Abstract 2643: Inhibition of calpain disrupts the autophagic response initiated by bortezomib resulting in increased death of myeloma cells

Abstract

The proteasome is a multi-subunit enzyme complex that plays a central role in the regulation of proteins controlling many important cellular functions including cell cycle progression, differentiation, gene transcription and apoptosis. Bortezomib is a potent proteasome inhibitor that has recently been approved for use in refractory multiple myeloma. Our previous work demonstrated that bortezomib induces an endoplasmic reticulum (ER) stress response ultimately leading to Ca2+-dependent apoptotic cell death. Intracellular Ca2+ has also been implicated in the cellular stress response known as autophagy. The biochemical events linking the cellular stress response with the induction of autophagy, and the relationship between autophagy and apoptosis are not well understood. In this study, we investigate the role of the Ca2+ dependent serine protease, calpain, as a mediator of the conversion from autophagic cell survival to accelerated cell death in the ER stress response. Using live cell imaging we demonstrate that bortezomib rapidly initiates autophagy in myeloma cells. Microtubule associated protein 1 light chain 3 beta (LC3) is an autophagic marker that is converted from the inactive form (LC3I) to the autophagosomal membrane-associated (LC3I) form. Simultaneous analysis of the LC3II/I ratio and the polyubiquitin-binding protein, p62/SQSTM1, demonstrates an early elevation in autophagosome formation with an increase in autophagic digestion. Inhibition of the Ca2+-dependent serine protease, calpain, dramatically increases the accumulation of non-functional autophagosomes illustrated by a rise in both the LC3II/I ratio and p62 levels. This effect is accompanied by a significant enhancement or acceleration of cell death. Similarly, elimination of the small catalytic subunit, CAPNS1 using siRNA significantly increased bortezomib-mediated cell death. Our data suggest that inhibition of calpain prevents autophagolysosome maturation, and subverts the survival response thereby enhancing cell death. These data suggest a new therapeutic strategy to enhance the activity of proteasome inhibitors and overcome drug resistance in refractory myeloma and other B cell malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2643. doi:10.1158/1538-7445.AM2011-2643