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Proapoptotic Activity and Chemosensitizing Effect of the Novel Akt Inhibitor (2S)-1-(1H-Indol-3-yl)-3-[5-(3-methyl-2H-indazol-5-yl)pyridin-3-yl]oxypropan2-amine (A443654) in T-Cell Acute Lymphoblastic Leukemia

Dipartimento di Scienze Anatomiche Umane (F.F., W.L.B., F.C., L.C., and A.M.M.) and Dipartimento di Ematologia e Scienze Oncologiche "L. e A. Seràgnoli" (G.M.), Università di Bologna, Italy; Centro Immunoematologia e Trasfusionale, Policlinico S. Orsola-Malpighi, Bologna, Italy (P.L.T.); Dipartimento di Scienze Motorie e della Salute, Università di Cassino, Italy (A.C.); Dipartimento di Biotecnologie ed Ematologia, Università degli Studi "La Sapienza," Roma, Italy (A.T.); and Department of Microbiology and Immunology, Brody School of Medicine at East Carolina University, Greenville, North Carolina (J.A.M.)

Constitutively activated AKT kinase is a common feature of T-cell acute lymphoblastic leukemia (T-ALL). Here, we report that the novel AKT inhibitor (2S)-1-(1H-indol-3-yl)-3-[5-(3-methyl-2H-indazol-5-yl)pyridin-3-yl]oxypropan2-amine (A443654) leads to rapid cell death of T-ALL lines and patient samples. Treatment of CEM, Jurkat, and MOLT-4 cells with nanomolar doses of the inhibitor led to AKT phosphorylation accompanied by dephosphorylation and activation of the downstream target, glycogen synthase kinase-3β. Effects were time- and dose-dependent, resulting in apoptotic cell death. Treatment of Jurkat cells with A443654 resulted in activation of caspase-2, -3, -6, -8, and -9. Apoptotic cell death was mostly dependent on caspase-2 activation, as demonstrated by preincubation with a selective pharmacological inhibitor. It is remarkable that A443654 was highly effective against the drug-resistant cell line CEM-VBL100, which expresses 170-kDa P-glycoprotein. Moreover, A443654 synergized with the DNA-damaging agent etoposide in both drug-sensitive and drug-resistant cell lines when coadministered [combination index (CI) = 0.39] or when pretreated with etoposide followed by A443654 (CI = 0.689). The efficacy of A443654 was confirmed using blasts from six patients with T-ALL, all of whom displayed low levels of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and constitutive phosphorylation of Akt on Ser473. At 1 µM, the inhibitor was able to induce apoptotic cell death of T-ALL blast cells, as indicated by flow cytometric analysis of samples immunostained for active (cleaved) caspase-3. Because activated AKT is seen in a large percentage of patients with T-ALL, A443654, either alone or in combination with existing drugs, may be a useful therapy for primary and drug-resistant T-ALL.

Address correspondence to: Prof. Alberto M. Martelli, Department of Human Anatomical Sciences, Cell Signaling Laboratory, University of Bologna, via Irnerio 48, 40126 Bologna, Italy. E-mail: alberto.martelli{at}gmail.com