Consequent Iron Chelation Therapy in Patients with Low Risk Myelodysplastic Syndrome Leads to up-Regulation of .CXCL12, JAK2 and HSF2: Possible Link Between Iron Chelation and Improvement of Erythropoiesis.

Abstract 2787

Poster Board II-763

Myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis and an increased risk of evolution to acute myeloid leukemia. The majority of MDS patients will depend on regular transfusions of packed red blood cells (PRBC) during their course of disease due to symptomatic anemia. Since recurrent transfusions of PRBC will result in iron overload with the risk of damage of organs such as heart, endocrine glands and the liver, consequent iron chelation therapy (IC) became an important element of supportive care in MDS patients. Recently, the availability of the oral iron chelator deferasirox provides a convenient management of iron overload in MDS. Since intensive IC has been shown to improve hematopoiesis in iron overloaded patients we performed gene expression profiling on patients with low or intermediate MDS prior and after IC, to elucidate wheter IC leads to alteration of genes involved in hematopiesis, in particular in erythropoiesis. Heparinized bone marrow samples were obtained after informed consent from 6 MDS patients (2 refractory anemia, 4 refractory anemia with ringed sideroblasts) upon initial diagnosis of iron overload (prior IC) and after a period of 1 year of iron chelation (after IC) with the oral iron chelator deferasirox. CD34+ hematopoietic progenitor as well as CD71+ erythroid progenitor cells were isolated by high gradient magnetic cell separation (Miltenyi Biotech, Bergisch Gladbach, Germany). RNA was extracted from CD34+ cells and CD71+ cells using TRIzol reagent (Invitrogen, Life Technologies, Grand Island, NY) according to the manufacturer's protocol. Quality controlled RNA was hybridized according to the standard Affymetrix protocol to HG-U133 Plus 2.0 microarrays. Data analysis was performed using the Gene Spring Software version 4.0 (Silicon genetics, San Carlos, CA). Restrictions were set as follows: only genes that were ‘present' in at least 75% of samples were used for further analyses, genes were considered as ‘differentially expressed' when they showed at least 3 fold change between the different groups. Statistical significance was calculated by non-parametric t-test, with P < 0.05. In a first step we compared gene expression patterns of CD71+ cells in MDS patients prior and after IC. In total 106 probe sets representing unique genes, hypthetical proteins and open reading frames matched the restriction settings. In an intensive survey on these genes we identified several genes that have been associated with erythropoiesis including Stromal derived factor-1 (CXCL12), Janus kinase 2 (JAK2), and Heat shock transcription factor 2 (HSF2). To exclude that these changes in gene expression where due to the natural course of the disease in specific patients, we compared gene expression of CD71+ cells from patients after IC to an independent test set of CD71+ MDS samples (n=12). Interestingly, we still found an aberrant expression of these genes, indicating that the observed gene expression changes were related to the IC in these patients rather than to the natural course of diesease. However, we were not able to find an altered expression of these genes in CD34+ progenitor cells prior and after IC, suggesting that the effect on gene expression is restricted to CD71+ cells. Iron overload is an inevitable side effect of regular blood transfusions in MDS patients. Intensive IC has been shown to improve erythropoiesis in iron overloaded patients. We found, that IC results in upregulation of Stromal derived factor-1, Janus kinase 2 and Heat shock transcription factor 2 all of them known to regulate hematopoiesis. Moreover, HSF2 and JAK2 have been closely involved in regulation of erythropoiesis. JAK2 deficiency has been shown to result in abrogated erythropoiesis and therefore increase of JAK2 expression after iron chelation might link IC to improvement of erythropoiesis and subsequently decrease of transfusion requirement in some patients receiving IC.