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. Author manuscript; available in PMC: 2021 Aug 1.
Published in final edited form as: Leukemia. 2020 May 18;35(2):550–561. doi: 10.1038/s41375-020-0865-2

Proteomic analysis identifies mechanism(s) of overcoming bortezomib resistance via targeting ubiquitin receptor Rpn13

Ting Du 1, Yan Song 1, Arghya Ray 1, Dharminder Chauhan 1,#, Kenneth C Anderson 1,#
PMCID: PMC7988682  NIHMSID: NIHMS1680009  PMID: 32424294

Abstract

Our prior study showed that inhibition of 19S proteasome-associated ubiquitin receptor Rpn13 can overcome bortezomib resistance in MM cells. Here, we performed proteomic analysis of Rpn13 inhibitor (RA190)-treated MM cells and identified an antioxidant enzyme superoxide dismutase (SOD1) as a mediator of Rpn13 signaling. SOD1 levels are higher in MM patient cells versus normal PBMCs; and importantly, SOD1 expression correlates with the progression of disease and shorter survival. Functional validation studies show that RA190-induced cytotoxicity in bortezomib-sensitive and -resistant MM cells is associated with decrease in SOD1 levels; conversely, forced expression of SOD1 inhibits RA190-induced cell death. Genetic knockdown and biochemical blockade of SOD1 with LCS-1 sensitizes bortezomib-resistant MM cells to bortezomib. SOD1 inhibitor LCS-1 decreases viability in MM cell lines and patient cells. LCS-1-induced cell death is associated with: (1) increase in superoxide and ROS levels; (2) activation of caspases, and p53/p21 signaling; (3) decrease in MCL-1, BCLxL, CDC2, cyclin-B1, and c-Myc; (4) ER stress response; and (5) inhibition of proteasome function. In animal model studies, LCS-1 inhibits xenografted bortezomib-resistant human MM cell growth and prolongs host survival. Our studies therefore show that targeting Rpn13 overcomes bortezomib resistance by decreasing cellular SOD1 levels, and provide the rationale for novel therapeutics targeting SOD1 to improve patient outcome in MM.

Introduction

Proteasome inhibitors (PIs) targeting the 20S proteasome holoenzyme [1-4] are a mainstream therapy for patients with multiple myeloma (MM) [5-8]. While PI therapies have significantly prolonged the survival of MM patients, they are associated with the emergence of drug resistance underlying relapse of disease [6-9]. Novel strategies to overcome PI resistance include targeting components of UPS localized upstream the 20S core proteasome. For example, we and others showed that targeting 19S proteasome-associated ubiquitin receptor (UbR) Rpn13 triggers cytotoxicity and overcomes bortezomib resistance in MM and in other cancers [10-14].

Rpn13 recognizes and chaperones ubiquitinated proteins (UbPs) for downstream proteasomal degradation. Specifically, Rpn13 recognizes the UbPs as substrate, followed by removal of ubiquitin moieties from the substrate by deubiquitinating enzymes UCH37 at the 19S proteasome; and then the target protein is unfolded by the AAA-ATPases for 20S proteasome-mediated degradation [15-17]. Our siRNA screening studies identified Rpn13 as a mediator of MM cell growth; conversely, biochemical inhibition of Rpn13 with RA190 triggers cytotoxicity, even in bortezomib-resistant cells, as well as activates anti-MM immune response [10, 18]. To date, however, the Rpn13-associated downstream substrates and signaling mechanism(s) in MM cells have not been delineated.

The UPS maintains normal cellular proteostasis via regulating expression and function of many proteins mediating cell-cycle progression, redox homeostasis, DNA repair, antigen presentation, or apoptosis. In particular, cellular redox balance is maintained through coordinated generation and elimination of reactive oxygen/nitrogen species (ROS/RNS) by antioxidant enzymes. Of note, various studies have reported that PIs including bortezomib increase ROS/RNS ratio and oxidative stress-related cell death in cancers, including MM [4, 19-22]. Nrf2-Keap1 antioxidant response pathway is regulated by UPS [23], and antioxidant enzymes are associated with bortezomib resistance [24]. Together, these findings indicate a role of UPS signaling in modulating cellular redox responses. The fact that Rpn13 is a component of 19S proteasome within the UPS, coupled with our prior findings that targeting Rpn13 blocks proteasome function and triggers apoptosis in MM cells [10, 18], suggests that Rpn13 inhibition, like bortezomib, may affect redox-associated signaling in MM cells.

