Imbalance between synthetic load and proteasomal capacity sensitizes normal and malignant plasma cells to proteasome inhibitors

INTRODUCTION: Multiple Myeloma (MM) is an aggressive, debilitating and still incurable hematological malignancy, arising from clonal expansion of plasma cells at multiple sites in the bone marrow. Recently, MM proved sensitive to a new class of drugs, proteasome inhibitors (PI), but the mechanisms of action and bases of individual susceptibility remain still largely unclear. Obviously, their clarification would improve the clinical application of PI, and open novel therapeutic strategies. Recent work linked PI sensitivity to protein synthesis and proteasome activity (1-3), raising the question whether different levels of proteasome expression and workload underlie PI sensitivity in normal and transformed plasma cells. RESULTS: To test this hypothesis we first adopted different models of mouse B cell activation in vivo, and observed that following polyclonal activation, proteasome activity decreases even more than previously reported in vitro. This decrease is linked to enhanced apoptosis after treatment with the first-in-class anti-myeloma proteasome inhibitor bortezomib (PS-341, Velcade™). Accordingly, in vivo treatment with bortezomib decreases Ab titres in T-dependent and -independent mouse immunization models, therefore providing the rationale for limiting the activity of Ab-secreting cells in vivo by impacting proteasome function. Next, to asses whether the exquisite sensitivity of certain MM cells (MMC) to PI is also due to an imbalance between limited proteasome capacity and high workload due to intense protein production, we directly assessed protein degradation by proteasomes using radioactive metabolic labeling and pulse-chase assays in two human MM cell lines - U266 and MM.1S - that display a differential apoptotic sensitivity to bortezomib. These two lines reveal a striking direct correlation between the degradative flux of proteins through proteasomes and the apoptotic response to bortezomib. In particular, proteasomal degradation within 30 minutes of chase is almost 20 times higher in MM.1S, the far more sensitive line, indicating that these cells are intensively degrading short-lived protein species through the ubiquitin-proteasome pathway. Paradoxically, cell extracts from MM.1S cells show 2.5 times lower proteasomal activity than U266 cells, suggesting a decreased pool of proteasomes as compared to the resistant line. In this scenario, accumulation of poly-ubiquitinated proteins and the accompanying decrease of free ubiquitin reveal proteasome stress in PI-sensitive MMC. Finally, to establish cause-effect relationships, we manipulated proteasome workload, by means of ER stressors, and proteasome capacity, via treatment with rapidly reversible PI, achieving profound alterations of PI sensitivity. Altogether, our data demonstrate that the balance between proteasome workload and degradative capacity represents a critical determinant of apoptotic sensitivity of MMC to PI, providing both a novel predictive tool of potential prognostic value and the framework for novel combination therapies