Akt expression in WM (Waldenstrom macroglobulinemia) cells. (A) Baseline phosphorylation expression of Akt serine 473 in freshly selected CD19 + bone marrow cells from patients with WM disease (N = 4) compared with bone marrow CD19 + cells from healthy donors (NBM) (N = 2). β-actin was used as a control. (B) Baseline phosphorylation expression of Akt serine 473 was assessed in BCWM.1 cell line (BC), MM.1S cell line (MM), and several IgM-secreting cell lines: WM-WSU (WSU), MEC-1 (MEC), RL, by Western blotting. α-tubulin antibody is always used as a control. Specific inhibition of Akt pathway affects survival of WM cells. (C) BCWM.1 cells were cultured with triciribine for 6 hours with doses that range from 1 to 10 μM. Whole cell lysates were subjecting to Western blotting using anti–p-Akt ser473, -Akt, –p-ERK, and α-tubulin antibodies. (D) Triciribine (1 to 50 μM) induces growth inhibition and cytotoxicity in BCWM.1 cells at 48 hours by the [3H]-thymidine uptake assay and the MTT assay. (E) BCWM.1 cells were transduced with 2 Akt shRNA for 48 hours. Mock: control plasmid; 62 and 63 represent shRNA that target 2 different sequences of Akt gene. Whole cell lysates were subjected to Western blotting using anti–p-Akt, -Akt, -p-S6R, –p-GSK3α/β, and α-tubulin antibodies.

Perifosine induces decrease in proliferation and cytotoxicity. (A) Thymidine uptake assay. MM.1S and BCWM.1 cells were cultured with perifosine (2 to 30 μM) for 48 hours. BCWM.1 (■), MM.1S (♦). (B) BCWM.1 cells were cultured with perifosine (2 to 50 μM) for 24 hours (♦) and 48 hours (■). (C) Several IgM-secreting cell lines, RL (♦), MEC-1 (■), WM-WSU (Δ) were cultured with perifosine (2 to 50 μM). (D,E) Freshly isolated BM CD19 + tumor cells from 3 patients with WM (D) and PBMCs from 3 healthy donors (E) were cultured with perifosine (5 to 50 μM). Cytotoxicity was assessed by the MTT assay (C-E). All results represent mean (± SD) of triplicate experiments. (F) Colony forming-cell assay. Nonadherent mononuclear cells were cultured using methylcellulose semisolid technique in absence and presence of perifosine (5 μM and 10 μM) for 14 days. Burst forming units-erythroid (BFU-E), colony-forming units-granulocyte/macrophage (CFU-GM), colony-forming units-macrophage (CFU-M), and colony-forming units-granulocyte/erythroid/macrophage/megakaryocyte (CFU-GEMM).

Perifosine inhibits Akt phosphorylation. (A) BCWM.1 cells were cultured with perifosine for 6 hours with doses that ranged from 2 to 20 μM. Whole cell lysates were subjecting to Western blotting using anti–p-Akt ser473, -Akt, –p-PDK1, –p-GSK3α/β, –p-S6R, and α-tubulin antibodies. (B,C) BCWM.1 cells were cultured with perifosine. Whole cell lysates were immunoprecipitated with anti-Akt antibody. Then the immunoprecipitated were washed and subjected to in vitro kinase assay according to manufacturer's protocol. Western blotting used -p-Akt, and fusion protein -p-GSK3α/β antibodies. (B) Dose effect of perifosine at 6 hours at doses that range from 5 to 20 μM. (C) Time-effect of perifosine at 10 μM for 2 to 6 hours.

Growth factors and coculture with BMSCs do not protect against perifosine-induced WM cell cytotoxicity. (A) BCWM.1 cells were cultured with control media and with perifosine (5 to 20 μM) for 48 hours in the presence or absence of BMSCs. Cell proliferation was assessed using the [³H]-thymidine uptake assay. (B) BCWM.1 cells were cultured with either perifosine (10 μM) alone and in presence of BMSCs for 6 hours. Whole cell lysates were subjected to Western blotting using anti–p-Akt, -Akt and α-tubulin antibodies. (C) BCWM.1 cells were cultured with perifosine (5 to 20 μM) in the absence and presence of IL-6 (25 ng/mL) or IGF-1 (50 ng/mL) for 48 hours. Cytotoxicity was assessed by the MTT assay. All data represent mean (± SD) of triplicate experiments (A,C). (D,E) BCWM.1 cells were cultured with perifosine (10 μM) for 6 hours in the absence and presence of (D) IGF-1 (50 ng/mL) or (E) IL-6 (25 ng/mL) for the last 10 minutes. Whole cell lysates were subjected to Western blotting using anti–p-Akt ser473, -Akt and α-tubulin antibodies.

