker suppresses hepcidin expression in hepatocytes in vitro. Depletion of GDF15 reversed hepcidin suppression High levels of GDF15 in thalassemia suppress expression of the iron regulatory protein hepcidin.. Another marker of innefective erithropoiesis is Non-transferrin-bound-iron (NTBI). NTBI appears in plasma when transferrin is almost completely saturated (saturation > 60–70%); it is taken up by cells and is highly toxic. NTBI is produced by the continued outpouring of iron derived from RBC catabolism in the face of saturated extracellular transferrin. The presence of NTBI was first documented in patients with thalassemia major with severe iron overload and complete saturation of circulating transferrin. The presence of NTBI has also been documented in the ineffective erythropoiesis of myelodysplastic syndrome with erythroid hyperplasia. The risks associated with the presence of NTBI in promoting the formation of toxic hydroxyl radicals are well documented Non-transferrin-bound iron in myelodysplastic syndromes: a marker of ineffective erythropoiesis?.
Transfusion dependency in MDS patients significantly reduces survival as Malcovati’s study demonstred:
Figure 4: Malcovati L et al. Haematologica 2006;91(12):1588–1590
Transfusion-dependent patients have a significantly shorter survival than transfusion-independent patients (p<0,001 overall). Survival curves do not take into account the time dependency of the transfusion requirement. The authors suggested that the reduced survival could be due to increased disease severity in the transfusion-dependent patients or to excess iron. To evaluate the latter, the authors examined the impact of iron overload (defined as serum ferritin >1000 ng/mL) and found that it negatively affected survival (HR=1.30; P=0.003). Iron overload was initially identified as an adverse risk factor in patients undergoing allogeneic HSCT for thalassemia:in a study by Gaziev et al. 15 thalassemic patients undergoing alloSCT received DFO (40 mg/kg/die as a 24 hr iv infusion administered before and early post-transplant). DFO didn’t affect the engraftment parameters or the incidence of infections or acute GVHD. Moreover, this therapy resulted in a significant decrease of serum ferritin at 6 months after transplantation with respect to untreated control group. These results suggest that the administration of DFO during and early-post-allo-SCT is safe and effective. Response to chelation therapy in MDS may not be the same as that in thalassaemia syndrome and other anemias, as the degree of ineffective erythropoiesis and thus the magnitude of chelatable iron pools may differ. Intravenous chelation therapy during transplantation for thalassemia.. However, since then, several studies have suggested a link between iron overload and specific causes of post-transplantation morbidity and mortality (including chronic liver disease, veno-occlusive disease, susceptibility to infections and overall survival). For example in Lim’s study a raised ferritin was significantly associated with an inferior overload survival, disease-free survival and transplant-related mortality. Impact of pre-transplant serum ferritin on outcomes of patients with myelodysplastic syndromes or secondary acute myeloid leukemia receiving reduced intensity conditioning allogenic haematopoietic stem cell transplantation.. It’s in fact established that the presence of free iron species in the serum or in the cells (the labile plasma iron and the labile iron pool,respectively) of patients generate ROS which result in increased parameters of oxidative stress that is deleterious to cells and may result in functional failure of vital organs such as heart, liver and endocrine system and increases ineffective erythropoiesis . In addition, NTBI has been shown to increase after intensive chemotherapy as well as both autologous and myeloablative HSCT, and hydroxyl free radicals catalysed by NBTI may cause oxidative cellular damage and tissue toxicity, thus resulting in post-transplant. The administration of iron chelators in multi-trasfused MDS patients decreases the intra and extracellular free iron species, so ameliorates parameters of oxidative stress such as ROS, glutathione’s levels and membrane peroxidation and avoid DNA instability Selective toxicity towards myelodisplastic hematopoietic progenitors- Another rationale for iron chelation in MDS.. In this way iron chelators causes a reduction in red blood cells transfusion requirement, as is shown in Jensen’s study about deferoxiamina. The hematologic response during the first year of chelation therapy is very good: the median time to hematologic response is 169 days (84 to 382).
Figure 5: List et al., J Clin Oncol. 2012 Apr 30.
It’s possible to notice an improved survival in patients with MDS receiving chelation therapy:
Figure 6: Rose C, et al. Leuk Res. 2010;34:864-70.
An adequate chelation is associated with better overall survival than weak chelation (124 vs 85 months; P<0.001), even after adjustment for IPSS and WHO-based prognostic scoring system, age, transfusion requirement and number of comorbidities.
