What signals are generated by anti-CD20 antibody therapy?

Immunotherapy with rituximab (a chimeric anti-CD20 monoclonal antibody), alone or in combination with chemotherapy, has improved the treatment outcome of patients with non-Hodgkin's lymphoma (NHL), but the in vivo mechanisms by which rituximab exerts its effects have not been elucidated. The mechanisms underlying resistance are not known. In addition to the proposed actions mediated by rituximab (such as complement-dependent cytotoxicity, antibody-dependent cellular cytotoxicity, and apoptosis), rituximab may signal the tumor cells and inhibit constitutively activated survival signaling pathways (Raf-1-MEK1/2-ERK1/2, p38 MAPK, NF-kappaB, and Akt), resulting in inhibition of cell growth and of selectively anti-apoptotic gene products such as Bcl-2 and Bcl-(xL). The inhibition of these anti-apoptotic gene products by rituximab sensitizes drug-resistant tumor cells to apoptosis induced by a variety of cytotoxic chemotherapeutic drugs. Also, rituximab sensitizes NHL cells to apoptosis resulting from upregulation of death receptors, implicating a novel in vivo role of host involvement in rituximab-mediated effects. We have developed rituximab-resistant clones that do not respond to rituximab-mediated cell signaling. The clones exhibited hyperactivated cell survival pathways and overexpression of anti-apoptotic gene products and could not be chemosensitized by rituximab. Inhibitors of the survival signaling pathways reverse drug resistance in both wildtype cells and resistant clones. These findings identify several novel intracellular pathways modifyed by rituximab that sensitize NHL cells to both chemotherapy and immunotherapy, as well as several therapeutic targets whose modifications reverse resistance. These findings have clinical relevance for both prognosis and novel treatment strategies for patients with NHL.

Enhanced expression of CD20 in human tumor B cells is controlled through ERK-dependent mechanisms.

Rituximab, a chimeric Ab directed against CD20, induces apoptosis in targeted cells. Although the majority of B cell malignancies express the CD20 Ag, only approximately 50% of patients will respond to single-agent rituximab. The available data suggest that a decreased CD20 expression could account for the lack of response observed in some patients treated with rituximab. Despite the potential critical role of CD20 in the biology of B cell malignancies, the mechanisms controlling its expression are poorly understood. We evaluated the effect of the immune modulator agent bryostatin-1 on the expression of CD20 in non-Hodgkin's lymphoma cells. Using the B cell lines, DB and RAMOS, as well as tumor cells derived from a chronic lymphocytic leukemia patient, we demonstrated that bryostatin-1 enhanced the expression of both CD20 mRNA and protein. The enhanced expression of CD20 was associated with increased transcriptional activity of the CD20 gene, whereas the stability of CD20 mRNA was not affected. The effect of bryostatin-1 on CD20 expression in non-Hodgkin's lymphoma cells was mediated through the MAPK kinase/ERK signal transduction pathway and involved protein kinase C, but was independent of p38 MAPK and was insensitive to dexamethasone. Cells pretreated with bryostatin-1 were more susceptible to the proapoptotic effect of anti-CD20 Ab. Overall, these data demonstrate for the first time that ERK phosphorylation is required for the up-regulated expression of CD20 on B cell malignancies. The findings also suggest that bryostatin-1 and rituximab could be a valuable combined therapy for B cell malignancies.

The association between CD20 and Src-family Tyrosine kinases requires an additional factor.

CD20 is a B cell surface protein which can initiate intracellular signals involving tyrosine kinase activation, and modify B cell growth and differentiation. CD20 is tightly associated with the Src-family kinases Lyn, Fyn and Lck; however, the mechanism of interaction remains to be determined. Association between CD20 and Src-family kinases has been detected in peripheral blood B cells and in 5 out of 8 unrelated B cell lines. The lack of CD20-associated kinase activity in some cell lines offered an opportunity to investigate the mechanism of CD20 associations. All 8 B cell lines were found to express Lyn, and, with one exception, all cell lines also expressed Fyn. Lck, however, was not detected in any of the cell lines in which CD20 failed to coprecipitate kinase activity. To test the possibility that Lck was required for assembly of the CD20 complex, Lck was transfected into one of the 3 CD20/kinase association-deficient lines, namely T51. CD20 did not coprecipitate kinase activity from the transfected T51 cells, despite their expression of high levels of exogenous Lck, as well as endogenous Lyn and Fyn. CD20 cDNA from T51 was sequenced and found to be normal. These data establish that association between CD20 and Src-family kinases requires an additional factor.