In the current study, we utilized multiplexed proteomics analysis with tandem mass spectrometry in MM cells treated with the Rpn13 inhibitor RA190 to identify proteomic alterations and specific downstream substrate proteins/signaling mechanisms triggered by Rpn13 blockade. Rpn13 inhibition markedly altered redox signaling network molecules in MM cells. In particular, Rpn13 blockade significantly altered the expression of antioxidant enzyme Copper/Zinc-superoxide dismutase (SOD1) [25], implicated in the regulation of mitochondrial unfolded protein response (mtUPR), in MM cells. To assess the functional significance of these findings, we utilized both in vitro and in vivo preclinical models of MM to show that: (1) RA190 overcome PI resistance in MM cells by downregulating SOD1; and (2) pharmacological inhibition of SOD1 triggers potent anti-MM activity even in bortezomib-resistant MM cells. These studies provide the preclinical rationale to target SOD1 to overcome PI resistance and improve patient outcome in MM.

Materials and Methods

Cell culture and reagents

Human MM cell lines MM.1S, MM.1R, INA6, RPMI-8226, RPMI-8226-LR5, RPMI-8226-DOX40, U266 were obtained from American Type Culture Collection (Manassas, MD), ANBL6-WT, ANBL6-BR were kindly provided by Dr Robert Orlowski (MD Anderson Cancer Center, TX), and normal PBMCs were cultured in RPMI1640 complete medium. All studies using MM patient samples were performed following IRB-approved protocols at Dana-Farber Cancer Institute/Brigham and Women’s Hospital, Boston, USA. Informed consent was obtained from all patients in accordance with the Helsinki protocol, and patient samples were de-identified prior to their use in experiments. MM patient cells were purified (>95% purity) by positive selection using CD138 Microbeads kit, as previously described [26]. Bone marrow stromal cells (BMSCs) and plasmacytoid dendritic cells (pDCs) from MM patients were isolated as described previously [27]. MM-pDCs and BMSCs were cocultured in complete RPMI1640 medium supplemented with IL-3 (Peprotech Inc., USA). Drug source: RA190 was purchased MedChemExpress LLC, NJ; LCS-1 was purchased from Sigma-Aldrich, St. Louis, MO, USA; bortezomib was purchased from Selleck Chemicals LLC, Houston, TX.

Proteomic analysis

MM.1S cells were treated with DMSO or RA190 (0.25 μM) for 24 h, and then subjected to multiplexed proteomics analysis using tandem mass spectrometry. Data were analyzed using uniprot composite database and SEQUEST-based software platform. Expression patterns for MM cells cultured with or without RA190 were compared, and a heat map was generated (>twofold change in protein level was considered significant, CI > 95%).

Cell viability and apoptosis analysis

Cell viability was determined by WST-1 (Takara Bio USA, Mountain View, CA, USA) and CellTiter-Glo Luminescent assays (Promega Corporation, Madison, WI, USA), as described previously [28]. MM.1S cells were treated with DMSO or LCS-1 (2 μM) for 16 h, and apoptosis was measured using Annexin V/PI staining [29].

Transient transfection

MM.1S and bortezomib-resistant ANBL6-BR cells were transiently transfected with scrambled (scr)-siRNA or SOD1-siRNA ON-TARGET plus SMART pool siRNA (Dharmacon, Inc. Lafayette, CO) using the cell line Nucleofector Kit V (Amaxa Biosystems, Cologne, Germany). pcDNA3.1 control vector and SOD1-WT (addgene, #26397) were transfected as above. Cells were harvested 24 h post transfection, followed by analysis using both immunoblotting and cell viability assay.

Measurement of SOD1 enzymatic activity and ROS/GSH levels

MM.1S cells were treated with DMSO or LCS-1 (1.25 μM) for 16 h, followed by analysis of cytosolic extracts for SOD1 enzyme activity using colorimetric assay kit (Cayman Chemical, MI, USA). MM.1S cells were treated with DMSO, LCS-1 (1.25 μM), or RA190 (0.3 μM) for 16 h; cells were then stained with membrane permeable DCFDA or DHE for the last 30 min, followed by measurement of ROS and superoxide anion levels using flow cytometry analysis. MM.1S cells were treated with DMSO, LCS-1 (1.25 μM), or RA190 (0.3 μM) for 16 h, followed by assessment for Glutathione (GSH)/Glutathione disulfide (GSSG) levels using GSH/GSSG-Glo Assay kit (Promega, WI, USA).

