ALBERT

Description: AMLs constitute an heterogeneous group of hematopoietic stem cell disorders of rather poor prognosis. Abnormal activation of signaling pathways including STAT, MAPK and NF-KB have been described in AMLs and could constitute new targets for therapy. In this study, we focused on the PI3K/AKT and mTOR/P70S6K activity in primary blast cells purified from the bone marrow of patients with primary (n = 54) or secondary (n =10) AMLs. Patients with de novo AMLs were issued from the AML2001 French Multicenter Trial directed by the GOELAMS. The FAB classification was the following: M0 (n = 8), M1 (n = 10) M2 (n = 21), M4 (n = 10), M4eo (n = 2), M5 (n = 11), unknown AML (n = 2). In 58% of these patients, a constitutive activation of PI3K/AKT was detected in blast cells by western blot analysis showing AKT and FOXO3a phosphorylation, and confirmed by immunofluorescence confocal microscopy and flow cytometry analysis in CD34+ blast cells. Constitutive GAB1/2 phosphorylation was detected in all PI3K+ patients. However, we did not find any correlation between PI3K activation and FLT3 gene mutations. PTEN and SHIP-1 expression was normal in all tested PI3K+ patients. Proliferation (3H thymidine incorporation) was significantly increased in PI3K+ samples compared to PI3K- samples (p=0.001). The mTOR/P70S6K pathway was activated and the mTOR inhibitor rapamycin selectively reduced proliferation of PI3K+ samples. We determined the expression of the alpha, beta and delta isoforms of the catalytic sub-unit of PI3K (p110) in leukemic cells and found that p110 delta was the only consistently expressed isoform. In 8 PI3K+ samples tested, IC87114 (ICOS Corporation), a specific p110delta inhibitor compound used at 10μM totally suppressed AKT and FOXO3a phosphorylation. IC87114 inhibited proliferation of PI3K+ leukemic cells whereas it did not induce apoptosis. IC87114 did not significantly impaired the proliferation of PI3K- blast cells. Interestingly, IC87114 did not inhibit the proliferation or clonogenicity of CD34+ cord blood cells cultured in SCF, FLT3L and TPO used as controls. Overall, our results report, a/ a constitutive activation of PI3K in 58% of AML, b/ no correlation between FLT3 gene mutations and PI3K positivity, c/ no implication of PTEN or SHIP-1 phosphatases in PI3K activation, d/ a major role of the p110 delta isoform of PI3K in leukemic cell proliferation, e/ a potential therapeutic value of inhibiting mTOR and P110 delta activity.