Targeting NF-kB by Perifosine, Bortezomib and Rituximab in Waldenstrom Macroglobulinemia (WM).

Background: The NF-kB pathway has been implicated in tumor survival and growth, and to induce resistance to conventional agents. Because of the natural ability of tumor cells to overcome single agent antitumor activity, we hypothesized that targeting differentially NF-kB pathway with combination of proteasome inhibitor bortezomib and of Akt inhibitor perifosine might lead to synergistic cytotoxicity on WM. Methods: The WM cell line BCWM.1 was used in these studies. Primary CD19+ WM cells were obtained from the bone marrow (BM) of patient after informed consent. Perifosine (P) was provided by Keryx (NY), Bortezomib (B) from Millennium (MA) and Rituximab (R) from Genentech (CA). NF-kB was studied using the Chromatin Immunoprecipitation (ChIP)-based assays, a new technology that allows for the specific analysis of activation/inhibition of one specific gene in the entire genome. IkB gene was used in this study as NF-kB target-activation gene. Quantitative real-time PCR (qPCR) for IkB in the p65NF-kB-DNA immunoprecipitated fragments was assessed and those results were confirmed at the transcriptional level using qPCR and immunoblotting on cytoplasmic/nuclear fractionation of cells. NF-kB activity assay was confirmed using the DNA-binding ELISA-based assay Active Motif kit on nuclear extract. Antibody-dependent cellular cytotoxicity assays (ADCC) was performed in presence of rituximab or human control IgG1. Results: We first showed that perifosine and bortezomib combination [P+B] induced synergistic cytotoxicity and inhibition of proliferation in WM cells. Similar cytotoxicity was observed in primary patient tumor cells and in presence of BM microenvironment, but spared normal hematopoietic cells. We next demonstrated that P and B inhibited p65NF-kB nuclear translocation and downstream IkB target gene using ChIP, and that the combination [P+B] showed additive inhibitory activity. We next sought to further dissect molecular mechanisms of synergy induced by the combination. B 5-10nM inhibited phosphorylation of ERK MAPK pathway and slightly upregulated Akt activity. On the other hand, P 10uM inhibited Akt phosphorylation, but induced activation of the ERK MAPK pathway. Interestingly, while either agent differentially upregulated one of those 2 signaling pathways, their combination [P+B] was able to overcome resistance induced by the other agent. This was demonstrated by significant inhibition of downstream target proteins of Akt, and by a significant decreased phosphorylation of fusion protein GSK3a/b using an in vitro Akt kinase assay. We next sought to examine the combination of R with [B+P], since R is known to inhibit Akt and NF-kB pathways. Pretreatment of WM cells with P, B or the combination [P+B] led to a significant increase in the ADCC cytotoxicity of R compared to R alone (p=0.025). We next confirmed that the combination [P+B+R] inhibited NF-KBp65 function in WM cells, using Active Motif assay and ChIP for IkB. Conclusion: Together these studies provide the framework for clinical studies of combination of perifosine with bortezomib and rituximab in a sequential approach to improve patient outcome in WM.