Synergistic Proapoptotic Activity of Recombinant TRAIL Plus the Akt Inhibitor Perifosine in Acute Myelogenous Leukemia Cells-a Novel Therapeutic Approach for Leukemia Displaying Elevated Akt Signaling.
Authors: Tazzari PierLuigi , Papa Veronica , Ricci Francesca , Chiarini Francesca , Evangelisti Camilla , Martinelli Giovanni , Bontadini Andrea , McCubrey James , Martelli Alberto M. 
Published: October 2019

Abstract
The Tumor Necrosis Factor (TNF) family member TNF-Related Apoptosis Inducing Ligand (TRAIL) was originally reported to induce apoptosis in many tumor cells but not in normal cells in vivo and thus represents a promising anticancer cytokine. The in vitro cytotoxic response of acute myelogenous leukemia (AML) cell lines to recombinant TRAIL varies from good to moderate, however, a number of in vitro studies have convincingly demonstrated that AML primary cells are resistant to the proapoptotic activity of TRAIL used as a single agent. To potentiate the response of AML cells to TRAIL cytotoxicity, we have adopted a strategy of combining perifosine, a novel Akt inhibitor, with recombinant TRAIL. The rationale for using such a combination is that perifosine was recently described to increase TRAIL-R2 receptor expression and decrease cellular FLICEinhibitory (c-FLIP, an inhibitor of caspase-8 activation) protein in human lung cancer cells. Both perifosine and TRAIL, when used alone, induced cell death by apoptosis in THP- 1 AML cells, which normally express constitutively active Akt and nonfunctional p53. Perifosine treatment, at concentrations well below the IC50 (0.5 μM), dephosphorylated Akt on Ser473 and increased TRAIL-R2 levels, as demonstrated by flow cytometry, western blot, and RT-PCR. Perifosine also downmodulated cFLIP-L and XIAP levels. However, perifosine did not affect expression of TRAIL-R1 and TRAIL-R4 receptors, or of other proteins which are critical for TRAIL-mediated proapoptotic signaling, including FADD and Mcl-1. Perifosine and TRAIL strongly synergized to induce cytotoxicity as suggested by calculation of the combination index (CI range: 0.15–0.37). The combined treatment resulted in upregulation of caspase-8 activity and apoptosis which was markedly reduced by a selective caspase-8 inhibitor (Z-IETD-FMK). While cFLIP-L and XIAP downregulation was dependent on inhibition of NF-κB activity caused by perifosine, upregulation of TRAIL-R2 expression was dependent on generation of reactive oxygen species (ROS) by perifosine which in turn sequentially activated protein kinase C (PKC)α, JNK2, and c-Jun. A ROS scavenger (N-acetylcysteine), siRNA downregulation of either PKCα or c-Jun, or a JNK1/2 selective pharmacological inhibitor (SP600125), all markedly impaired perifosine-dependent TRAIL-R2 upregulation. Perifosine synergized with TRAIL by inducing apoptosis exclusively in primary AML cells displaying constitutive activation of the Akt pathway. Also in primary AML blasts, perifosine upregulated TRAIL-R2 levels, downmodulated the expression of both c-FLIP and XIAP, and increased Ser 63 p-c-Jun levels, without affecting the expression of FADD. Remarkably, perifosine increased p-JNK2 levels and TRAIL-R2 expression in primary AML patient blasts (CD34+, CD38Low/Neg, CD123+) enriched in putative leukemic stem cells. Perifosine and TRAIL combined treatment was effective in inducing apoptosis (55–60%) in this immature blast population, as documented by a quadruple staining flow cytometric technique for CD34+, CD38Low/Neg, CD123+, Annexin V+ blast cells. The combined treatment negatively affected the clonogenic activity of CD34+ cells from AML patients with Akt activation. In contrast, CD34+ cells from healthy donors and AML patients without Akt activation were resistant to perifosine plus recombinant TRAIL treatment. Our findings suggest that a combination consisting of perifosine plus recombinant TRAIL might offer a novel therapeutic strategy for AML displaying enhanced Akt signaling by overcoming critical mechanisms of apoptosis resistance. Moreover, this kind of combination therapy could be effective also in AML cases exhibiting nonfunctional p53 or low levels of p53.