The Association Between STAT5a Activation, LEF-1 Down Regulation and Leukemic Transformation in Patients with Severe Congenital Neutropenia

Congenital hematological disorders are excellent models for investigating the regulation of hematopoiesis in humans. Severe Congenital Neutropenia (CN) is a heterogeneous syndrome characterized by a maturation arrest of granulopoiesis at the level of promyelocytes with no mature neutrophils in the peripheral blood. Even though G-CSF treatment results in increased neutrophil numbers in more than 90% of CN patients, G-CSF-dependent granulocytic differentiation is severely affected in these patients. CN patients are found to be at increased risk of developing acute myeloid leukemia or myelodysplastic syndrome (AML/MDS) (cumulative incidence ˜ 20%). Since AML is not observed in cyclic neutropenia patients treated with G-CSF, an underlying defect down stream of G-CSF signaling rather than G-CSF therapy per se predisposes to malignant transformation in CN. Activated STAT3 and STAT5 are known to be the key factors involved in the G-CSF – G-CSFR mediated signaling cascades. Also STAT5 has been found to be activated in AML. Recently we found that downregulation of LEF-1 transcription factor and its target gene C/EBPα are a common pathomechanism for CN. Therefore, we investigated the effects of G-CSF on the phosphorylation status of STAT5a in CN and whether it leads to the down modulation of LEF1 expression and functions. Indeed, we detected elevated phosphoSTAT5 levels in CD34+ cells from CN patients before G-CSF stimulation in vitro, as compared to these cells from healthy individuals. Moreover, treatment with G-CSF resulted in a significantly higher phosphorylation of STAT5a in CN, as compared to the control cells. Intriguingly, levels of phosphoSTAT5 in myeloid blast cells from one CN patient who developed AML was even higher and was in line with undetectable LEF-1 protein expression. Transduction of constitutive active STAT5a (STAT5a 1*6) in CD34+ cells from healthy individuals resulted in significant downregulation of LEF-1 gene expression. A screen of 10 kb upstream region of LEF-1 gene revealed two putative STAT5 binding sites [(-3891bp to -3909bp) and (-3714bp to -3732bp)] and the specificity of this binding was confirmed in the nuclear extracts of CD34+ cells by chromatin immunoprecipitation assay. We found enhanced and prolonged STAT5a binding to the LEF-1 promoter in G-CSF treated CD34+ cells from CN patients, as compared to healthy individuals. Additionally, transfection of CD34+ cells with LEF-1 cDNA resulted in elevation of LEF-1 promoter activity, which suggests a strong LEF-1 autoregulation. Co-transfection with STAT5a 1*6 significantly disrupted LEF-1-dependent activation of LEF-1 promoter. The significance of STAT5 binding and inhibition of LEF-1 autoregulation was further analyzed by using LEF-1 gene promoter constructs having mutated STAT5 binding sites or LEF-1 binding sites and there by performing reporter gene assays. Our preliminary data indicates that the deletion of STAT binding sites upstream of LEF-1 gene promoter or disruption of the LEF-1 autoregulation abrogated the STAT5a dependent inhibition of LEF-1 gene expression. Moreover STAT5a 1*6 severely abrogated the LEF-1 dependent regulation of C/EBPα gene promoter. Taken together phosphorylation of STAT5 is upregulated in hematopoietic progenitors from CN patients which lead to subsequent down regulation of LEF-1.