Figure 7: Rose C, et al. Leuk Res. 2010;34:864-70.
This study suggests that chelation therapy could improve survival in heavily transfused lower-risk patients with MDS, who have a long history of transfusion and are therefore at high risk of iron overload and iron related tissue damage. For MDS patients eligible for a transplant procedure,the available evidence and consensus-based therapeutic guidelines recommend that iron chelation therapy should be early considered. In transplant survivors, iron overload may persist long after the procedure because of the limited iron excretion capability of human body. In patients with MDS and iron overload after transplant, iron removal through phlebotomy is the first choice therapy (6 ml/kg blood withdrawal at 14-day interval), in relationship with the phlebotomy program for thalassemic patients after allo-SCT,described by Angelucci’s group. Phlebotomy to reduce iron overload in patients cured of talassemia by bone marrow transplantation. Italian Cooperative Group for phlebotomy Treatment of Transplanted Thalassemia Patients.. For those patients who cannot be phlebotomized due to low hemoglobin level or cardiac impairment, deferoxamine or deferasirox should be considered. The optimal strategy, however, remains to be defined. In patients receiving phlebotomy, target iron status is serum ferritin inside the normal laboratory range and transferrin saturation <45%. Inpatients receiving iron-chelating agents therapy should be continued until ferritin has reached levels below 500 ng/ml; periodic biochemical evaluation of body iron status should be performed in order to avoid toxicity by overchelation. "Iron overload and iron chelation therapy in patients with myelodisplastic syndrome treated by allogenic stem-cell transplantation: report from the working conference on iron chelation of the Gruppo Italiano Trapianto di Midollo Osseo.":http://www.ncbi.nlm.nih.gov/pubmed/?term=IrIron+overload+and+iron+chelation+therapy+in+patients+with+myelodysplastic+syndrome+treated+by+allogeneic+stem-cell+transplantation%3A+report+from+the+working+conference+on+iron+chelation+of+the+Gruppo+Italiano+Trapianto+di+Midollo+Osseo Several international guidelines based on expert panel agreements strongly support iron chelation therapy for MDS patients undergoing chronic transfusional therapy with a sufficiently long life expectancy in order to improve patient survival and clinical conditions. *MDS foundation guidelines* on management of iron overload in MDS establish that iron chelation therapy should be administrated in patients with: ● Transfusion dependence (2 units/month for >1 year) ● Serum ferritin >1000 ng/mL ● Ineligibility for or unresponsiveness to primary therapy (e.g. hormonal or hypomethylation) ● IPSS: Low or Int-I: WHO RA, RARS, and 5q- ● Life expectancy ≥1 year ● No comorbidities that would limit prognosis ● Potential to receive allograft Chelation therapy should be continued as long as the patient is receiving transfusion therapy Chelation therapy can be withheld when ferritin level declines to <1000 ng/mL and no additional transfusions are needed. The choice of chelation agent is at the discretion of the physician.
The available iron chelators are: • Deferiprone (DFP, Ferriprox): is an oral iron chelator, but its use is rare because can give agranulocitosis. Other collateral effects are: gastrointestinal disturbances, arthralgia, elevated liver enzymes. In the study of Kersten have been analyzed 38 patients (18 with MDS, 4 with myelofibrosis, 7 with aplastic anaemia, 4 with β-thalassaemia, 5 others), to whom has been administrated deferiprone 3–6 g/day for 1 year. Only 20 patients completed the study. Chelation was effective in 20 patients (56%), ineffective in 3 patients; 1 patient developed agranulocytosis. In conclusion, this study didn’t observed an improvement in transfusion requirement with deferiprone Long-term treatment of transfusional iron overload with the oral iron chelator deferiprone (L1): a Dutch multicenter trial.. • Deferoxamina (DFO, Desferal): is an iron chelator that required a subcutaneous infusion. Some collateral effects of DFO are: local reactions, ocular and auditory abnormalities, allergic reactions. DFO induces urinary iron excretion and decreases levels of serum ferritin, liver iron and heart iron in MDS. DFO has been used clinically for more than 40 years, although the difficulties in complying with treatment are well recognised. Subcutaneous infusions may be particularly troublesome in patients with MDS, who are typically elderly and may have decreased platelet counts or platelet dysfunction, leading to bruising at the infusion site. In the study of Jensen have been analyzed 11 MDS patients followed for up to 60 months (during and after treatment with DFO). Reduction in RBC transfusion requirement (≥ 50%) has been observed in 7/11 (64%) patients. 5 patients became RBC transfusion-independent (46%). All patients in whom iron chelation therapy was highly effective showed improvement of erythropoietic output The effect of iron chelation on haemopoiesis in MDS patients with transfusional iron overload.. • Deferasirox (DFX, Exjade): is a new oral iron chelator. DFX is the most chelator used now for the better compliance of the patients and for its benefical effects. Some collateral effects of DFX are: non-progressive increase in serum creatinine, gastrointestinal disturbances, skin rash, elevation in liver transaminases, high-frequency hearing loss and lenticular opacities. An important study called EPIC (Evaluation of Patients’ Iron Chelation with Exjade) explaines the change in transfusion requirements, haemoglobin level and platelet and neutrophil counts using the hematologic response criteria outlined by the International Working Group (IWG) 2006. EPIC study included 341 MDS: 176 chelation experienced patients and 165 chelation naïve patients. EPIC study shows a reduction of ferritin serum level regardless of prior chelation history only starting from third month of therapy.
