Purpose Bortezomib, a first generation proteasome inhibitor, induces an endoplasmic reticulum (ER) stress response which ultimately leads to dysregulation of intracellular Ca2+ and apoptotic cell death. This study investigated the role of the Ca2+-dependent enzyme, calpain, in bortezomib cytotoxicity. A novel therapeutic combination was evaluated in which HIV protease inhibitors were used to block calpain activity and enhance bortezomib cytotoxicity in myeloma cells in vitro and in vivo. Methods Bortezomib-mediated cell death was examined using assays for apoptosis (Annexin V staining), total cell death (trypan blue exclusion) and growth inhibition (MTT). The effects of calpain on Bortezomib-induced cytotoxicity were investigated using siRNA knockdown or pharmaceutical inhibitors. Enzyme activity assays and immunofluorescence analysis were used to identify mechanistic effects. Results Inhibition of the Ca2+ dependent cysteine protease calpain, either by pharmacologic or genetic means, enhances or accelerates bortezomib-induced myeloma cell death. The increase in cell death is not associated with an increase in caspase activity, nor is there evidence of greater inhibition of proteasome activity, suggesting an alternate, calpain-regulated mechanism of brtezomib-induced cell death. Bortezomib initiates an autophagic response in myeloma cells associated with cell survival. Inhibition of calpain subverts the cytoprotective function of autophagy leading to increased bortezomib-mediated cell death. Combination therapy with bortezomib and the calpain-blocking HIV protease inhibitor, nelfinavir, reversed bortezomib resistance and induced near-complete tumor regressions in a SCID mouse xenograft model of myeloma.

Multiple myeloma (MM) is an incurable malignancy of plasma cells. Although multiple myeloma patients often respond to initial therapy, the majority of patients will relapse with disease that is refractory to further drug treatment. Thus, new therapeutic strategies are needed. One common mechanism of acquired drug resistance involves a reduction in the expression or function of the drug target. We hypothesized that the cytotoxic activity of topoisomerase II (topo II) poisons could be enhanced, and drug resistance overcome, by increasing the expression and activity of the drug target, topo II in myeloma cells. To test this hypothesis, we evaluated the cytotoxicity of the anthracenecontaining topo II poison, ethonafide (AMP-53/6-ethoxyazonafide), in combination with the proteasome inhibitor bortezomib (PS-341/Velcade). Combination drug activity studies were done in 8226/S myeloma cells and its drug resistant subclone, 8226/Dox1V. We found that a 24-hour treatment of cells with bortezomib maximally increased topo IIα protein expression and activity, and consistently increased the cytotoxicity of ethonafide in the 8226/S and 8226/Dox1V cell lines. This increase in cytotoxicity corresponded to an increase in DNA double-strand breaks, as measured by the neutral comet assay. Therefore, increasing topo IIα expression through inhibition of proteasomal degradation increased DNA double-strand breaks and enhanced the cytotoxicity of the topo II poison ethonafide. These data suggest that bortezomib-mediated stabilization of topo IIα expression may potentiate the cytotoxic activity of topo II poisons and thereby, provide a strategy to circumvent drug resistance.

The 26S proteasome is a key regulator of proteins controlling many important cellular functions, including cell cycle progression, differentiation, gene transcription and apoptosis. Proteasome inhibition is a new therapeutic strategy that has shown promise in the treatment of B cell malignancies, primarily multiple myeloma. We and others have demonstrated that proteasome inhibitors induce endoplasmic reticulum (ER) stress and activate an unfolded protein response (UPR) in transformed cells. Our previous work demonstrated that bortezomib induces an endoplasmic reticulum (ER) stress response ultimately leading to calcium-dependent apoptotic cell death. Co-treatment of myeloma cells with the mitochondrial uniporter inhibitor ruthenium red (RuR) abrogated bortezomib mediated cell death, indicating that the cytotoxic effects of proteasomal inhibition requires dysregulation of intracellular Ca2+. Intracellular Ca2+ has also been implicated in the cellular stress response known as autophagy or “self-eating”. Macroautophagy (hereafter referred to as autophagy) is induced by various cellular stresses including nutrient deprivation, metabolic insufficiency, interruption of growth factor signaling, elevated ROS, accumulation of intracellular Ca2+, and the UPR. The biochemical events linking the cellular stress response with the induction of autophagy, and the relationship between autophagy and apoptosis is 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. We demonstrate that the proteasome inhibitor, bortezomib, initiates autophagy in myeloma cells, and protection from bortezomib-mediated cell death by mitochondrial Ca2+ inhibitors is associated with a promotion and stabilization of the autophagosome. This response can be reversed, and indeed, accelerated, leading to enhanced cell death, by blockade of calpain activity. Inhibition of calpain activity with the tri-peptide zLLY-FMK (Calpain Inhibitor IV, (CiIV) or the non-peptide inhibitor, PD150606, demonstrated a significant increase in the cytotoxic activity of bortezomib. Similarly, elimination of the small catalytic subunit, CAPNS1, using siRNA, enhanced bortezomib-mediated cell death, and prevented autophagosome-lysosomal progression. Furthermore, inhibition of calpain by clinically approved HIV protease inhibitors including Nelfinavir, Ritonavir, Saquinavir, and Indinavir sulfate, significantly increased the cytotoxic activity of bortezomib in vitro. We suggest that disregulation of Ca2+ by bortezomib-mediated ER stress activates the autophagic survival response. Inhibition of mitochondrial Ca2+ uptake by the uniporter inhibitor RuR promotes autophagy, and confers resistance to bortezomib. Conversely, inhibition of the Ca2+-dependent serine protease, calpain, prevents autophagolysosome maturation, and subverts the survival response to cell death. These data are likely to have important clinical implications for the treatment of refractory myeloma and other B cell malignancies.

Multiple myeloma is a hematological cancer of plasma cells. Proteasome inhibitors, such as Bortezomib (BZ, Velcade/PS‐341) are used to treat myeloma because they induce apoptosis via activation of ER stress. We showed that ER stress results in a transient increase in cytosolic calcium ([Ca2+]c), and subsequent initiation of apoptosis. Since inhibition of mitochondrial Ca2+ ([Ca2+]m) uptake with ruthenium red blocked apoptotic initiation and activation of caspase activity, we analyzed [Ca2+]m in the myeloma line 8226 using Rhod‐2. Although BZ elicits a transient increase in [Ca2+]c, [Ca2+]m was found to rapidy (< 1 min) decrease. Following this initial decrease, two distinct responses were identified; either [Ca2+]m returned to then exceeded baseline, or [Ca2+]m remained below baseline levels. Our results indicate that there are two discrete sub‐populations of myeloma cells with respect to response to BZ which is consistent with the observed efficacy of BZ on <70% of the myeloma population. We propose that the population in which [Ca2+]m remains low is refractory to BZ induced apoptosis. Current studies will test this hypothesis, and aim to further characterize these two sub‐populations of myeloma cells. These data will allow us to identify targets for initiating apoptosis in the BZ refractory population with the goal of increasing the efficacy and specificity of drug treatment.

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