Phenotypic and Functional Effects of Novel Akt Inhibitor Perifosine on Immune System.

Perifosine is a synthetic novel alkylphospholipid, a new class of antitumor agent which targets cell membranes and inhibits Akt activation. Perifosine inhibits multiple myeloma (MM) cell growth in vitro and in vivo. Currently perifosine is under phase II clinical evaluation in MM. Although perifosine has shown significant direct antitumor effects, its effect on immune system has not yet been clarified. The objective of this study was to investigate the effects of perifosine on immune system including the activity of antigen presenting cells (APCs), T cells and NK cells. Perifosine was used at a clinically relevant concentrations of 2.5 μM, 5 μM and 10 μM. Monocyte-derived dendritic cells (DCs) from healthy donors were used as the APCs. We observed that the perifosine up to 48 hours had no effect on viability (>90%), as assessed by annexin V and PI staining. Alteration of DC phenotype by perifosine was further examined by flow cytometry. Our results demonstrated that perifosine treatment led to a dose-dependent downregulation of surface antigen expression, associated with costimulation (CD40, CD80 and CD86), antigen presentation (HLA-ABC, HLA-DQ) and maturation (CD83) on immature DCs at 24 and 48 hours. The significant downregulation of CD40, CD83, HLA-ABC and HLA-DQ was also observed on mature DCs. Perifosine also inhibited immature DC uptake of antigens (40-kDa Dextran-FITC, 45-kDa protein A-Alexa Fluor 488 and 20-kDa protein G-Alexa Fluor 488) in a dose-dependent manner. Since DCs play a crucial regulatory role via cytokine production, we next determined IL-12p70 and IL-10 secretion by LPS-induced DCs with and without perifosine treatment. Compared to controls, perifosine treatment at 24 hours significantly inhibited LPS-induced-IL-12p70 production by DCs (trt vs. untrt = 166 pg/ml (2.5μM), 111 pg/ml (5μM) and 45 pg/ml (10μM) vs. 192 pg/ml), as well as inhibited IL-10 production (trt vs. untrt = 371 pg/ml (2.5μM), 306 pg/ml (5μM) and 179 pg/ml (10μM) vs. 472 pg/ml). These data suggest that perifosine is able to affect both immature and mature DCs and could contribute to inhibition of DC-mediated immune responses. Furthermore, we evaluated effect of perifosine on T cells obtained from healthy donor peripheral blood mononuclear cells (PBMCs) by negative selection. Although perifosine treatment, up to 10μM, had no effect on T cell survival, it inhibited IFN-gamma production by T cells stimulated with PMA and ionomycin compared to that of the control (intracellular flow cytometry, trt vs. untrt= 11.7% vs. 21.3%). The IFN-gamma inhibition by perifosine treatment (10μM) was further confirmed by ELISA upon stimulation with anti- CD2/CD3/CD28 activation beads (trt vs. untrt =11340 pg/ml vs. 18150 pg/mL). Similarly, viability of NK cells obtained from PBMCs by negative selection was not significantly affected by perifosine; however, decreased cytotoxicities were observed by NK cells treated with perifosine (10μM) against the U266 myeloma cell line (trt vs. untrt=30% vs. 51% (E:T=10:1) and 17% vs 30% (E:T=5:1) respectively). In addition, the IFN-gamma production by IL-2 and IL-12- induced NK cells was significantly inhibited following the perifosine treatment (trt vs. untrt=286 pg/ml (2.5μM), 210 pg/ml (10μM) vs. 439 pg/ml). These studies demonstrate that perifosine treatment significantly affects phenotype and function of human DCs, T cells and NK cells. Our pre-clinical data therefore indicates the need to monitor immune functions in patients under the Akt inhibitor treatment.