Proteasome inhibition regulates survival and homing in Waldenstrom Macroglobulinemia (WM)
Authors: Aldo Roccaro, Xavier Leleu, Antonio Sacco, Anne-Sophie Moreau, Xiaoying Jia, Hai Ngo, Abdelkareem Azab, Feda Azab, Teru Hideshima, Michael Palladino, Kenneth Anderson, Dharminder Chauhan, Irene Ghobrial
Journal: Cancer Research (2008)

3259     Background:  WM is a biologically unique low grade B-cell lymphoma characterized by high protein turnover. Protein metabolism is a tightly regulated process, and inhibition of its turnover may lead to apoptosis in malignant cells, such as with proteasome inhibitors. We dissect the biological role of proteasome inhibitors NPI-0052 ( N ) and bortezomib ( B ) in WM.   Methods:  WM cell lines (BCWM1,WSU-WM) and IgM secreting cell lines (MEK1,Namalwa) were used. Bone marrow (BM) primary CD19 +  cells and BM stromal cells (BMSC) were obtained from patients with WM, after informed consent. Cytotoxicity and DNA synthesis were measured using MTT assay and [ 3 H]-thymidine uptake, respectively. Synergistic effect of  N+B  combination was calculated using the CalcuSyn software. Cell signaling and apoptotic pathways were determined by Western Blot. The effect of  N + B  combination on NF- k B activity was determined using the Active Motif’s-TransAM kit. p65NF- k B nuclear translocation was monitored by immunofluorescence. The chymotrypsin-, trypsin- and caspase-like proteolytic activities of the 20S proteasome were determined by detecting the release of the fluorophore AMC, after cleavage from the labeled substrates specific for each enzymatic activity. The effect of  N  and  B  on WM cells in the co-culture with BMSCs was evaluated, as well as their effect on adhesion to fibronectin and migration  in vitro  and  in vivo .   Results: N  induced cytotoxicity and inhibited DNA synthesis in WM cell line (IC 50  15nM). Similar effects were obtained in all IgM secreting cell lines and in primary CD19 +  WM cells (IC 50  18-30nM). No cytotoxicity was observed on peripheral blood mononuclear cells at similar doses. The combination of  N + B  induced synergistic cytotoxicity in WM cells, leading to inhibition of nuclear translocation of p65NF- k B and synergistic induction of caspases-3, -8, -9 and PARP cleavage.  B  and  N  inhibited the canonical and non-canonical NF- k B pathways, inhibited p65NF- k B translocation to the nucleus; acted synergistically through their differential effect on the 20S proteasome enzymatic activities and on Akt activity. The inhibition of the caspase-like activity synergistically increased when  N  and  B  were used in combination on BCWM.1 cell and CD19+ primary cells.  N  inhibited PI3K/Akt pathway and Akt activity in BCWM.1 cells, even when combined with  B , which induced an increase of Akt phosphorylation.  N -induced cytotoxicity was completely abrogated in an Akt-knockdown BCWM.1 cells indicating that its major activity is mediated through the Akt pathway. Moreover,  N  and  B  inhibited migration, adhesion  in vitro  and homing of WM cells  in vivo ; and overcame resistance induced by mesenchymal cells or by the addition of IL-6 in a co-culture system.   Conclusion:  These data enhance our understanding of the biological role of the proteasome pathway in WM, and provide the preclinical basis for clinical trials of combinations of proteasome inhibitors in WM.