Potent Synergistic Activity of the NAD+ Synthesis Inhibitor APO866 and of the Apoptosis Inducer TRAIL in in Vitro and Ex Vivo Cellular Models of Non Hodgkin's Lymphoma and Chronic Lymphocytic Leukemia.
Authors: Zoppoli Gabriele , Cea Michele , Fruscione Floriana , Bruzzone Santina , Soncini Debora , Bedognetti Davide , Moran Eva , Balleari Enrico , Bruzzone Andrea , Garuti Anna , Cirmena Gabriella , Rocco Ilaria , Gobbi Marco , Ghio Riccardo , Pommier Yves , Carella Angelo Michele , Ballestrero Alberto , Parodi Silvio , Patrone Franco , Nencioni Alessio 
Published: October 2019

Abstract
Abstract 2733
Poster Board II-709

Introduction:
APO866, a reversible inhibitor of nicotinamide phosphoribosyltransferase (Nampt), acts by severely depleting intracellular NAD+ content and thus eliciting mitochondrial dysfunction, ATP depletion, and autophagic cell death. TNF-Related Apoptosis Inducing Ligand (TRAIL) binds to plasma membrane receptors DR4 and DR5 and causes apoptosis through activation of caspase-8 and caspase-10. These, in turn, activate effector caspases either directly or via Bid cleavage and subsequent initiation of the mitochondrial apoptotic pathway. In the present work, we have explored the interaction between APO866 and TRAIL in cellular models of hematological malignancies.

Materials and Methods:
The lymphoma cell lines Jurkat, PEER, MOLT-4, H9, and Namalwa, primary B-cell chronic lymphocytic leukemia (B-CLL) cells, as well as normal peripheral blood mononuclear cells (PBMCs) were treated with increasing concentrations of APO866, TRAIL, or with 1:1, 1:3, or 1:10 combinations of both agents for 72 hours (48 h APO866 + 24 h TRAIL). Cell death was assessed by propidium iodide staining and flow cytometry (FC). Apoptosis was assessed by Annexin V-propidium iodide staining and FC. Combination indices were calculated according to the Chou and Talalay method. DR5 membrane expression was assessed with a FITC-conjugated anti-DR5 antibody and FC after 12, 24, and 48 h exposure to APO866. Mitochondrial transmembrane potential (Δψm) and cell cycle analysis after single agent or combination treatment were determined by tetramethylrhodamine (TMRE) and Nicoletti staining, respectively, and FC. Intracellular NAD+ and ATP contents were quantified by cyclic assays and HPLC, respectively. The role of autophagy and of caspase activity in the cytotoxic activity of APO866, TRAIL, and of their combination was assessed using the PI3K inhibitors 3-methyladenine, LY2940002, and wortmannin, and the caspase inhibitors Z-VAD-FMK (pan-caspase inhibitor), Z-LEHD-FMK (caspase-9 inhibitor), and Z-IETD-FMK (caspase-8 inhibitor), respectively.

Results/Discussion:
APO866 induced NAD+ depletion, Δψm dissipation, and ATP exhaustion in the absence of caspase activation as detected by caspase-3 cleavage. Accordingly, autophagy but not caspase inhibitors blocked APO866 activity. Vice versa, TRAIL-induced apoptosis was blocked by the caspase inhibitors and enhanced by the PI3K inhibitors. Remarkably, APO866 and TRAIL showed a potent synergistic interaction in lymphoma cell lines and in primary B-CLL cells, consistently exhibiting combination indices below 1 upon exposure to a wide range of drugs concentrations. On the other hand, the two drugs and their combinations were poorly active and failed to show any cooperation in healthy PBMCs, indicating a specific activity against malignant cells. As a metabolic correlate of their interaction, TRAIL enhanced APO866-induced NAD+ depletion and Δψm. dissipation. As a result, lymphoma cells underwent massive ATP depletion and autophagic cell death. No DR5 upregulation at the cell surface of lymphoma cells was observed in response to APO866.

Conclusions:
APO866 and TRAIL show potent synergistic interaction in preclinical cell models of Non Hodgkin's Lymphoma and B-CLL. Due to the mild and manageable side effects of these two drugs in clinical trials, the addition of TRAIL to APO866 appears as an interesting option for the treatment of refractory hematological malignancies.

Disclosures:
No relevant conflicts of interest to declare.