Downregulation of IRS2 in Myelodysplastic Syndrome (MDS) May Be Related to Impaired Cell Differentiation,

Abstract 3814

Myelodysplastic syndromes (MDS) encompass a heterogeneous group of clonal hematopoietic stem cell disorders, characterized by ineffective hematopoiesis, refractory cytopenia and a tendency to progress towards acute myeloid leukemia. Abnormalities in cell differentiation are probably involved in the pathogenesis of MDS. Insulin receptor substrates (IRS) are adaptor proteins that link signaling from upstream activators to downstream effectors to modulate normal growth, metabolism, survival and differentiation. IRS2, a member of the IRS family, binds to Insulin Grow Factor 1 receptor (IGF1R), the Erythropoietin receptor (EPOR) and Thrombopoietin receptor (MPL). Recently, we reported that IRS2 was downregulated in MDS cells compared to normal cells, and in high risk compared to low-risk MDS. Additionally, IRS2 was significantly upregulated during erythroid differentiation of normal CD34⁺ cells, but not in MDS CD34+ cells. In view of the role of IRS2 in EPO, IGF1 and TPO signaling, and possibly in erythroid, granulocytic, and megakaryocytic differentiation, we hypothesized that IRS2 might be related to the impaired hematopoiesis of MDS cells. The aim of the present study was to correlate the mRNA expression levels of IRS2 in bone marrow from MDS patients with laboratorial and clinical data. We also elucidated the expression levels of IRS2 during megakaryocytic, granulocytic and erythroid differentiation of established leukemia cell line models. We studied 12 healthy donors and 32 patients with MDS at the time of diagnosis (16 low-risk [RA/RARS] and 13 high-risk [RAEB/RAEBt] according to FAB classification). Gene expression was evaluated by Q-PCR in total cells from bone marrow samples and cell lines. IRS2 protein expression and phosphorylation was evaluated by Western blot and immunoprecipitation. K562 cells were induced to megakaryocytic differentiation by treatment with 20nM PMA for 4 days. NB4 cells were induced to granulocytic differentiation by treatment with 10⁻⁶ M ATRA for 4 days. KU812 cells were induced to erythroid differentiation by treatment with 50μM HE and 100 μM HU for 4 days. Spearman correlation analysis showed that IRS2 expression demonstrated a significant positive correlation with peripheral blood neutrophils (r =0.43, P =0.01) and platelets counts (r =0.36, P =0.04), and bone marrow granulocytes percentages (r =0.40, P =0.02). In contrast, a significant negative correlation was observed between IRS2 expression with bone marrow erythroblasts (r =-0.360, P =0.04) and myeloid precursor percentages (grouping myeloblast, promyelocytes, myelocytes, metamyelocytes and blasts) (r =-0.60, P <0.01). There was no correlation between IRS2 expression with age, hemoglobin, number of erythrocytes in the peripheral blood, and number of dysplasias in bone marrow. Finally, since IRS2 is involved with TPO, IGF1 and EPO signaling, and IRS2 expression was correlated with platelet, granulocyte and erythroblast numbers in MDS patients, we monitored IRS2 expression during hematopoietic cell differentiation. Interestingly, a significant increase in IRS2 expression and phosphorylation was observed in K562, NB4 and KU812 after megakaryocytic, granulocytic and erythroid differentiation, respectively. In summary, the lower expression of IRS2 in MDS and its upregulation during differentiation may suggest that defective IRS2 expression in MDS contributes to disease progression and impaired hematopoietic cell differentiation, a hallmark of the disease.