Baseline NF-κB expression in WM cells. (A) NF-κBp65 DNA binding activity was assessed in vitro using nuclear extracts using the Active Motif assay. We compared BM isolated CD19⁺ cells from 4 patients with WM to one healthy donor. Jurkat nuclear extracts provided as a positive control in the kit were used as a control in the first 3 conditions. P indicates patient; NBM, healthy donor normal BM. WT and Mut are wild-type and mutated consensus competitor oligonucleotides, respectively. Data represent mean plus or minus SD of triplicate experiments. (B) Immunofluor-escence for phospho-NF-κBp65 on BM isolated CD19⁺ cells from one patient with WM compared with one healthy donor (NBM). Immunocytochemical analysis was assessed using anti–p-NF-κBp65 antibody (ii). DAPI was used to stain nuclei (i). (iii) The merge of i and ii panels.

Perifosine and bortezomib inhibit NF-κB function in WM cells. (A) Chromatin immunoprecipitation (ChIP)-based assay. BCWM.1 cells were cultured with either perifosine (P, 10 μM), bortezomib (B, 10 nM), or the combination (B + P) overnight, then TNFα (10 ng/mL) was added for the last 30 minutes, and then anti–p65NF-κB immunoprecipitation was performed. Quantitative real-time PCR (Q-PCR) for IκB in the p65NF-κB-DNA immunoprecipitated fragments was assessed using SYBR. Experimental Q-PCR values were normalized against values obtained for 25 ng of input DNA with the same primer set. (B) NF-κB activity assay using Active Motif. BCWM.1 cells were cultured with either perifosine (10 μM), bortezomib (10 nM), or the combination for 6 hours, and then TNFα (10 ng/mL) was added for the last 30 minutes. NF-κBp65 transcription factor-binding to its consensus sequence on the plate-bound oligonucleotide was studied from nuclear extracts. WT and Mut are wild-type and mutated consensus competitor oligonucleotides, respectively. Data represent mean plus or minus SD of triplicate experiments. (C) BCWM.1 cells were cultured with either perifosine (10 μM), bortezomib (10 nM), or the combination for 6 hours, then TNFα (10 ng/mL) was added for the last 30 minutes, and then the effect on NF-κB pathway was studied using immunobloting on cell fractionation. Cytoplasmic and nuclear fractions were subjected to Western blotting using anti-IκBα, –NF-κBp50, –p-NF-κBp65, -nucleolin, and –α-tubulin antibodies.

The combination of perifosine and bortezomib induces a decrease in proliferation and survival in WM tumor cells. (A) Normalized isobologram produced by Calcusyn software. BCWM.1 cells were cultured with either perifosine (5 μM and 10 μM), bortezomib (5 nM and 10 nM), or the combination. The table shows affected fractions and combination indices. Cytotoxicity was assessed using MTT assay. (B) Cytotoxicity induced with several agents known in WM disease therapy. Combination indices (CI) and fractions affected (FA) produced by Calcusyn software. BCWM.1 cells were cultured with the following agents: fludarabine (1 and 5 μg/mL), melphalan (1 μM and 5 μM), and doxorubicin (0.1 nM and 0.5 nM) in combination with perifosine (5 μM and 10 μM) for 48 hours. (C) Cytotoxicity was assessed on several IgM-secreting cell lines, MEC-1 (■), WM-WSU (Δ), and RL (x). Cells were cultured with either perifosine (10 μM), bortezomib (5 nM and 10 nM), or the combination for 48 hours. (D) Freshly isolated BM CD19⁺ samples from 3 patients with WM treated with the combination of perifosine (10 μM), bortezomib (5 nM and 10 nM), and the combination for 48 hours. Data represent mean plus or minus SD of triplicate experiments.

Differential targeting of perifosine and bortezomib on Akt, Erk, and proteasome pathways. (A) BCWM.1 cells were cultured with the combination perifosine (10 μM) and bortezomib (10 nM) for 6 hours and rituximab (10 ng/mL) in the last hour, and then the effect on Akt and ERK MAPK pathways was assessed by immunobloting. Whole cell lysates were subjected to Western blotting using anti–p-Akt, -Akt, –p-GSK3α/β-p-S6R, -ERK1/2, –p-ERK, and –α-tubulin antibodies. (B) BCWM.1 cells were cultured with the combination perifosine (10 μM) and bortezomib (10 nM) for 6 hours, and then whole cell lysates were immunoprecipitated overnight with anti-Akt antibody. Then the immunoprecipitates were subjected to in vitro kinase assay according to manufacturer's protocol. Western blotting used -Akt and fusion protein -p-GSK3α/β antibodies. (C) BCWM.1 cells were cultured with either triciribine (5 μM and 10 μM), U0126 (5 nM and 10 μM), or the combination. Cytotoxicity was assessed using MTT assay. (D) 20S proteasome activity assay. BCWM.1 cells were cultured with either perifosine (10 μM), bortezomib (10 nM), or the combination for 4 and 12 hours, and then whole cell lysates were subjected to proteasome activity measurement. Data represent mean plus or minus SD of triplicate experiments.

