Abstract
Introduction: Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in North America. Despite many therapeutic advances over the past decade, drug resistance and disease recurrence are common. Novel therapeutic approaches are therefore required to treat CLL. One novel target identified in a variety of cancers, including acute myeloid leukemia, is the lysosome. In transformed cancerous cells, lysosomes were found to be sensitive to permeabilization by lysotropic agents in a process called lysosome membrane permeabilization. Permeabilization of lysosomes releases their acidic and proteolytic contents into the cytoplasm causing lysosome-mediated cell death. The therapeutic strategy of targeting lysosomes has yet to be determined in CLL.
Methods: Primary CLL cells were purified from patient peripheral blood using negative selection and separated on a ficoll gradient. Three different lysosome-targeting drugs used in the clinic for other purposes were investigated: a quinolone, a fluoroquinolone antibiotic, and a cationic drug (CAD). To determine the mechanism of action, various dyes were used to stain lysosomes, mitochondria, and reactive oxygen species. Fluorescence was visualized under the confocal microscope and quantified using flow cytometry. To determine the role of reactive oxygen species (ROS) the antioxidants α-tocopherol, lycopene, N-acetyl cysteine, and glutathione were added to cells. To determine the role of proteases the inhibitors zVADfmk, Ca-074-Me, Chymostatin, and E64 were added to cells.
Results: All the lysotropic agents except the antibiotic effectively killed CLL cells isolated from patients. The CAD had the greatest activity and was significantly more cytotoxic to the CLL cells as compared to T cells from the same patients and peripheral blood mononuclear cells from non-CLL donors. Treatment was equally effective in p53-proficient and p53-deficient CLL cells, demonstrating that the most aggressive and drug-resistant CLL cells were sensitive to this CAD. Mechanistic studies revealed that lysosome membrane permeabilization occurred within minutes and led to an increase in ROS and loss of mitochondrial membrane potential. The permeabilization of lysosomes was further confirmed by the translocation of transcription factor EB (TFEB) into the nucleus indicating promotion of lysosomal biogenesis. Lipid ROS were necessary to induce cell death, as only lipophilic antioxidants prevented cell death. Lipophilic antioxidants did not prevent lysosomal permeabilization, but did prevent downstream mitochondrial dysfunction. Inhibitors of caspases and lysosomal cathepsins failed to prevent cell death in CLL cells.
Conclusions: Results show that the lysosome-targeting quinolone and CAD effectively permeabilize lysosomes and induce lysosome-mediated cell death in primary human CLL cells. This unique mechanism of cell death in CLL is dependent on the generation of lipid ROS, but not on the action of caspases or cathepsins. Overall, targeting lysosomes may be an effective strategy to selectively kill CLL cells regardless of p53 expression. Future studies are focused on the lysosomal differences in B cells and CLL cells.
Disclosures
No relevant conflicts of interest to declare.
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