381 BiP knockdown decreases antibody production in malignant and non-malignant plasma cells

Abstract

OBJECTIVES/GOALS: Numerous diseases, including AL amyloidosis, are due to expression of aberrant antibodies. Significant effort has gone into plasma cell toxic therapies with varying degrees of success, but no therapies preventing antibody synthesis have been developed. The goal of this study is to assess BiP targeting to prevent antibody secretion in plasma cells. METHODS/STUDY POPULATION: Using 4 multiple myeloma cell lines (KMS11, RPMI8226, ANBL-6, U266), we knocked down BiP expression with RnaseH dependent siRNA or subA toxin, a bacterial toxin that specifically cleaves BiP, and measured changes in unfolded protein and intracellular light chains by flow cytometry during drug induced ER stress created by the intracellular calcium depleting agent thapsigargin. BiP is the master regulator of the unfolded protein response (UPR), an ER stress pathway important for protein folding. BiP is also an ER resident protein folding chaperone important for proper antibody folding. We hypothesized that BiP downregulation will lead to decreased folded antibody in the cell, increased unfolded antibody and constitutive activation of the UPR. RESULTS/ANTICIPATED RESULTS: 1 to 4 hours after treatment with thapsigargin plus siRNA against BiP, levels of BiP are significantly decreased. The levels of intracellular light chains decrease, and the level of unfolded protein within the cells increases dramatically. Interestingly, in alignment with the UPR literature, 24 hours post treatment, these levels have normalized again in surviving cells. SubA treatment increased BiP expression by 4 hours, contrary to our hypothesis, and minimally increased unfolded proteins and minimally decreased intracellular light chains. We expect that further functional testing of antibody secretion by ELIspot assays will show decreased secretion of antibody with BiP siRNA treatment. Combination therapies with other UPR stressing agents may act synergistically to affect antibody production. DISCUSSION/SIGNIFICANCE: BiP knockdown reduces antibodies and boosts unfolded proteins. SubA toxin ineffectiveness likely stems from increased BiP due to feedback loops. Combining anti-BiP treatments with UPR stressing drugs like bortezomib may halt antibody synthesis and induce cell death. These findings support BiP as a viable drug target for antibody-related diseases.