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Myelodysplastic syndromes (MDS) result from a malignant stem cell clone characterized by ineffective hematopoiesis, manifested as peripheral cytopenia with a cellular bone marrow. A leading hypothesis is that MDS results from a breakdown in the control of myeloid cell proliferation and apoptosis. Through its generation of 3′-phosphoinositides and subsequent activation of effectors such as Akt, phosphatidylinositol 3-kinase (PI 3-kinase) drives cell proliferation, apoptosis, differentiation, and motility. We show here that PI 3-Kinase is profoundly deregulated in high-risk MDS. Bone marrow cells from high-risk MDS patients displayed a 3–30 fold increase in constitutive activation of the Src kinase Lyn. Constitutive serine/threonine phosphorylation of Akt, a surrogate of PI-3 kinase activity, was seen in all specimens. Protein levels of PTEN, which dephosphorylates the D3-position phosphate of the inositol ring, were variably decreased. Since PTEN is frequently silenced by hypermethylation, we treated U937, THP-1, and Mo7e cells with 5-azacytidine. However, real-time PCR showed no increase in PTEN transcripts in these cell lines. More significantly, protein expression of SHIP-1, which dephosphorylates the D5-position phosphate of the inositol ring, was markedly decreased or absent in bone marrow cells from MDS patients, whereas they were present in AML or ALL blasts. Real-time PCR showed SHIP-1 transcripts in MDS cells to be 50% of normal CD34+ stem cells. Although 5-azacytidine treatment resulted in an increase in SHIP-1 transcripts, as measured by quantitative PCR, protein levels did not increase. Because SHIP-1 contains three PEST-rich regions, we hypothesized that the low or absent level of expression of SHIP-1 may due to protein degradation. Neither the cryopreservation of cells nor lysis buffer could explain the absence of SHIP-1 protein. Instead, when U937 and HL60 leukemic cell lines were treated with the proteastome inhibitor lactacystin (1–5 uM), protein levels of SHIP-1 increased. These results suggest that constitutive activation of Akt is likely due to decreased PTEN and absent SHIP-1 protein levels in primary MDS cells. Consistent with these findings, mice deficient in PTEN and SHIP-1 suffer from anemia, thrombocytopenia, leukocytosis and impaired function of myeloid progenitors (BLOOD103:4503, 2004). These results also suggest that a combination of a Src kinase inhibitor and a proteasome inhibitor may be a therapeutic approach for the treatment of high-risk MDS.