Perifosine, an Oral Bioactive Novel Alkyl-Phospholipid, Inhibits Akt and Induces In Vitro and In Vivo Cytotoxicity in Human Multiple Myeloma (MM) Cells.

Perifosine (NSC 639966; Keryx Biopharmaceuticals, New York, NY) is a synthetic novel alkyl-lysophospholipid, a new class of anti-tumor agents which targets cell membranes and inhibits Akt activation. In phase I studies of Perifosine in solid tumor, dose-limiting toxicity was not reached. Importantly, no hematologic toxicity was observed, and maximum-tolerated dose was defined at 200 mg/day, achieving plasma concentrations of Perifosine of 7.5 μg/ml (16.2 μM). In this study, we showed that Perifosine, an oral bioactive novel alkyl-phospholipid, inhibits Akt and induces in vitro and in vivo cytotoxicity in human MM cells. Baseline phosphorylation of Akt was completely inhibited by Perifosine in a time- and dose-dependent fashion. Phosphorylation of FKHRL1 and GSK3α/β, downstream target proteins of Akt, was also markedly inhibited; in contrast, phosphorylation of PDK-1, a known upstream molecule of Akt, was not affected by Perifosine. Interestingly, phosphorylation of MEK and ERK was significantly increased by Perifosine. In vitro Akt kinase assays confirmed the direct inhibitory effect of Perifosine on Akt activity. The growth of MM cell lines and freshly isolated patient MM cells was significantly inhibited by Perifosine (IC₅₀of 1.5–15 μM at 48h); in contrast, Perifosine did not induce cytotoxicity in peripheral blood mononuclear cells. Neither exogenous IL-6 nor IGF-1 overcomes Perifosine-induced cytotoxicity. Perifosine triggered c-Jun NH2-terminal kinase (JNK) activation followed by caspase-8, caspase-9, and PARP cleavage; conversely, JNK inhibitor SP600125 abrogated Perifosine-induced apoptosis, suggesting that JNK plays a crucial role in Perifosine-induced apoptosis. Since phosphorylation of MEK/ERK was enhanced and ERK pathway is known cascade which promotes MM cell proliferation, we hypothesized that MEK inhibitor could augment cytotoxicity triggered by Perifosine. MEK inhibitor U0126 synergistically (CI = 0.29–0.58) enhances Perifosine-induced cytotoxicity in MM cells. Furthermore, Perifosine augments dexamethasone. doxorubicin, melphalan, and bortezomib-induced MM cell cytotoxicity. Importantly, Perifosine induces apoptosis even of MM cells adherent to BMSCs. In vivo activity of Perifosine was also assessed by xenograft murine (beige-nude-xid) model. Both oral daily (36 mg/kg/day) and weekly (250 mg/kg/week) administration of Perifosine significantly reduced MM tumor growth and increased survival, compared to control animals treated with PBS vehicle only. In contrast, there was no significant difference between the Perifosine treatment groups. Using Kaplan-Meier curves and log-rank analysis, the mean overall survival (OS) was 31 days (95% confidence interval [CI], 18–44 days) in the control cohort versus 59 days (95% CI, 45–73 days) and 63 days (95% CI, 49–76 days) in groups treated with daily and weekly Perifosine, respectively. There was no statistically significant difference in the mean OS of mice treated with the two doses of Perifosine. Importantly, tumor tissue from responding mice demonstrated significant inhibition of Akt phosphorylation whereas tumor from non-responding mice showed relatively higher phosphorylation of Akt. Taken together, our data provide the rationale for clinical trials of Perifosine to improve patient outcome in MM.