Treatment with the Anti-CD20 Antibody (B1) and Irradiation Result in Synergistic Cytotoxicity That Is Dependent on MAPK Activation.

Radioimmunotherapy using radiolabeled anti-CD20 antibodies (mAb) is an effective new treatment in non-Hodgkin lymphoma with high response rates. However, the molecular mechanisms behind these impressive clinical responses are poorly understood. To elucidate these mechanisms we studied the signaling events evoked in a panel of lymphoma cell lines following treatment with anti-CD20 mAb alone or in combination with irradiation. In all three lymphoma cell-lines tested a synergistic cytotoxic effect was observed when the anti-CD20 mAb B1 was combined with irradiation. The additive effect seen with B1 mAb and radiation was not observed with Rituximab and could be reversed with MEK inhibitors U0126 and PD98059 as well as siRNA targeting MEK1 or 2. Moreover, addition of U0126 reversed the decrease in clonogenic survival triggered by treatment with B1 and irradiation. To further probe the mechanism of this synergistic cell death we used cell lines over-expressing BCL₂ or crmA, to block mitochondrial and death receptor pathways, respectively. Although BCL₂ and crmA over-expression mediated protection against radiation alone, it had no impact on the increased cytotoxicity induced by B1+irradiation. Morphological studies revealed gross vacuolization of the cytoplasm, yet relatively well preserved nuclei in cells treated with B1+irradiation. Taken together our data indicate that activation of the MAPK cascade is an important factor that contributes to the synergistic effect of anti-CD20 (B1) antibody and irradiation and provides important new insights into how this treatment may work in the clinic.