The effects of secondary iron overload and iron chelation on a radiation-induced acute myeloid leukemia mouse model.

BACKGROUND: Patients with myelodysplastic syndrome (MDS) require chronic red blood cell (RBC) transfusion due to anemia. Multiple RBC transfusions cause secondary iron overload and subsequent excessive generation of reactive oxygen species (ROS), which leads to mutations, cell death, organ failure, and inferior disease outcomes. We hypothesize that iron loading promotes AML development by increasing oxidative stress and disrupting important signaling pathways in the bone marrow cells (BMCs). Conversely, iron chelation therapy (ICT) may reduce AML risk by lowering iron burden in the iron-loaded animals. METHODS: We utilized a radiation-induced acute myeloid leukemia (RI-AML) animal model. Iron overload was introduced via intraperitoneal injection of iron dextran, and iron chelation via oral gavage of deferasirox. A total of 86 irradiated B6D2F1 mice with various levels of iron burden were monitored for leukemia development over a period of 70 weeks. The Kaplan-Meier estimator was utilized to assess AML free survival. In addition, a second cohort of 30 mice was assigned for early analysis at 5 and 7 months post-irradiation. The BMCs of the early cohort were assessed for alterations of signaling pathways, DNA damage response and gene expression. Statistical significance was established using Student's t-test or ANOVA. RESULTS: Iron loading in irradiated B6D2F1 mice accelerated RI-AML development. However, there was a progressive decrease in AML risk for irradiated mice with increase in iron burden from 7.5 to 15 to 30 mg. In addition, ICT decreased AML incidence in the 7.5 mg iron-loaded irradiated mice, while AML onset was earlier for the 30 mg iron-loaded irradiated mice that received ICT. Furthermore, analysis of BMCs from irradiated mice at earlier intervals revealed accelerated dysregulation of signaling pathways upon iron loading, while ICT partially mitigated the effects. CONCLUSIONS: We concluded that iron is a promoter of leukemogenesis in vivo up to a peak iron dose, but further iron loading decreases AML risk by increasing cell death. ICT can partially mitigate the adverse effects of iron overload, and to maximize its benefit this intervention should be undertaken prior to the development of extreme iron overload.

Intracellular ROS profile in hematopoietic progenitors of MDS patients: association with blast count and iron overload.

Objectives: Reactive oxygen species (ROS) are under scrutiny as a participant in the pathophysiology of myelodysplastic syndrome (MDS) and the progression of MDS to acute myeloid leukemia (AML). Measurement of intracellular ROS (iROS) is particularly important since iROS is a direct indicator of cellular health and integrity.Methods: We developed a technique to measure standardize iROS (siROS) level in lymphocytes and bone marrow (BM) CD34+ hematopoietic progenitors using the fluorescent probe dichlorofluorescein (DCF). We then quantified the siROS in 38 consecutive BM specimens from 27 MDS patients over the course of 10 months. Disease outcome of these patients were also assessed.Results: High serum ferritin, high blast count and poor IPSS were associated with inferior survival and AML progression in this cohort. High blast MDS patients had lower siROS in their BM CD34+ cells than those of low blast patients, consistent with increased reliance on glycolysis and enhanced ROS defense in high blast MDS. We also observed narrower siROS distribution in the BM CD34+ cells of high blast patients, suggesting that loss of heterogeneity in ROS content accompanies the clonal evolution of MDS. Furthermore, we observed a strong correlation between CD34+ cells siROS and serum ferritin level in high blast patients. In one case, iron chelation therapy (ICT) resulted in parallel decreases in serum ferritin and CD34+ cells siROS.Conclusion: Our findings established the siROS profile in early hematopoietic cells of MDS patients and its relationship with blast count and iron overload.

The orphan nuclear receptor EAR-2 (NR2F6) inhibits hematopoietic cell differentiation and induces myeloid dysplasia in vivo.

