Assessing Proteostasis and Proteasome Stress In Light Chain Amyloidosis

Abstract 3992

Recently, proteasome inhibitors (PI) proved powerful against multiple myeloma (MM), the neoplastic transformation of plasma cells. The balance between proteasome expression and degradative workload (mainly contributed by protein synthesis) proved a crucial determinant of apoptotic sensitivity of MM cells to proteasome inhibition (Bianchi et al, Blood 2009). Light chain amyloidosis (AL) is a plasma cell dyscrasia caused by a bone marrow plasma cell clone synthesizing structurally unstable, misfolded, monoclonal immunoglobulin (Ig) light chains, which polymerize into amyloid fibrils. Interestingly, AL is proving even more sensitive than MM to PI in clinical trials with unprecedented response rates (>80%) rapidly achieved in previously untreated patients (Kastritis et al, J Clin Oncol 2010), raising the question as to whether, and if so why, AL cells are intrinsically more sensitive than MM to PI. We hypothesized that AL cells suffer from intense proteasome stress linked to the synthesis of the misfolded Ig light chain, thereby facing constitutive proteotoxicity.

To test this hypothesis, we set out to optimize purification of primary bone marrow plasma cells from AL patients, and determine: intrinsic sensitivity to the PI bortezomib (by FACS); proteasome activity (by fluorogenic assays); accumulation of ubiquitinated (Ub) proteins and Ig light chain (by immunofluorescence).

Our ex vivo studies demonstrated twofold higher PI sensitivity in AL plasma cells as compared to primary MM cells (EC50 in 24 hr apoptosis assays: AL, 8.3 ± 2.2 nM; MM, 15.1 ± 3.0 nM). We also found that, similar to MM cells, proteasome activity of primary AL plasma cells varies greatly among different patients (5.2 ± 3.6 nM substrate specifically cleaved by the chymotryptic β-peptidase activity per cell per min). Furthermore, accumulation of Ub proteins strongly correlates with light chain content, suggesting a crucial role for paraprotein synthesis and/or retention on proteasome stress. Interestingly, unlike MM cells, we failed to detect a clear correlation between proteasome activity and ex vivo assessed PI sensitivity, possibly due to intracellular toxicity of the misfolded light chain. The resulting hypothesis that different mutations could result in different intrinsic proteotoxicity in AL cells is currently being tested.

In conclusion, our integrated approach indicates that AL cells are intrinsically more sensitive to PI than MM cells, providing a potential explanation for the excellent clinical responses. Moreover, we established a technological platform to investigate proteostasis and proteotoxic stress in primary AL cells. This strategy may help investigate the efficacy of proteostasis regulators on plasma cell dyscrasias, including MM, and identify molecular markers of clinical use to predict disease severity and response to therapy.