Figure 8: Schmid M et al. Presented at ASH 2009 [Blood 2009;114(22):abst 3806]
DFX reduces transfusion requirements and increases bone marrow response in a minor time (months) as compared with other iron chelators. The table below shows this aspect.
Table 3: Cilloni D, et al. Blood. 2011;118;[abstract 611].
All iron chelators improve erithropoiesis by reduction of ROS as described Messa et al. Deferasirox is a powerful NF-κB inhibitor in myelodysplastic cells and in leukemia cell lines acting independently from cell iron deprivation by chelation and reactive oxygen species scavenging.. The available iron chelators can: enter cells and complex iron, reduce LIP (Liver Iron Pool) ROS promotion, remove iron from cells and restore affected functions. However, DFX appears independent of effective iron chelation therapy; improvement occurs very early during treatment. In fact the hematologic responce with this drug appears after 3 months, instead of 6 and 9 months respectively with DFO and DFO-DFX together. Which are additional mechanism of Deferasirox?
The off target effects of Deferasirox include: effectively blocks NF-кB activity, inhibits mTOR activity, increase of chemosensitivity, decrease of fungal infections.
NF-Кb inactivation In the early stages of MDS deferasirox doesn’t act on NF-Кb , because this target isn’t activated. Instead in the later stages of MDS NF-Кb is activated and its inhibition by the drug induces apoptosis and reduces the formation of blast cells, that would increase in the later stages characterized by anti-apoptosis. Nuclear factor- кB is a key regulator of many cellular processes and its impaired activity has been described in different myeloid malignancies including MDS. It consists of a small group of proteins that, if associated with the inhibitory complex IKK, maintains an inactive state of the p65 subunit and a cytoplasmic localization. After proteasomal degradation of this negative regulatory protein, NF-кB is able to enter the nucleus. In MDS patients increased activity is strictly related to the clonal population in the bone marrow. The most important stimulo for NF-кB activation is the tumor necrosis factor (TNF) receptor signalling pathway. However, TNF signaling in cells results in a subtle balance between survival and death. Infact, NF-κB activation mediated by TNF leads to an antiapoptotic effect through both caspase and JNK (Jun N-terminal Kinase) cascade inhibition, while reactive oxygen species accumulation induced by the same stimulus leads to cell death through JNK activation. The activation of NF-кB can be observed in both polytransfused MDS and MDS non-transfused patients. In the first group the amount of the iron intake leads to ROS generation, in the second group this event is explained with involvement of the ROS in the pathogenesis of the disease (as demonstrated by Padua et al. in a mouse model) BCL-2 and mutant NRAS interact physically and functionally in a mouse model of progressive myelodysplasia.. Messa et al. Deferasirox is a powerful NF-κB inhibitor in myelodysplastic cells and in leukemia cell lines acting independently from cell iron deprivation by chelation and reactive oxygen species scavenging.. demonstrated the activity of Deferasirox as an NF- кB inhibitor. They tested the drug in two types of leukemia cell line (K562 and HL60) characterized by high basal NF-κB activity. They then tested NF-κB activity in 40 peripheral blood samples of MDS and AML secondary to MDS (sAML) patients. In 28 of 40 peripheral blood samples with high basal NF-κB activity, deferasirox incubation induced a significant inhibition of NF-κB activity and a cytoplasmic sequestration of its active subunit p65 in an inactive form. Finally, they investigated if other commercially available oral chelators share the same effect: neither deferiprone nor deferioxamine incubation with either the HL60 or K562 cell lines reduced NF-κB activation despite a similar reactive oxygen species clearance. Two leukemia cell lines (K562 and HL60) were incubated with 50 μM deferasirox for 18h and were subsequently evaluated for NF-κB activity and p65 localization. Immuno-fluorescence assays (Figure 9) using a p65 antibody (green fluorescence) show both a cytoplasmic and nuclear localization of the active NF-κB subunit in basal conditions whereas; after drug incubation, p65 localization was mainly cytoplasmic (inactive form). Western blotting (Figure 10) shows a significant decrease in the amount of p65 in nuclear extracts after deferasirox incubation.
