Abstract
Abstract 289MDS are characterized by a high risk of evolution into acute myeloid leukemia (AML). The pathogenesis of this evolution is still unclear. Some studies indicate that aberrant activation of survival signaling pathways is involved. The 90-kDa heat shock protein (HSP90) is implicated in the conformational maturation and stabilization of protein kinases and has key roles in signal transduction, protein folding, and protein degradation. HSP90 levels are increased in AML cells, and associated with resistance to chemotherapy induced apoptosis. Moreover, HSP90 is involved in the formation of focal adhesions. Focal Adhesion Kinase (FAK), a non-receptor tyrosine kinase, and a client of HSP90, is a member of the integrin-mediated signal transduction pathway. FAK was found over-expressed and constitutively activated in solid tumors. In AML, FAK expression is associated with enhanced blast migration and poor prognosis. FAK also exerts a potent antiapoptotic effect through adhesion to extracellular matrix and stromal cells. Finally 27-kDa heat shock protein (HSP27), a small HSP, prevents apoptosis by interfering with the mitochondrial pathway of apoptosis. In a cancer cell line, HSP27 has been shown to regulate cell adhesion via modulation of FAK. The aim of our study was to investigate the role of HSP90 in high-risk MDS and its potential role on focal adhesion. The expression of HSP90, HSP27, phosphorylated FAK (pFAK), and phosphorylated Akt (pAkt) was assessed by multicolor flow cytometry in bone marrow (BM) mononuclear (MNC) and CD34+ cells from 177 MDS samples at diagnosis: 96 refractory anemias with excess of blasts (RAEB), 58 refractory anemias (RA) and 23 chronic myelomonocytic leukemias (CMML). The levels of HSP90, HSP27, FAK, pFAK, and pAkt in MNC from RAEB patients, were significantly higher than in those measured in RA or CMML patients (p<10−5) (Table). The same difference was observed in CD34+ cells, showing that the increased levels observed in RAEB MNC were not a consequence of increased percentage of blasts. In control marrows (n=5), the percentages of positive cells in MNC and CD34+ populations were similar to that in RA. The percentages of HSP90, HSP27, FAK, pFAK and pAKT were significantly correlated with those of blasts and CD34+ cells, and tended to be higher in cases with high-risk cytogenetics. Consequently the risk of transformation into AML was significantly higher when these proteins were highly expressed. The effects of inhibition of HSP90 were evaluated in 30 RAEB samples by incubating MNC with 17-Alyl-amino-gelgenamycin (17-AAG) at concentrations of 1 and 5μM for 24 hours in liquid culture. A downregulation of HSP90, pFAK and pAKT was observed in CD34+ cells at 12 hours, followed by increased apoptosis at 24h as assessed by activated caspase 3 and annexin V staining. Our results suggest that FAK, HSP90 and Akt activation are associated with cell survival and may contribute to the progression of MDS to leukemia. Moreover this signaling network could be a therapeutic target through HSP90 inhibition by 17-AAG.HSP90HSP27FAKpFAKpAktMean percentage of MNC positive cellsRAEB (n=96)3937343227RA (n=58)811546CMML (n=23)2229201716Mean percentage of CD34+ positive cellsRAEB (n=96)6954537052RA (n=58)15157108CMML (n=23)3543262820 Disclosures:No relevant conflicts of interest to declare.
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