The AKT Inhibitor, A443654, Induces Cell Cycle Arrest, Apoptosis and Synergizes with Chemotherapeutic Drugs in Multi-Drug Resistant T-Cell Acute Lymphoblastic Leukemia - A Novel Agent for Therapy of Drug Resistant ALL.
Authors: McCubrey James A. , Blalock William L. , Tazzari Pier Luigi , Cappellini Alessandra , Iacobucci Ilaria , Chiarini Francesca , Abrams Steve L. , Ottaviani Emanuela , Martinelli Giovanni , Tafuri Agostino , Cocco Lucio , Martelli Alberto M. , Fala Federica 
Published: October 2019

Abstract
The PI3K/Akt pathway is a key mediator of proliferation and survival; its constitutive activation is implicated in pathogenesis and progression of a wide variety of hematological malignancies. Constitutively-activated Akt is a common feature of the T-ALL cell lines (MOLT-4, Jurkat, CEM). Hyperactivity of Akt leads not only to their growth and resistance to apoptosis but also to drug-resistance. In this study, we sought to examine the potential of a novel Akt inhibitor, A443654, in the therapy of T-ALL. A443654 led to rapid cell death of CEM, Jurkat, and MOLT-4. All three lines were sensitive to nanomolar (nM) doses of A443654 (IC50=60 nM, 120 nM and 900 nM for MOLT-4, CEM, and Jurkat, respectively). Effects were dose dependent and resulted in G2/M arrest. Indeed, approximately 39% of Jurkat cells treated with A443654 were in G2/M whereas only 11% of the untreated cells were in G2/M. Cell cycle arrest was followed by apoptotic cell death as determined by annexin V-PI and trypan blue staining. Treatment of CEM and Jurkat cells led to de-phosphorylation of the downstream Akt substrate GSK-3beta. Treatment of Jurkat cells with A443654 resulted in activation of caspase-2, -3, -8, and -9. Apoptotic cell death was greatly reduced by caspase-3 and -9 selective inhibitors. Additionally, A443654 was shown to be highly effective against the drug-resistant cell line CEM-VBL100 (CEM-R), which overexpresses Pgp. This Akt inhibitor initially (1 to 4 hour treatments) decreased Pgp activity, but not protein levels, further documenting a link between Akt and Pgp activity. While CEM-R cells displayed decreased sensitivity to A443654, treatment of CEM-R cells with sub-lethal doses of A443654 for 24 hours, reduced the surface expression of Pgp. Moreover, A443654 synergized with the DNA damaging agent etoposide, a substrate of Ppg, in both drug sensitive and resistant lines. At etoposide concentrations between 25 mM to 100 mM, A443654 enhanced the extent of cell killing from 25% to 45%. We then confirmed the effect of A443654 on human leukemia samples using blasts from 6 patients with T-ALL, all of whom displayed constitutive Akt activation. In a dose dependent fashion, A443654 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. In contrast, this Akt inhibitor was determined to be minimally cytotoxic on normal CD34+ hematopoietic precursor cells isolated from cord blood. Taken together, our findings indicate that the Akt inhibitor, A443654, either alone or in combination with existing drugs, may in the future be a useful therapeutic option for primary and refractory T-ALL displaying activated Akt signaling. Furthermore, this novel Akt inhibitor was effective in suppressing the growth of multidrug resistant ALL cells while having minimal effects on normal hematopoietic precursor cells documenting its poteintial in the treatment of drug resistant leukemias.