ROLE OF INOSITIDE-DEPENDENT SIGNAL TRANSDUCTION PATHWAYS ON EPO-INDUCED EYTHROID DIFFERENTIATION IN LOW-RISK MDS PATIENTS

Introduction. An impaired regulation of the PI3K/Akt axis is often associated with hematologic malignancies. Our group previously demonstrated PI-PLCbeta1 and Akt are inversely correlated in high-risk MDS. EPO treatment is currently used in the therapy of low-risk MDS, to compensate and counteract their ineffective erythropoiesis, although some patients do not respond to this treatment, or lose response. The activation of the EPO receptor has been linked to the activation of the PI3K/Akt/PI-PLCgamma1 axis, so that EPO could affect cell proliferation and apoptosis. Methods. Here we studied 16 MDS patients (IPSS risk: low or intermediate-1) and quantified the expression of several genes implicated in inositide signalling, that is PI-PLCbeta1 splicing variants and its downstream target Cyclin D3, as well as PI-PLCgamma1 and Beta-Globin, in order to assess the effect of EPO therapy. Moreover, we also studied the effect of EPO on Akt activation. Finally, to further investigate the role of PI-PLCbeta1 in erythroid differentiation, we exposed normal human CD34+ cells to EPO and analyzed the effect of PI-PLCbeta1 overexpression on Beta-Globin and PI-PLCgamma1 expression, as well as on Akt phosphorylation. Results. 8 out of 16 patients (50%) showed a favourable response to EPO, which was associated with a specific induction of Beta-Globin gene expression within the first two months of therapy. As for PI-PLCbeta1 and Cyclin D3 gene expression levels, the PIPLCbeta1/Cyclin D3 axis is down-regulated in EPO responder patients after 3-4 months of therapy. Moreover, not only Akt phosphorylation, but also PI-PLCgamma1 gene and protein expression increased during EPO treatment, therefore confirming the activation of the Akt/PIPLCgamma1 pathway in EPO responder patients. Finally, in normal CD34+ cells induced to erythroid differentiation, PI-PLCbeta1 overexpression abrogated both EPO-induced Akt phosphorylation and BetaGlobin expression. Conclusions. In our EPO responder patients the PIPLCbeta1/Cyclin D3 axis is down-regulated after 3-4 months of therapy. This is consistent with previous findings showing that PI-PLCbeta1, after an early transient increase, is down-regulated in primary human erythroblasts treated with EPO for up to 96 hours, therefore suggesting that PI-PLCbeta1 could be required at the beginning of erythroid differentiation but is dispensable, if not inhibitory, at later stages. These results, along with the fact that EPO responders displayed a specific phosphorylation of Akt, suggest that PI-PLCbeta1 can act as a negative regulator of erythroid differentiation and confirm the involvement of the Akt/PI-PLCgamma1 pathway in EPO signalling, therefore contributing to the comprehension of the effect of EPO in low-risk MDS and possibly paving the way to the identification of MDS patients at higher risk of refractoriness to EPO treatment.