Abstract P1184: Spatiotemporal Proteomics Approach To Discern New Features Of Carfilzomib-mediated Cardiotoxicity

Abstract

Background: Proteasome inhibitors including carfilzomib have led to remarkable improvement in the survival of multiple myeloma patients, but are associated with cardiovascular adverse events including heart failure. A better understanding of the mechanism of toxicity is needed to improve oncogenic and cardiovascular outcomes. Rationale: Proteasome inhibition is known to impair the degradation rates as well as the subcellular distribution of proteins, processes that are not fully reflected by differential gene expression analysis. We hypothesize that cardiomyocyte proteins show impaired spatiotemporal distributions upon carfilzomib exposure, and that these changes constitute a crucial under-examined aspect of cardiotoxicity mechanism. Approach: To test this hypothesis, we developed a new mass spectrometry-based proteomics strategy that can simultaneously trace the turnover kinetics and subcellular localizations of proteins in cardiac cells. Results: In preliminary work, we applied this method to human iPSC-derived cardiomyocytes under 0.5 μM carfilzomib vs. control (n=2). We quantified the localization and turnover rates of >3,500 proteins in control and carfilzomib-treated cells. Unexpectedly, no significant difference was identified in the global rate of protein turnover between control and carfilzomib treated cells (p = 0.24 Wilcoxon-Mann-Whitney test), where the median protein half-lives of of control and carfilzomib treated cells were 31.0 vs. 30.7 hours, respectively. However, when we used a T-augmented Gaussian mixture model to predict protein localizations and assess compartment-specific turnover changes, we observed a significant reduction in turnover in the nuclear fraction, and a concomitant increase in that of proteasomal fraction under carfilzomib. The data also revealed a change in assigned localization in many myosin-binding proteins toward lysosomal co-sedimenting compartments, consistent with sarcomeric disarray and degradation in carfilzomib-exposed cardiomyocytes. Finally, we show a decrease in turnover of both fatty acid and glucose metabolism associated proteins. This method has potential for discovering unique molecular features of proteasome inhibitor mediated cardiotoxicity.