The Role of the AKT Inhibitor Perifosine in Migration and Adhesion in Multiple Myeloma (MM).

Homing of plasma cells to the bone marrow microenvironment supports the cells’ survival and growth. We have previously demonstrated that MM cells migrate in response to the chemokine SDF-1 and that the PI3K/AKT pathway is an important regulator of migration in MM. The AKT inhibitor, perifosine (KRX-0401, Keryx, NY, NY, provided by the NCI) is an alkylphosphocholine, a member of a new class of potent AKT inhibitors that is being tested preclinically as a therapeutic agent in MM. The objective of this study was to determine the effect of this novel inhibitor on the migration and adhesion of MM cells and to determine differences in response between MM cells with high AKT activity (OPM2 cells) and lower AKT activity (MM.1S). MM cell lines were treated for 16 hrs with serial concentrations of perifosine 2–50uM or vehicle (sterile water). Apoptosis was determined using Annexin V/PI flow cytometry analysis (BD Biosciences, CA). Migration was determined using a transwell migration assay (Costar, Corning, NY). Treated cells were washed, placed in serum free medium for two hours, then placed in the upper chambers of the migration assay with 1% FCS medium in the presence of the chemoattractant SDF-1 in the lower chambers (SDF-1 20nM, a concentration previously determined to induce maximum migration in MM cells). After 4 hours of incubation, cells that migrated to the lower chambers were counted. Similarly, adhesion was determined using an adhesion assay (EMD Biosciences, San Diego, CA) with 96 well plated coated with fibronectin in the presence or absence of serial concentrations of SDF-1 (10–30nM). A two-tailed student t-test was used for statistical analysis. Perifosine induced a dose dependent inhibition of migration in both MM.1S and OPM2. In MM.1S, 2uM perifosine inhibited migration to 82% while 5uM inhibited migration to 44% of control. There was no further significant reduction of migration at 20nM of perifosine (40% migration). In OPM2, 2uM perifosine inhibited migration to a greater extent than MM.1S at 59% of control (p=0.03), and 5uM induced 41% migration similar to the effect observed in MM.1S (p=0.7). There was no induction of apoptosis up to 48hrs of treatment with the low concentrations of perifosine (2–5uM) indicating that the effect of low dose perifosine on MM cells is specific to migration and not due to apoptosis of cells. We have previously demonstrated that SDF-1 increases adhesion of MM cells to fibronectin indicating activation of alpha4beta integrin receptor, with 30nM SDF-1 inducing 296% increase in adhesion. Perifosine alone (2–20uM) inhibited adhesion of MM cells (38% with 2uM, and 12% with 5–20uM as compared to control). Perifosine 2–20uM inhibited adhesion even in the presence of 30nM SDF-1 (55% to 12% respectively). There was no difference in the adhesion response of MM.1S and OPM2 to perifosine. We demonstrate that low doses of the AKT inhibitor perifosine inhibit migration and adhesion of MM cells in response to SDF-1. These doses do not induce apoptosis, and therefore are specific to inhibiting migration. Future studies to test the AKT inhibitor perifosine as a modulator of MM homing, migration and adhesion in vivo are warranted. Supported in part by an ASH scholar award and an MMRF grant.