Figure 9: Messa E et al. Haematologica 2010;95:1308 - 1316
Figure 10: Messa E et al. Haematologica 2010;95:1308 - 1316
In low-risk MDS patients DFX leads to a reduction in ineffective erythropoiesis induced by a malignant clone, while in sAML or high-risk MDS, the drug reduces the percentage of blast cells through an increase in apoptosis. A positive correlation was found between NF-κB activity and both blast percentage (r=0.75, P<0.0001) and reactive oxygen species levels in mononuclear cells (r=0.82, P<0.0001), but not between NF-κB and serum ferritin amount (r=0.12, P=0.42). Among patients with increased basal NF-κB activity (n=28), the incubation with deferasirox induced a significant NF-κB inhibition (P=0.0002). This effect has been observed both in patients with or without iron overload. In summary, NF-κB inhibition is a particular and iron independent effect of deferasirox, not shared by other chelators and not relying on the reactive oxygen species scavenging properties of the drug. This observation offers new insights into iron chelation therapy in myelodysplastic patients, which is not only essential for secondary hemosiderosis prevention, but also acts as a targeted therapy on malignant clones. This could be associated with other therapeutic options.
mTOR inhibition Deferasirox might have benefit for not only iron chelation but also be an antiproliferative agent in some myeloid leukemia cells, especially in patients with myelodysplastic syndrome who need both iron chelation and reduction of leukemia cells. Junko H. Ohyashiki’s group study focus on the pathway involved in the anticancer effect of deferasirox. Iron is critical for DNA synthesis and energy production and neoplastic cells require more iron for their rapid proliferation. Iron depletion inhibits iron-containing enzymes, ribonucleotide reductase, and up regulates proapoptotic proteins, Bax, caspase-3, 8 and 9. The antiproliferative effect of iron chelating agents has been well recognized. Chantrel-Graussard et al. further demonstrated that deferasirox induced cell cycle blockade in the G2-M phase and inhibited polyamine biosynthesis by decreasing ornithine decarboxylase and spermidine N1-acethyltransferase activities and decreasing ornithine decarboxylase mRNA level. They concluded that deferasirox has powerful antineoplastic effects and blocks cell proliferation in neoplastic cells by a pathway different from that of other iron chelators. However, they only refer to a limited number of reports regarding antiproliferative effect on human leukemia cells. The new orally active iron chelator ICL670A exhibits a higher antiproliferative effect in human hepatocyte cultures than O-trensox.. In Ohyashiki’s study at first have been examined the effects of deferasirox in vitro in various myeloid leukemia cells by a cell-counting assay. To determine whether or not the cell death induced by deferasirox was due to apoptosis in myeloid leukemia cell lines, K562, U937, and HL60, they measured the activity of caspase-3/7 by a Caspase-Glo 3/7 kit (Promega). The number of viable cells were counted after 24h exposure to deferasirox, in order to normalize the caspase-3/7 activity with respect to the number of cells per well. In all three leukemia cell lines tested, the activity of caspase-3/7 significantly increased after 50 μM deferasirox exposure. To further understand how deferasirox induced cell death in human myeloid cells, K562 cells were treated with deferasirox or control for short time-periods, and microarray analysis was performed using a GeneChip (GEO, GPL570). Differential expression was analyzed using a GeneSifter®. All the microarray data was deposited in GEO. The salient features of up-regulated genes are summarized as follows: first, up-regulation of genes related to cell-cycle regulation was evident; cyclin G2 and cyclin-dependent kinase inhibitor 1A (CDKN1A) encoding p21, CDK-interacting protein1 (Cip). Second, genes regulating interferon were also up-regulated: interferon-induced protein with teteratricopeptide repeat 1 (IFIT1, ISG56), IFIT3 (ISG 60), and interleukin 23 A (IL23A), which stimulate the production of interferon-γ. Third, genes related to apoptosis, such as inhibin-β, B-cell lymphoma (BCL6), pleckstrin homolog-like domain family A member 1 (PHLDA1), Bcl2/adenovirus E1B19-kDa proteininteracting protein 3-like (BNIP3L), tribbles homolog 3 (TRIB2),a negative regulator of NF-κB, were up-regulated. Fourth, growth differentiation factor 15 (GDF15), which is currently known as a negative regulator of the iron regulatory protein hepcidin, is