Perifosine induces SAPK/JNK-dependent MM cell apoptosis. (A) MM.1S and BCWM.1 cells were cultured with perifosine (5-50 μM) for 48 hours. Then the percentage of cells undergoing apoptosis was studied using Apo2.7 staining and flow cytometry. (B) BCWM.1 cells were cultured with perifosine (10 μM) for the indicated periods. Whole cell lysates were subjected to Western blotting using anti-caspase 9, -caspase 8, -PARP, and α-tubulin antibodies. Horizontal lines have been inserted to indicate a repositioned gel lane. (C) BCWM.1 cells were cultured with perifosine (2-20 μM) for 6 hours. Whole cell lysates were subjected to Western blotting using anti–p-SAPK/JNK and α-tubulin antibodies. (D) BCWM.1 was cultured with control media, perifosine (10 μM), SP600125 (5-20 μM), or perifosine (10 μM) plus SP600125 (10 μM and 20 μM) for 10 hours. Whole cell lysates were subjected to Western blotting using anti–p-SAPK/JNK, -caspase 9, -caspase 8, -PARP, and α-tubulin antibodies. Horizontal lines have been inserted to indicate a repositioned gel lane. (E) BCWM.1 cells were cultured for 48 hours with media and with perifosine (5-20 μM) in the absence or presence of 5 to 20 μM of JNK inhibitor SP600125. Cytotoxicity was assessed by the MTT assay. All data represent mean (± SD) of triplicate experiment (A,E).

Perifosine inhibits human WM cell growth in vivo. (A) Immunodeficient irradiated SCID mice were inoculated subcutaneously in the flank with 3 × 10⁶ BCWM.1 cells in 100 μL RPMI 1640 medium. Oral perifosine was administered daily (35 mg/kg per day) starting after the development of measurable tumors (N = 11). Perifosine significantly inhibited WM cell growth at week 12 (P = .04) compared with the control group (N = 8) treated with vehicle only (water). Error bars represent the variation in tumor volume for the mice per group. (B) Tumor tissues from mice treated with control vehicle (N = 2) or daily perifosine (N = 2) were harvested, processed, and subjected to Western blotting using anti-Akt, –p-Akt, –p-S6R and –β-actin antibodies. Adhesion and migration in vitro and homing in vivo. (C) Transwell migration assay showing inhibition of migration of BCWM.1 in the presence of perifosine 1 to 5 μM. SDF-1 30 nM was placed in the lower chambers and induced migration as compared with control with no SDF-1 (CTL, control). SDF-1 was placed in the lower chambers of the perifosine-treated wells. (D) Adhesion assay with BCWM.1 in the presence or absence of perifosine 2 to 10 μM. BCWM.1 demonstrated increased adhesion in fibronectin-coated wells (CTL, control) as compared with BSA-coated wells. Perifosine inhibited adhesion in fibronectin-coated wells in a dose-dependent manner. All data represent mean (± SD) of triplicate experiments (C,D). (E) In vivo flow cytometry. DID-labeled cells, either treated with perifosine 5 μM () or untreated control (), were injected in the tail vein of 2 balb/c mice. Cells were counted every 5 minutes for 1 hour. The cell count decreased by 75% in the control and only by 40% in the treated mouse, P = .001. (F) Histogram showing that the number of cells present in the perivascular bone marrow niches of the skull was significantly higher in the control mice compared with the perifosine-treated group at 24 hours after injection (P = .039), using in vivo confocal imaging of quadrants 3 and 4 of the skull of mice.

Inhibition of ERK signaling augments perifosine-induced cytotoxicity. (A) BCWM.1 cells were cultured with perifosine for 6 hours at doses that range from 2 to 20 μM. Whole cell lysates were subjected to Western blotting using anti–p-ERK, –ERK1/2, –p-c-Raf Ser259, –pan-p-PKC, and α-tubulin antibodies. (B) BCWM.1 cells were cultured for 48 hours with media and with perifosine (5-20 μM) in the absence or presence of 5 and 10 μM of MEK1/2 inhibitor U0126. Cytotoxicity was assessed by the MTT assay. Data represent mean (± SD) of triplicate experiments. (C) BCWM.1 cells were cultured with control media and either perifosine (10 μM), U0126 (5 μM and 10 μM), or the combination for 10 hours. Whole cell lysates were subjected to Western blotting using anti–p-ERK, -ERK1/2, -caspase 9, -caspase 8, -PARP and –α-tubulin antibodies. Horizontal lines have been inserted to indicate a repositioned gel lane. (D) BCWM.1 cells were transduced with 2 Akt shRNA for 48 hours. Mock: control plasmid; 62 and 63 represent shRNA that target 2 different sequences of Akt gene. Whole cell lysates were subjected to Western blotting using anti–p-ERK, -ERK1/2, and α-tubulin antibodies. (E) BCWM.1 cells were cultured with triciribine for 6 hours with doses that ranged from 1 to 10 μM. Whole cell lysates were subjected to Western blotting using anti–p-ERK and α-tubulin antibodies. (F) BCWM.1 was cultured with perifosine (20 μM, 4 hours) in the absence or presence of 25 μM of PI3K inhibitor LY294002. BCWM.1 was cultured with control media, perifosine (20 μM), LY294002 (25 μM, 15 minutes), LY294002 for 15 minutes (1) followed by perifosine for 4 hours (2), or perifosine for 4 hours (1) followed by LY294002 for 15 minutes (2) Whole cell lysates were subjected to Western blotting using anti–p-ERK, -ERK1/2, –p-Akt Ser473, -Akt, –p-c-Raf Ser259, –pan-p-PKC and α-tubulin antibodies.