IRS2 Is Dowregulated In Primary MDS Cells and During MDS Erythroid Differentiation

Abstract 1886

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 (AML). The abnormal differentiation of myeloid and erythroid cells is 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) and Erythropoietin receptor (EPOR). It is upregulated and phosphorylated by EPO in normal bone marrow erythroblasts and in UT-7 leukemic cells, as well as in HL60 leukemic cells during granulocytic-differentiation upon DMSO induction. IGF1 signaling is capable of inducing differentiation in several cell types and plays an important role in the regulation of human erythropoiesis. EPO functions primarily as an erythroblast survival factor, and its antiapoptotic actions have been proposed to involve predominantly PI3-kinase and BCL-X pathways. In view of the role of IRS2 in the erythroid and granulocytic differentiation process, we hypothesized that IRS2 might be related to the deficient differentiation of MDS cells and disease progression. The aim of the present study was to characterize the mRNA and protein expression levels of IRS2 in cells of MDS patients and normal donors and to analyze the IRS2 expression levels between low-risk and high-risk of MDS. We also elucidated the expression levels of IRS2 transcripts during erythroid differentiation of CD34+ cells from normal donors and MDS patients. We studied 12 healthy donors and 29 patients with MDS at the time of diagnosis (16 low-risk [RA/RARS] and 13 high-risk [RAEB/RAEBt] according to FAB classification; 15 low-risk [RCUD/RCMD/RARS] and 11 high-risk [RAEB-1/RAEB-2] according to WHO classification; 22 low/INT-1 risk and 7 INT-2/high risk according to IPSS; 25 low risk and 4 intermediate/high risk cytogenetic). RT-PCR was performed in total cell from bone marrow samples for gene expression studies. Protein expression was evaluated by Western blot in bone marrow mononuclear cells from MDS patients or in peripheral blood CD34+ from normal donors. Erythroid-differentiation was performed in CD34+ bone marrow cells from 4 normal donors and 4 MDS patients. IRS2 gene expression was significantly decreased in primary MDS cells compared with normal cells (0.74 [4.06-0.15] vs. 4.71 [11.78-0.66], P<0.0001). Western blot analysis corroborated these findings. According to FAB and WHO classifications, real time RT-PCR demonstrated a significantly lower expression of IRS2 in high-risk MDS samples when compared with low-risk: FAB, 0.34 [0.15–1.56] vs. 1.05 [0.19–4.06], P=0.0172; WHO, 0.30 [0.15–1.44] vs. 1.00 [0.19–4.06], P=0.0204. Based on the IPSS classification and cytogenetic risk group, the expression levels of IRS2 were similar between the low and high-risk groups. During erythroid differentiation, we evaluated IRS2 gene expression of CD34+ cells on days 6, 8 and 12 of culture. On day 12 of normal CD34+ erythroid differentiation, there was an 8.25-fold increased in IRS2 expression, compared to day 6. Interesting, MDS CD34+ cells showed a lower increment in IRS2 transcripts at the same time point (3.89-fold increase only). In conclusion, the down regulation of IRS2 in primary MDS cells and the lower increase in its expression during MDS erythroid differentiation, suggest a role of IRS2 to maintain the effective hematopoiesis. Moreover, IRS2 lower expression in high-risk group, suggests that IRS2 plays a role in the MDS pathophysiology and disease progression. Supported by FAPESP and CNPq.