Antibody-Induced Non-Apoptotic Cell Death in Human Lymphoma and Leukemia Cells Is Mediated Through NADPH Oxidase-Derived Reactive Oxygen Species

Anti-CD20 monoclonal antibodies (mAbs), most notably rituximab, have revolutionized the treatment of B-cell malignancies with substantially improved clinical outcome for patients. However, a proportion of patients still relapse and become refractory to rituximab. Therefore, several next-generation mAbs are being developed to improve responses further and provide novel therapies for refractory patients. In addition to classical Fc-dependent mechanisms such as antibody-dependent cellular cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC), certain mAbs can eliminate target cells by triggering intracellular signaling upon antigen ligation to directly induce programmed cell death (PCD). The role of direct PCD and its underlying mechanisms remain under-investigated and poorly understood. We recently demonstrated that certain mAbs (specifically type II anti-CD20 and anti-HLA DR mAbs) potently evoked PCD which was dependent on homotypic adhesion and the rearrangement of the actin cytoskeleton, which in turn triggered lysosome membrane permeabilization (LMP) and cathepsin-mediated cell death (Ivanov et al J Clin Invest 2009, Alduaij et al Blood 2011). Here, we further probe the mechanisms involved, specifically investigating the contribution of reactive oxygen species (ROS).

The generation of ROS was detected using dihydroethidium (HE) and 5-(and-6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA) monitored using flow cytometry and fluorescence microscopy. Cell death was quantified using propidium iodide (PI) and annexin V staining, and Chromium-51 release assays. For primary B-cell chronic lymphocytic leukemia (B-CLL) cells, cell death was detected using Annexin V-Cy5.5 and 7-aminoactinomycin. Mitochondrial depolarization was monitored using the JC-1 dye.

The extent of ROS generated by a range of mAbs, including type II anti-CD20 mAbs (tositumomab and GA101) and anti-HLA DR mAbs (L243 and 1D10/apolizumab), was positively correlated with their ability to induce PCD, in human B-lymphoma cell lines and primary B-CLL cells. The ROS scavengers tiron and tempol abrogated mAb-induced PCD indicating that ROS are required for its execution. ROS was found to be generated downstream of the mAb-induced actin cytoskeletal reorganization and LMP. Although GA101 potently induced mitochondrial membrane permeabilization, the ability of mitochondrial respiration-deficient Raji subclones to efficiently produce ROS and undergo cell death upon mAb treatment was unaffected, strongly suggesting that mitochondria are not essential for mAb-induced ROS production and cell death. Instead, ROS generation and PCD were blocked by inhibitors of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases and reduced by siRNA-mediated knock down of the NADPH oxidase NOX2.

These findings provide further novel insights into the non-apoptotic PCD pathway evoked by mAbs in B-cell malignancies, highlighting a previously unrecognized role for NADPH oxidase-derived ROS in the terminal effector phase of the death pathway. This newly characterized cell death pathway may be exploited to eliminate malignant lymphoid cells which are refractory to conventional chemotherapy and immunotherapy.

No relevant conflicts of interest to declare.