Immunoblotting

Cellular protein extracts were prepared using RIPA lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5% sodium deoxycholate, and 0.1% SDS). Protein lysates were subjected to immunoblotting using antibodies against SOD1, poly ADP ribose polymerase (PARP, BD Bioscience Pharmingen, San Diego, CA), caspase-3, caspase-8, p53, p21, MCL-1, BclxL, c-Myc, cytochrome-c (Santa Cruz Biotechnology), caspase-9, p-eIF2α (Abcam, Cambridge, MA), cyclin-B1, CDC25C, CDC2, HSP60, CLPP, COX IV, PERK, BIP, Calnexin, GFP (Cell Signaling, Beverly, MA), polyubiquitin (Enzo Life Sciences, Inc., Farmingdale, NY), GAPDH, or β-actin (Sigma-Aldrich).

Proteasome activity assays

MM.1S cells were treated with LCS-1 (1 μM) or bortezomib (1 μM) for 3 h; cells were then harvested and lysed in lysis buffer, followed by removal of debris by centrifugation. Total protein (25 μg) was analyzed for proteasome activity using 20S proteasome assay kit (Calbiochem, San Diego, CA, USA), as previously described [30].

Proteasome disassembly

MM.1S cells were treated with LCS-1 (1.25 μM), RA190 (0.3 μM), or bortezomib (2.5 nM) for 5 h and then analyzed for proteasome disassembly, as previously described [31]. Briefly, cell lysates were subjected to native PAGE gel, followed by incubation of gel at 37 °C for 30 min in 10 ml of fluorogenic proteasome substrate Suc-LLVY-AMC (0.1 mM) in buffer A (25 mM Tris pH7.4, 10 mM MgCl2, 10% Glycerol, 1 mM ATP, 1 mM DTT). Proteasome bands were visualized under UV light (360 nm) and documented using Gel Doc machine (BioRad, CA, USA).

Human plasmacytoma and SCID-hu xenograft models

Animal model studies were performed, as described previously [27, 30, 32]. Briefly, 5-week-old female CB17 severe combined immunodeficiency (SCID) mice were subcutaneously inoculated with 5.0 × 106 MM.1S cells. When tumors were measurable (100 mm3) at ~3 weeks after MM cell injection, mice were randomized to treatment group (nine mice/group) and treated on an every other day schedule for 14 days with vehicle alone or LCS-1 (20 mg/kg, diluted in saline). Mice were euthanized when tumor volume reached institutional limit (2000 mm3). In the SCID-human (hu) model, human fetal bone grafts were subcutaneously implanted into SCID mice. Two weeks after bone implantation, bortezomib-resistant (ANBL6-BR) MM cells (5.0 × 106) were injected directly into the fetal bone implant in SCID mice (seven mice/group), and MM cell growth was assessed by serial measurements of circulating levels of soluble human IL-6R in mouse serum using ELISA (R&D Systems, Minneapolis, MN, USA). Upon detection of soluble human interleukin 6-receptor (shIL-6R), mice were randomized to treatment group (seven mice/group) and treated with vehicle or LCS-1 (10 mg/kg), and mouse serum was analyzed for alterations in shIL-6R levels. All animal protocols were approved by and conformed to the relevant regulatory standards of the Institutional Animal Care and Use Committee at the Dana-Farber Cancer Institute.

Statistical analysis

Student’s t test was utilized to derive statistical significance. The minimal level of significance was p < 0.05 (Graph Pad PRISM version 6, La Jolla, CA, USA). Survival of mice was analyzed by GraphPad Prism software.

