Assessing proteasome workload to predict response and overcome resistance to proteasome inhibitors in multiple myeloma

The natural history of Multiple Myeloma (MM), a common and aggressive plasma cell cancer, has been radically changed by the use of bortezomib, a first in class proteasome inhibitor (PI) that has been demonstrated to significantly improve overall survival in relapsed/refractory patients. Nevertheless, less than half of the patients (38% in all patients, 45% with bortezomib as second line-treatment) enrolled in the APEX phase 3 clinical trial achieved a satisfactory response after treatment with bortezomib alone. The reasons for such diverse susceptibility of MM patients to bortezomib are still unclear. Given the remarkable extent of protein synthesis in multiple myeloma cells (MMCs) and the key role of the proteasome in coping with the inevitable defective products of this process, we hypothesized that the workload on the proteasome could be a main determinant of the toxicity of PIs. Thus, figures of proteasome load and capacity could predict sensitivity to proteasomal blockade in MMCs. To test this hypothesis, we set out to investigate proteasome load and capacity in two MM cell lines–U266 and MM.1S – with differential sensitivity to bortezomib. Indeed, in our hands, MM.1S proved at least one log more sensitive than U266 to 48- h treatment with bortezomib or other PIs. In this experimental model, we directly assessed protein degradation by proteasomes (load) using radioactive metabolic labeling and pulse-chase assays, and proteasome capacity as the total proteasome activity using specific fluorogenic peptides in cell extracts. We found a striking direct correlation between the degradative flux of proteins through proteasomes and the apoptotic response to PIs. In particular, proteasomal degradation within 30 min of chase was eighteenfold higher in MM.1S. Moreover, cell extracts from MM.1S cells showed less than half proteasomal activity of U266 cells, suggesting a decreased pool of proteasomes as compared to the resistant line. Taken together, the data suggest that an unfavorable load/capacity ratio may predispose cells to PI-mediated apoptosis. Finally, U266 cells, which rely less on proteasomes and are relatively resistant to proteasomal blockade, can be strongly sensitized to bortezomib by means of cytotoxic stressors, such as the protein N-glycosylation inhibitor tunicamycin, which increase the need for proteasomal degradation of proteins deriving from the endoplasmic reticulum. Altogether, these observations suggest that the direct assessment of proteasomal degradation may serve to predict responsiveness of MM cells to bortezomib and help inform the design of customized therapies by combining cytotoxic stressors with bortezomib to overcome drug-resistance.