Blocking Protein Secretion and Degradation Is a Novel Treatment Strategy For Malignant Cells With High Protein Load

Multiple myeloma (MM) is the result of deregulated proliferation in malignant plasma cells that secrete an overabundance of a monoclonal immunoglobulin (Ig). The high level of Ig production in MM cells places a unique burden on the ubiquitin proteasome system (UPS) to maintain protein homeostasis and may explain why MM cells are among the most sensitive cell type to proteasome inhibition. We hypothesized that blocking protein secretion would further increase the protein load in MM cells and induce an anti-tumor response. In H929 and U266 cells, exposure to the proteasome inhibitors carfilzomib (CFZ) or bortezomib for 1 hour resulted in ubiquitination of several components of the Sec61 translocon, a hetero-trimeric channel present in the ER membrane central to protein secretion. Recently, cotransins were identified as a class of selective Sec61 inhibitors that block the translocation of specific nascent peptides into the ER. We determined the sensitivity to dual inhibition of protein secretion and degradation in 3 MM lines (NCI-H929, RPMI-8226, U266) and two plasma cell leukemia lines (JJN3, ARH-77) using the combination of CFZ and the cotransin CT08. We observed that 8226, H929, and JJN3 cells were sensitive to single agent CT08, with cell viability IC₅₀ values ranging from 120 to 170 nM, while U266 and ARH-77 showed little sensitivity. CFZ IC₅₀ values ranged from 1.5 to 6 nM across all 5 cell lines. CT08-sensitive cells treated with both agents, across a matrix of concentrations, showed a significant decrease in cell viability. For most concentrations, combination index (CI) values fell below 0.9, indicating synergistic activation of cell death, and reached levels of 0.19 (H929), 0.277 (JJN3), and 0.536 (8226). In resistant cells, CT08 potentiated (CI not evaluable) an anti-myeloma response when co-treated with CFZ, suggesting a significant interaction effect. CT08 also potentiated the cytotoxic effect of CFZ in the small cell lung cancer line DMS114 despite these cells being 10-fold less sensitive to CFZ than MM cell lines. In addition, CT08/CFZ combination treatment of U266 cells in co-culture with the bone marrow stromal cell line HS-5 overcame the protective effect of the stromal cells, as measured by Annexin V and propridium iodide staining. Taken together, these data suggest that dual Sec61 and proteasome inhibition represents a novel treatment strategy for malignant cells with high protein secretion like multiple myeloma.