Lysosomal Disruption Augments Obinutuzumab-Induced Direct Cell Death

Anti-CD20 mononclonal antibodies (mAbs) have a well-established role in the treatment of B-cell lymphoid malignancies. In addition to classic Fc-dependent mechanisms, including antibody-dependent and complement-mediated cytotoxicity, the so-called type II mAbs induce direct cell death. It has been shown that obinutuzumab, without Fc-crosslinking agents or effector cells, triggers non-apoptotic, lysosomal-dependent programmed cell death (PCD). The mechanism of PCD is characterized by actin reorganization, followed by permeabilization of the lysosomal membrane and subsequent generation of reactive oxygen species (ROS) through NADPH oxidase. Although, mechanisms of PCD are well-described, little is known about factors influencing sensitivity of malignant B-cells to obinutuzumab-mediated direct cell killing. Strategies to improve PCD could be potentially exploited to eliminate malignant cells, which are refractory to conventional immunotherapy. In this study, we aimed to investigate the influence of lysosomotropic agent, chloroquine, on the efficacy of obinutuzumab-mediated cytotoxicity. As PCD is dependent on lysosomal destabilization, we hypothesized that combination of obinutuzumab with lysosome-destabilizing agent would result in increased cell death.

In our study, we used a Burkitt lymphoma Raji cell line that is widely employed as a model to assess the efficacy of obinutuzumab. Raji cells were incubated with obinutuzumab, alone or in combination with increasing concentrations of chloroquine, followed by annexin V/PI staining. Chloroquine, significantly increased direct cell death induced by obinutuzumab, without being toxic alone. Those observations were further corroborated by cell staining with other viability dies - TO-PRO-3 iodide and 7-AAD.The efficacy of the tested combination was completely abrogated by cytochalasin D - an inhibitor of actin polymerization and concanamycin A - inhibitor of vacuolar ATPases that activate acidic vacuoles including lysosomes, suggesting that chloroquine and obinutuzumab share a common mechanism of action. Since chloroquine has been reported to promote ROS generation, we analyzed the production of ROS with DCFDA staining. We observed that chloroquine potentiated the oxidative effect of obinutuzumab. Consistently, the effect of the combination was completely abrogated by a cell-permeable ROS scavenger - Tiron. As obinutuzumab has been shown to induce ROS production via activation of NADPH oxidase, we investigated the influence of NADPH oxidase inhibitor - diphenylene iodonium (DPI) on the combination's efficacy. DPI only partially reversed the effect of obinutuzumab, while strongly decreasing the effect of the combination.

Altogether, we show for the first time that chloroquine sensitizes cell to lysosomal cell death induced by obinutuzumab. The results of our study provide a strong rationale for combining obinutuzumab with lysosomotropic agents. These findings may have particular importance given the fact that another lysomotropic agent - siramesine has recently been shown to induce selective cytotoxicity in chronic lymphocytic leukemia (CLL).