Treatment by PI3K/mTOR Inhibitor BEZ235 Combined with TLR-7/8 Agonist Interfere with Immune Evasion Mechanisms of WEHI-3 Mouse Leukemia Cells.

Abstract

Several PI3K/Akt/mTOR pathway inhibitors and TLR agonists induce tumor cell death. However, the mechanisms of these therapeutic approaches in acute myeloid leukemia (AML) cells are still unknown. To investigate the effects of BEZ235, as a dual inhibitor of PI3K and mTOR pathways, and TLR7/8 agonist R848 on the expression and regulation of the immune inhibitory molecules in myeloid leukemia cells. WEHI-3 leukemia cells were incubated with dual PI3K and mTOR inhibitor BEZ235 and TLR7/8 agonist R848 for 48 hrs. Firstly, cell viability was assessed by MTT method. The semi-quantitative relative mRNA expression of Galectin-9 (Gal-9), PD-L1, PVR, and STAT3 was assessed according to HPRT as a housekeeping gene. Finally, the protein expression of phosphorylated STAT3 was evaluated by western blotting analysis. WEHI-3 cells showed growth inhibition following treatment with BEZ235 and R848 whose combination exerted more proliferation arrest. The mRNA expression of Gal-9, PD-L1 and PVR immune checkpoint molecules significantly reduced in treated cells with BEZ235 and R848. Combined treatment indicated more reduction compared with the single treatment. Finally, the expression and phosphorylation of STAT3 were down-regulated after a single or dual treatment with BEZ235 and R848. Our results conclude that treatment with the combination of BEZ235 and R848 interferes with immune evasion mechanisms through STAT3-signaling pathway in WEHI-3 leukemia cells.