Growth factors and coculture with BM microenvironment cells do not protect WM cells against the combined perifosine and bortezomib-induced cytotoxicity. (A) BCWM.1 cells were cultured with the combination perifosine (5 μM and 10 μM) and bortezomib (10 nM) 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), bortezomib (5 nM and 10 nM), or the combination in presence of IL-6 (50 ng/mL) or CD40L (3 μg/mL) for 48 hours. Cytotoxicity was assessed by the MTT assay. (C) PBMCs from 3 healthy donors with either perifosine (10 μM), bortezomib (5 nM and 10 nM), and the combination for 48 hours. Cytotoxicity was assessed by MTT assay. (D) Colony-forming cell assay. Nonadherent mononuclear cells were cultured using methylcellulose semisolid technique cultured with either single agents or the combination. The plates were read at days 14 to 16. BFU-E indicates burst-forming units–erythroid; CFU-GM, colony-forming units–granulocyte/macrophage; CFU-M, colony-forming units–macrophage; CFU-GEMM, colony-forming units–granulocyte/erythroid/macrophage/megakaryocyte. Data represent mean plus or minus SD of triplicate experiments.

Perifosine and bortezomib-induced apoptosis and cell-cycle arrest were measured in WM cells. (A) BCWM.1 cells were cultured with either perifosine (5 μM and 10 μM), bortezomib (5 nM and 10 nM), and the combination 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 either perifosine (10 μM), bortezomib (5 nM and 10 nM), or the combination for 10 hours. Whole cell lysates were subjected to Western blotting using anticaspase 9, -caspase 8, -caspase 3, -PARP, -MCL1, Bcl-xl, and –α-tubulin antibodies. (C) Cell cycle was then studied using PI staining by flow cytometry. BCWM.1 cells were cultured with either perifosine (10 μM), bortezomib (10 nM), or the combination for 24 hours. Percentages indicate cells in sub-G₁ phase, G₀/G₁ phase, and G₂/M phase. (D) BCWM.1 cells were cultured with either perifosine (10 μM), bortezomib (5 nM and 10 nM), or the combination for 12 hours. Whole cell lysates were subjecting to Western blotting using anti-CDK4, -CDK6, -CDK2, -p-CDK2, -p27kip1, -p21Waf1/Cip1, -p53, -p-Rb, and –α-tubulin antibodies. Data represent mean plus or minus SD of triplicate experiment (A,C).

The combination of perifosine with bortezomib-induced cytotoxicity is enhanced in combination with the anti-CD20 monoclonal antibody, rituximab. (A,B) BCWM.1 cells were cultured with the combination of perifosine (10 μM) and bortezomib (10 nM) for 4 to 6 hours and with rituximab (10 μg/mL) during the last hour. (A) NF-κB activity assay using Active Motif. NF-κBp65 transcription factor-binding to its consensus sequence on the plate-bound oligonucleotide was studied from nuclear extracts. WT and Mut are wild-type and mutated consensus competitor oligonucleotides, respectively. (B) Relative quantitative PCR of IκB gene. (C) Whole cell lysates were subjected to Western blotting using anti–p-Akt, -Akt, –p-S6R, and –α-tubulin antibodies. (D) BCWM.1 cells were treated similarly to that in panel A, and then whole cell lysates were immunoprecipitated overnight with anti-Akt antibody. Then the immunoprecipitates were subjected to in vitro kinase assay according to the manufacturer's protocol. Western blotting used -Akt and fusion protein -p-GSK3α/β antibodies. (E) Antibody-dependent cell-mediated cytotoxicity assay (ADCC). BCWM.1 cells were pretreated with either perifosine (10 μM), bortezomib (10 nM), and the combination overnight then washed then treated with rituximab (10 μg/mL) for 4 hours in the presence of the effector cells. Results are reported in terms of mean percentage of specific lysis characterized by measurement of release of calcein-AM with different effector:target ratios (E/T ratio). Data represent mean plus or minus SD of triplicate experiments (A,B,E).