title: Targeting Thioredoxin Reductase in Multiple Myeloma
Creators: name: Sze, Jun Hui
Publisher: name: Griffith University
Year: 2020
Subjects: subject: Multiple myeloma
subject: therapeutic strategies
subject: Gold compounds
subject: gold chloride
subject: diphosphine ligands

description: Multiple myeloma (MM) is a clonal plasma B-cell neoplasm characterised by the presence of uncontrolled proliferation of antibody-secreting plasma cells in the bone marrow. Despite therapeutic advancements, MM remains incurable due to its low median survival rate and nearly all patients will eventually relapse regardless of the frontline regimen they receive. Thus, alternative therapeutic strategies that target the pathogenic and resistance mechanisms of MM need to be developed to improve overall survival in MM patients. Cancer cells generally have higher metabolic demands due to their high proliferative nature and as such, production of intracellular by-products including free reactive oxygen species (ROS) are also increased, which can cause oxidative cell death. Hence, cancer cells exploit antioxidant molecules to maintain intracellular redox homeostasis and to prevent further cell damage from the anticancer drugs that induce oxidative stress. One of the major antioxidant systems is the thioredoxin (Trx) system, which is comprised of thioredoxin (Trx), thioredoxin reductase (TrxR), and nicotinamide adenine dinucleotide phosphate (NADPH), all of which as one can scavenge ROS, reduce oxidised proteins and redox-sensitive molecules, and regulate transcription factors. Myeloma cells have been shown to have high Trx and TrxR expression, which correlates with increased cell proliferation, evasion of programmed cell death and chemoresistance. Gold compounds have a high affinity for thiol and selenol groups, which makes the Trx system vulnerable to these compounds. In this study, TrxR was targeted in myeloma cells with gold(I) compounds, including auranofin and [Au(d2pype)2]Cl. [Au(d2pype)2]Cl exhibited significant inhibition on TrxR activity in both in vitro and in vivo models, which led to induction of ROSmediated cell death in myeloma cells. [Au(d2pype)2]Cl also completely abrogated the tumourigenic capacity of myeloma cells in clonogenic assays, whereas auranofin was less effective. The expression of the MYC oncogene, which drives myeloma progression, was also downregulated in both in vitro and in vivo models when treated with [Au(d2pype)2]Cl. Auranofin and [Au(d2pype)2]Cl were further evaluated in 2D co-culture assays to determine their efficacy in overcoming environment mediated drug resistance (EMDR) in myeloma cells that are co-cultured with bone marrow stromal cells (BMSCs) in normoxic (20% O2) and hypoxic (1% O2) conditions. [Au(d2pype)2]Cl generally displayed much better efficacy by exhibiting lower IC50 values as compared to auranofin when used on either the attached or non-attached myeloma cells in both normoxia and hypoxia. Based on the potency that [Au(d2pype)2]Cl displayed over auranofin in these co-culture assays, [Au(d2pype)2]Cl was selected as a tool for subsequent studies on the role of the Trx system in the molecular mechanisms that contribute to drug resistance in MM. Cell adhesion mediated drug resistance (CAM-DR) occurs from the intercellular adhesion between myeloma cells and the bone marrow microenvironment (BMME). Several studies have indicated CAM-DR is associated with the activation of NF-ĸB and causes a subset of myeloma cells attached to BMSCs and/or extracellular matrix (ECM) to be chemo-resistant prior to MM therapy. In this study, inhibition of TrxR with [Au(d2pype)2]Cl decreased the adhesive capabilities of myeloma cells to BMSCs and was accompanied by downregulation of integrin Very Late Activation Antigen-4 (VLA-4), which is an important cell adhesion molecule responsible for CAM-DR in MM. [Au(d2pype)2]Cl treatment also significantly downregulated protein levels of the p65 subunit of NF-ĸB as well as expression of NF-ĸB-regulated genes. Moreover, the expression of VLA-4 was markedly decreased when NF-ĸB was inhibited by BAY 11-7082, inhibitor of IĸB kinase (IKK), similar to results obtained when [Au(d2pype)2]Cl was used. These findings suggest that the Trx system may affect the expression of VLA-4 in myeloma cells by regulating the NF-ĸB signalling pathway. Based on the results obtained with VLA-4, the role of another cell adhesion molecule, mucin 1 (MUC1) was evaluated in this study. MUC1 is aberrantly expressed in MM and is involved in oncogenesis as well as regulation of ROS. Since there is a possible link between the redox cellular balance and MUC1, this study investigated if inhibition of TrxR could affect MUC1 expression in myeloma cells. Results showed that inhibition of TrxR decreased the expression of MUC1 in myeloma cells that were co-cultured with BMSCs. Moreover, the adhesion of myeloma cells to BMSCs was significantly decreased following the inhibition of MUC1 with either siRNA or MUC1 peptide inhibitor, GO201. Treatment of myeloma cells with [Au(d2pype)2]Cl resulted in decreased expression of NF-ĸB and Sp1, which coincided with a decreased expression of MUC1. In conclusion, targeting TrxR with improved gold(I)-based compounds may provide an effective anti-myeloma approach for the future development of MM therapies. Moreover, [Au(d2pype)2]Cl has been shown to be useful for future functional studies that will focus on investigating the mechanisms that are involved in the cellular changes related to the effects from TrxR inhibition.