Augmenting Immune Mediated Cytotoxicity

Considering TNF-a and FasL, there are major problems of toxicity associated with the direct administration of these death ligands to animals. By contrast, TRAIL is cytotoxic to a number of tumor cells in vitro and in vivo in the absence of major associated toxicities (Almasan and Ashkenazi 2003). Therefore, we have used TRAIL as a model to test our hypothesis that the response of tumor cells to death ligand-mediated apoptosis can be augmented by the administration of sensitizing agents. The proapoptotic activities of TRAIL on cancer cells are known to be enhanced by both radiation and chemotherapy. However, it is hard to envisage how radiation or chemotherapy could be effectively used in combination with immuno-therapy, as both radiation and chemotherapy are quite immunosuppressive. We have therefore chosen to study the efficacy of combining the proteasome inhibitor bortezomib (Velcade) and TRAIL in promoting tumor cell apoptosis. We and others have shown that bortezomib can sensitize a number of mouse and human tumor cells to TRAIL-mediated apoptosis (Sayers and Murphy 2006). The effects of the combination of bortezomib and TRAIL on various human renal cell carcinomas (RCCs) were tested. These RCCs were resistant to the apoptotic effects of low dose of TRAIL. However, treatment with bortezomib dramatically sensitized some of these carcinoma cells such as A498, ACHN, and UO-31 to the antitumor effects of TRAIL (Figure 1). This dramatic reduction in cell number was due to apoptotic death of the cancer cells (data not shown). We also tested a panel of 60 human cancer cells of different origin and found essentially the same pattern. That is, bortezomib at 5-20 nM concentrations sensitized about 25-30% of the panel of cancer cells to the apoptotic effects of TRAIL. None of the human RCCs we tested were intrinsically resistant to TRAIL-mediated apoptosis, since treatment with cycloheximide dramatically sensitized all seven cell lines to TRAIL. Thus, the apoptotic pathways in response to TRAIL could potentially be triggered in all the RCCs. Therefore, differences between the sensitization of ACHN and Caki-1 were specifically manifest in response to bortezomib (Figure 1).

A498 ACHN CAKI-1 SN12C TK-10 UO-31 786-0

Figure 1. Effects of bortezomib and TRAIL on human renal carcinoma cells. Viable cell counts were estimated 18 h after TRAIL addition by MTS staining, and percent growth inhibition was then calculated.

The molecular mechanism underlying this selective sensitization of tumor cells to TRAIL-mediated apoptosis remains unclear. It was initially thought that the ability of bortezomib to block activation of the transcription factor NF-kB would be crucial for sensitizing tumor cells to TRAIL. However, this activity of bortezomib does not seem to be crucial for the sensitization of a variety of tumor cells to TRAIL. Other possibilities include increases in the levels of TRAIL death receptors, increases in proapototic Bcl-2 family members, as well as decreases in cellular levels of an-tiapoptotic proteins such as cellular FADD-like IL-1-converting enzyme-inhibitory protein (cFLIP) or inhibitors of apoptosis proteins (IAPs). It was recently demonstrated that bortezomib treatment of tumor cells resulted in an enhanced activation of caspase-8 following TRAIL binding. Therefore, it is possible that the major effects of bortezomib in sensitizing tumor cells to TRAIL may occur at a proximal level of the apoptotic signaling cascade (Ganten et al. 2005). Further work is required to identify the precise molecular mechanism(s), whereby bortezomib sensitizes tumor cells to TRAIL apoptosis. As yet, there is very little information available on whether the combination of bortezomib and TRAIL can display therapeutic benefit in an in vivo setting. We first reported that this combination was superior to either agent alone in purging C1498 murine acute myeloid leukemia cells from a bone marrow inoculum (Sayers et al. 2003). Furthermore, more recent studies have shown that bortezomib can be combined with allogeneic bone marrow transfer allowing for a reduction of graft-versus-host disease while maintaining beneficial graft-versus-tumor effects (Sun et al. 2004). Current studies from our laboratory also indicate that the combination of bortezomib with an agonist antibody to TRAIL death receptor 5

can provide therapeutic benefit in tumor-bearing mice in the absence of detectable toxicity (Shanker and Sayers, in preparation). It seems likely that bortezomib could also be effectively combined with agents that induce TRAIL production in vivo, particularly if this induction could be localized to the vicinity of the tumor or its metastases. Human genome sciences have recently commenced with a clinical trial investigating any therapeutic benefit of combining bortezomib and agonist antibodies to TRAIL death receptors in patients with multiple myeloma.

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