Proteasome Inhibitors Sensitize Myeloma Cells to T Cell-Mediated Killing

Graft-versus-myeloma (GvM) effect following allogeneic stem cell transplantation (allo-SCT) is well established and often accompanied by graft-versus-host disease (GVHD). Unfortunately, most patients still relapse following allo-SCT, underscoring the need for new transplant strategies. Interestingly, studies evaluating the proteasome inhibitor bortezomib combined with donor lymphocyte infusions (DLI) in myeloma patients suggest enhanced efficacy of the combination. In contrast, other reports demonstrate that bortezomib has activity in preventing GVHD in various malignancies, including myeloma. Here, we investigate the effects of bortezomib on T cell-mediated myeloma cell killing in vitro.

Tumor cell compartment-specific bioluminescence imaging (CS-BLI) technology was used to monitor the cytotoxic effects of T cells on myeloma cells in the presence or absence of antimyeloma agents. To this end, myeloma cell lines were stably transduced with a firefly luciferase construct. Two different T cell clones were used: 1) the alloreactive CD8+-T cell clone allo-A2, which kills all HLA-A2-positive cells, including the myeloma cell line U266; and 2) the CD4+ T-cell clone 3AB11, generated from a myeloma patient with clinical GvM/GVHD after allo-SCT. 3AB11 cells recognize a hematopoietically restricted minor antigen, also presented by the myeloma cell line UM9. Protein expression was determined by western blot analysis.

The CD4+-T cell clone 3AB11 and the allo-A2 specific CD8+- T cell clone displayed a time- and dose-dependent lysis of myeloma cell lines UM9 and U266, respectively. Concurrent treatment of myeloma cells with the T cell clones and bortezomib resulted in synergistic enhancement of myeloma cell death, compared to T cells alone or bortezomib alone. This synergism was observed at 24, 48, and 72 hours (but not after 4 hours) of co-treatment. Dexamethasone, which significantly inhibited myeloma cell death induced by T cells, functioned as control.

When myeloma cells were treated for 12 hours with bortezomib and then extensively washed, there was still enhancement of T cell-mediated killing. However, pre-incubation of T cells with bortezomib significantly impaired their ability to induce myeloma cell lysis. These washout experiments indicate that bortezomib sensitizes myeloma cells to T cell-mediated killing, but does not enhance the intrinsic cytotoxic activity of T cells.

Mechanistic studies demonstrated that antibodies blocking FAS ligand partially impaired myeloma cell lysis by 3AB11 and allo-A2 clones. More interestingly, however, blockade of FAS/FAS ligand interactions completely abrogated the synergism between bortezomib and T cells. Antibodies against TRAIL had no such effects. Furthermore, western blot analyses revealed that in UM9 and U266 cells bortezomib reduced protein levels of FLICE inhibitory protein (cFLIP), which is a potent inhibitor of FAS ligand-induced apoptosis. These results support the idea that bortezomib enhances the sensitivity of myeloma cells toward T cell-mediated lysis by restoring apoptosis signaling via the FAS/FAS ligand pathway.

Proteasome inhibition sensitizes myeloma cells to T cell-mediated killing via a FAS/FAS ligand-dependent mechanism. Conversely, bortezomib impairs T cell function. However, the net effect is a synergistic killing of myeloma cells when T cells and bortezomib are combined. These data support further exploration of the use of bortezomib in preclinical models, with derived clinical trials in the allogeneic setting (e.g. in combination with DLI or following allo-SCT) or in the autologous setting (e.g. in combination with a tumor vaccine) potentially warranted. (CSM and TM are joint senior authors)

van de Donk:Celgene: Research Funding. Lokhorst:Celgene: Research Funding; Jansen-Cilag: Research Funding. Hayes:Pfizer: Research Funding; Amgen: Research Funding. Munshi:Celgene: Consultancy; Millenium: Consultancy; Novartis: Consultancy; Onyx: Consultancy. Richardson:Millennium:; Celgene:; Johnson & Johnson:; Novartis:; Bristol Myers Squibb:. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Acetylon: Membership on an entity's Board of Directors or advisory committees. Mitsiades:Millennium Pharmaceuticals: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Novartis Pharmaceuticals: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Merck: Consultancy, Honoraria; Kosan: Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; Centocor: Consultancy, Honoraria; Amnis Therapeutics: Consultancy, Honoraria; PharmaMar: Licencing royalties; OSI Pharmaceuticals: Research Funding; Amgen: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis: Research Funding; Gloucester Pharmaceuticals: Research Funding; Genzyme: Research Funding; Johnson & Johnson: Research Funding.