BACKGROUND: In patients with myelodysplastic syndrome (MDS), bone marrow cells have an increased predisposition to apoptosis, yet MDS cells outcompete normal bone marrow (BM)-- suggesting that factors regulating growth potential may be important in MDS. We previously identified v-Erb A related-2 (EAR-2, NR2F6) as a gene involved in control of growth ability. METHODS: Bone marrow obtained from C57BL/6 mice was transfected with a retrovirus containing EAR-2-IRES-GFP. Ex vivo transduced cells were flow sorted. In some experiments cells were cultured in vitro, in other experiments cells were injected into lethally irradiated recipients, along with non-transduced bone marrow cells. Short-hairpin RNA silencing EAR-2 was also introduced into bone marrow cells cultured ex vivo. RESULTS: Here, we show that EAR-2 inhibits maturation of normal BM in vitro and in vivo and that EAR-2 transplant chimeras demonstrate key features of MDS. Competitive repopulation of lethally irradiated murine hosts with EAR-2-transduced BM cells resulted in increased engraftment and increased colony formation in serial replating experiments. Recipients of EAR-2-transduced grafts had hypercellular BM, erythroid dysplasia, abnormal localization of immature precursors and increased blasts; secondary transplantation resulted in acute leukemia. Animals were cytopenic, having reduced numbers of erythrocytes, monocytes and granulocytes. Suspension culture confirmed that EAR-2 inhibits granulocytic and monocytic differentiation, while knockdown induced granulocytic differentiation. We observed a reduction in the number of BFU-E and CFU-GM colonies and the size of erythroid and myeloid colonies. Serial replating of transduced hematopoietic colonies revealed extended replating potential in EAR-2-overexpressing BM, while knockdown reduced re-plating ability. EAR-2 functions by recruitment of histone deacetylases, and inhibition of differentiation in 32D cells is dependent on the DNA binding domain. CONCLUSIONS: This data suggest that NR2F6 inhibits maturation of normal BM in vitro and in vivo and that the NR2F6 transplant chimera system demonstrates key features of MDS, and could provide a mouse model for MDS.

Initial transfusion intensity predicts survival in myelodysplastic syndrome.

We evaluated 52 patients with myelodysplastic syndrome (MDS) who had received at least one red blood cell (RBC) transfusion. In the 4-week period following the first transfusion, 24 patients (group 1) required no transfusion, while 28 (group 2) required transfusion of two or more units of RBCs. Survival was greater in group 1 (440 weeks vs. 167 weeks, p < 0.01), even when only international prognostic scoring system (IPSS) low and intermediate-1 risk patients were analyzed (median overall survival 491 vs. 170 weeks, p < 0.05), independent of age, IPSS and progression to acute myeloid leukemia (AML). The intensity of transfusion required in the first few weeks after the first transfusion predicts disease severity and correlates with survival.

Cross-Talk between PPARs and the Partners of RXR: A Molecular Perspective.

The PPARs are integral parts of the RXR-dependent signaling networks. Many other nuclear receptor subfamily 1 members also require RXR as their obligatory heterodimerization partner and they are often co-expressed in any given tissue. Therefore, the PPARs often complete with other RXR-dependent nuclear receptors and this competition has important biological implications. Thorough understanding of this cross-talk at the molecular level is crucial to determine the detailed functional roles of the PPARs. At the level of DNA binding, most RXR heterodimers bind selectively to the well-known "DR1 to 5" DNA response elements. As a result, many heterodimers share the same DR element and must complete with each other for DNA binding. At the level of heterodimerization, the partners of RXR share the same RXR dimerization interface. As a result, individual nuclear receptors must complete with each other for RXR to form functional heterodimers. Cross-talk through DNA binding and RXR heterodimerization present challenges to the study of these nuclear receptors that cannot be adequately addressed by current experimental approaches. Novel tools, such as engineered nuclear receptors with altered dimerization properties, are currently being developed. These tools will enable future studies to dissect specific RXR heterodimers and their signaling pathways.