Antifungal therapy with itraconazole impairs the anti-lymphoma effects of rituximab by inhibiting recruitment of CD20 to cell surface lipid rafts.

Immunotherapy with rituximab alone or in conjunction with chemotherapy has significantly improved the treatment outcome of B-cell lymphoma patients. Nevertheless, a subpopulation of patients does not respond to rituximab. The reason for treatment failure as well as the exact mechanism of action is still uncertain. The function of rituximab has long been associated with the partitioning of CD20 molecules to membrane microdomains. Here, we show that concomitant antifungal treatment with itraconazole impairs the rituximab anti-lymphoma effect both in vitro and in vivo. At the molecular level, recruitment of CD20 to lipid rafts is inhibited in the presence of itraconazole. Furthermore, calcium influx, which is crucial for rituximab-mediated cell death, was nearly completely abolished by itraconazole treatment. In contrast, the antifungal drug caspofungin did not inhibit CD20 recruitment to lipid rafts, nor did it affect calcium influx or the cytotoxic effect of rituximab. The finding that itraconazole also abolished the cytotoxic effects of other therapeutic antibodies directed against lipid raft-associated molecules (i.e., CD20 and CD52) but not those against the non-raft-associated molecule CD33 further supported our proposed mechanism of action. Our results argue that concomitant medications must be adjusted carefully to achieve optimal antitumor effects with monoclonal antibodies.

GM1 expression of non-Hodgkin's lymphoma determines susceptibility to rituximab treatment.

Immunotherapy with rituximab alone or in conjunction with chemotherapy has significantly improved the treatment outcome of B-cell lymphoma patients. Nevertheless, a subpopulation of patients does not respond to rituximab. The reason for treatment failure as well as the exact mechanism of action is still uncertain. The function of rituximab has long been associated with the partitioning of CD20 molecules to lipid microdomains. We now show that the extent of CD20 recruitment to lipid rafts correlates with response to rituximab. In addition, expression of the raft-associated sphingolipid GM1 on lymphoma cells is associated with the susceptibility of lymphoma cells to rituximab. Furthermore, we show substantially different GM1 expression in various primary non-Hodgkin's lymphomas. Whereas chronic lymphocytic leukemia (CLL) cells have a low GM1 expression, marginal zone lymphoma cells exhibit much higher levels. Differences were not only detected among various lymphoma subgroups but also within one lymphoma subtype. Interestingly, whereas CLL cells from patients with high GM1 expression responded to rituximab, patients with low GM1 expressing CLL cells did not. These data show the importance of membrane microdomains in the effect of rituximab and may offer a predictive factor for the responsiveness of lymphoma cells to rituximab.