remarkably up-regulated – the measure of this marker is difficult, so results are not correct probably–. Finally, it is notable that genes closely related to the oxygen regulatory system, including those regulated in development and DNA damage responses 1 (REDD1, also known as a HIF-1 responsive protein, RTP801), and phosphoglycerate dehydrogenase (PHGDH), which is related to NO metabolism, are up-regulated. Finally has been found down-regulation of solute carrier family 5 member 6 (SLC5A6), which is related to iron-transport. This gene expression profiling in Deferasirox-treated K562 cells clarified up-regulation of several pathways which may reflect molecular mechanisms of iron chelator in human myeloid leukemia cells. Based on the results obtained from the differential expression pattern, the study particularly focused on a gene closely related to oxygen regulation, REDD1. To determine whether upregulation of REDD1 takes place ubiquitously in the antitumor activity of deferasirox, has been examined the change of REDD1 expression by real-time RT-PCR in three human myeloid leukemia cell lines. Cells were treated with or without deferasirox (10 μM and 50 μM) for 24 h, and total RNA was collected. The REDD1 expression remarkably increased after deferasirox treatment with a more than 2-fold increase of REDD1 expression at 50 μM deferasirox, in all three leukemia cell lines. The study also examined REDD1 expression in four samples obtained from AML patients. Although the degree of increased REDD1 expression varies among the samples, REDD1 expression was up-regulated after deferasirox treatment in some freshly obtained samples from AML patients. What about REDD1/TSC (tuberous sclerosis complex) pathway, which modulate mTOR signalling? REED1 is a recently identified stress response gene and it’s strongly induced by hypoxia. It can activate TSC2 protein. TSC is composed of two proteins: TSC1 (also known as hamartin) and TSC2 (also known as tuberin), which function to integrate growth factors and cell stress responses. It has been shown that the major function of the TSC1/2 complex is to inhibit the checkpoint protein kinase mTOR, a major regulator of cell death and proliferation. So an important mechanism through which mTOR signaling is regulated involves the Tuberin-Hamartin complex. Has been found up-regulation of TSC2 (tuberin) in accordance with REDD1 in deferasirox-treated K562 cells. Since TSC1 is regulated by V-AKT murine thymoma viral oncogene homolog 1 (AKT), the study examined the AKT expression in deferasirox-treated K562 cells. However, AKT protein expression was not altered after deferasirox treatment. This indicates that TSC2 is up-regulated through the REDD1/TSC2 pathway, rather than the AKT/TSC2 pathway. Subsequently, phosphorylated mTOR, and phosphrylated-p70S6kinase, decreased in a dose dependent manner. Has been noted a dosedependent decrease of phophorylated-S6 protein, which is known as a downstream effecter of mTOR, in K562 cells treated with 50 μM of deferasirox , indicating that deferasirox inhibits ribosomal S6 via mTOR pathway in K562 cells. Downregulation of phosphorylated ribosomal S6 protein was also found in deferasirox-treated U937 and HL60 cells. To assess whether or not the enhanced expression of REDD1 mRNA was necessary for repression of mTOR signaling, siRNAs direct against the human REDD1 mRNA were used to reduce its expression in K562 cells in the presence or absence of deferasirox. Treatment of REDD1 siRNA caused a reduction in REDD1 expression to ~50% of the value observed in untreated cells or cells that had been treated with control siRNA. Moreover, treatment of REDD1 siRNA prior to deferasirox dramatically attenuated the drug-induced expression of REDD1. Notably, deferasirox induced decrease in S6K1 phosphorylation was blocked by REDD1 siRNA treatment. In contrast, the control siRNA had no effect on the deferasirox-induced decrease in S6K1 phosphorylation. Deferasirox moreover supresses heterotransplated tumor growth in nudemice bearing myeloid leukemia cells. To further study the activity of deferasirox on tumor growth in vivo, they tested a mouse model of human myeloid leukemia. Subcutaneous injection of U937 cells into nude mice resulted in an aggressive malignancy resembling acute leukemia, characterized by tumor, splenomegaly, and invasion of leukemia cells into hematopoietic and non-hematopoietic tissue: deferasirox-treated mice tended to survive longer than those with saline (P = 0.2450). The tumor volume of the subcutaneous tumors was significantly smaller in mice treated by deferasirox compared to those with vehicle alone (P < 0.0001). No deferasirox-treated mice showed any adverse events. Histopathological analysis of xenotransplant mice revealed infiltration of the spleen and bone marrow with leukemic blasts. In contrast, deferasirox-treated mice demonstrated distinct morphological changes, including condensed nucleoli and an increasing number of apoptotic cells detected by the TUNEL method. These results indicate that deferasirox yields a desirable therapeutic index that can reduce the in vivo growth of myeloid leukemia cells in an efficacious manner. Blockage of the REDD1 expression by siRNA resulted in restoration of mTOR and phosporylation of S6 protein in deferasirox treated leukemia cells, indicating that the pathway involving mTOR might be important for cytotoxicity in the presence of iron chelating agents. These data provide valuable insights for novel therapeutic approaches aimed at the REDD1/mTOR pathway (in fact mTOR pathway is altered in many kinds of cancer) in human myeloid leukemia cells by means of iron chelation. The oral iron chelator deferasirox repress signalling through the mtor in myeloid leukemia cells by enhancing expression of REDD1..
Figure 11: Ohyashiki et al. Cancer Science 2009;100: 970-977
Increase of chemosensitivity DFX with inhibition of NF-κB enhances the activity of chemotherapeutic agents. This effect is demonstrated in the same study of Messa et al. previously mentioned. K562 cells were incubated with either 10 μM etoposide alone or preceded by deferasirox 50 μM for 18h. The percentage of apoptotic cells increased in a statistically significant manner in the sample incubated simultaneously with both drugs (P=0.003). Apoptosis was evaluated by flow cytometry for the detection of annexin V-positive cells.
Decrease of fungal infections Iron is an important metal, essential for the organisms’ development, for this reason, many pathogens have developed a system of uptake of iron. In this way iron contributes to the pathogenesis of infections. Patients included in Maltuzzi’s prospective observational study have AML or High-risk MDS and have undergone induction or first salvage chemotherapy. Serum ferritin levels were measured at baseline, and considered normal if they fell between 10 and 291 ng/mL. To the authors’ knowledge, this is the first report that shows that serum baseline ferritin can be used as predictor of infection, including bacterial, fungal and non-microbiologically documented infections in patients with acute leukemia. Acute pulmonary failure during remission induction chemotherapy in adults with acute myeloid leukemia or high-risk myelodysplastic syndrome.. Iron chelation therapy with Deferasirox in MDS patients is able to reduce iron, id est pathogens’s fuel, and so decreases the risk of infections, especially fungal infections.
In conclusion, it’s possible saying that iron chelation therapy is not a simple iron removal, but something more in consideration of off target effects of Deferasirox. This effects could improve survival of MDS patients. Despite all these evident benefits of chelation therapy,however, there is still a question whether iron chelation has an effect on the long term survival of MDS patients, since the available data on the effect of iron chelation on survival are based above on retrospective studies on low risk patients, who have a longer life expectancy and so the cumulative effects of transfusion dependence may develop overtime. As a consequence, prospective studies, also incorporating alternative biomarkers of iron metabolism, are needed to improve our understanding of the role of iron chelation in MDS patients. For example, the TELESTO clinical study is an ambitious industry-sponsored, randomized, prospective, placebo-controlled trial of deferasirox Iron Chelation Therapy (ICT) in transfusion-requiring patients who have lower-risk MDS, an elevated serum ferritin level, and preserved renal and hepatic function. The TELESTO study is projected to enroll 630 patients, who will be treated with either placebo or deferasirox for up to 5 years. Although convincing patients to commit to a lengthy placebo-controlled trial may be challenging, legitimate clinical equipoise exists about the role of ICT in MDS, and the data obtained will be critical for answering questions about the potential benefits and risks of ICT in MDS. Additional studies required to further determine the impact of iron chelation in MDS on: overall survival, leukemia free-survival and impact on hematopoiesis.
Sara Lo Pumo e Sara Leoncini