Results and discussion

Blockade of ubiquitin receptor (UbR) Rpn13 triggers alterations in proteins mediating redox homeostasis

MM.1S cells were treated for 24 h with DMSO control or Rpn13 inhibitor RA190 (IC50: 250 nM; Fig. 1a, bar graph, upper panel), and subjected to multiplexed proteomics analysis using tandem mass spectrometry. Data were analyzed using uniprot composite database and SEQUEST-based software platform. Expression patterns for MM cells cultured with or without RA190 were compared, and a heat map was generated (>twofold change in protein level was considered significant, CI > 95%). The results show that RA190 alters several proteins in MM cells including those mediating redox-homeostasis pathways (Fig. 1a heat map). For example, RA190-treated MM cells showed significant downregulation of SOD1 (Fig. 1a): 2.6-fold decreased expression was noted in RA190- versus DMSO-treated MM cells (Fig. 1a bottom bar graph, CI > 95%). SOD1 is an essential enzyme which protects cells from free radical-induced damage by eliminating superoxide radicals (O2−) [33, 34]. Although RA190 altered other redox homeostasis-associated signaling molecules, we focused on examining the role of SOD1 since: (1) a marked downregulation of SOD1 versus other molecules was observed in RA190-treated cells; (2) other studies have linked SOD1 to oxidative stress resistance and tumorigenesis [35]; (3) tumor cells produce high levels of superoxide radicals, which render them more dependent on SOD1 for survival and sensitive to SOD1 inhibition; (4) superoxide radicals can trigger deleterious mutations in DNA, which are linked to cancer and other diseases, such as amyotrophic lateral sclerosis [36]; (5) the assessment of functional significance of SOD1 is feasible due to availability of a specific biochemical inhibitor; and (6) we have previously shown that Rpn13 inhibition overcome bortezomib resistance [10], but the role of SOD1 in this context is undefined.

Fig. 1. Ubiquitin Receptor Rpn13 targets Superoxide dismutase.

Fig. 1

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a (left upper panel and heat map) MM.1S cells were treated for 24 h with DMSO or RA190 (0.25 μM) and subjected to cell viability analysis using WST assays, as well as proteomic analysis by multiplexed proteomics with tandem mass spectrometry (mean ± s.d.; n = 3; p < 0.05). Expression patterns for MM cells cultured with or without RA190 were compared, and a heat map was generated (>twofold change in protein level was considered significant, CI > 95%). a (left lower panel) The fold change in SOD1 expression level in RA190- and DMSO-treated cells was quantified and shown in bar graph. b (upper panel) MM.1S cells were treated for 24 h with DMSO, RA190 (0.25 μM), or bortezomib (BTZ, 2.5 nM); total protein lysates were subjected to immunoblot analysis using anti-SOD1 or anti-GAPDH Abs. Densitometry was utilized to quantify SOD1 levels after normalization with GAPDH control to obtain fold change in SOD1. b (bar graph) MM.1S cells were transiently transfected with control pCDNA3.1 vector or SOD1-WT plasmid. After 24 h transfection, cells were treated with indicated concentrations of RA190 for an additional 48 h, followed by assessment for cell viability using WST assay (mean ± s.d.; n = 3) (bar graph). Inset: protein extracts from RA190-treated transfected cells were analyzed for SOD1 using immunoblotting. c ANBL6-WT and ANBL6-BR cells were treated for 48 h with DMSO or indicated concentration of RA190, followed by assessment for cell viability using WST assay (p < 0.05 for both cell lines; n = 3). Inset: protein extracts from ANBL6-WT and ANBL6-BR cells were analyzed for SOD1 using immunoblotting. d ANBL6-WT and ANBL6-BR cells were treated for 24 h with DMSO or RA190 (1.25 μM); total protein lysates were subjected to immunoblot analysis using SOD1 or β-actin Abs. Densitometry was utilized to quantify SOD1 levels after normalization with β-actin control to obtain fold change in SOD1 (p < 0.05; n = 3). e ANBL6-BR cells were transfected with scr-siRNA or SOD1-siRNA, followed by IC50 measurement 24 h post transfection using WST assay (mean ± s.d.; n = 3; p < 0.05). Inset: immunoblot showing SOD1 expression in cells transfected with scr-siRNA or SOD1-siRNA.

Functional significance of SOD1

MM.1S cells were treated with RA190 and analyzed for SOD1 protein expression using immunoblotting. In concert with our proteomic results, RA190 markedly decreased SOD1 levels (47% decrease versus untreated cells); conversely, forced expression of SOD1-WT attenuated RA190-induced cell death (Fig. 1b, immunoblot and bar graph). Interestingly in contrast to RA190, bortezomib treatment significantly increased SOD1 expression (56% increase versus untreated control) (Fig. 1b). Based on these results, we next examined whether (1) SOD1 levels correlate with bortezomib resistance; and (2) whether RA190 overcomes bortezomib resistance by downregulating SOD1. As in our prior study [10], RA190 overcomes bortezomib resistance, evident by an equipotent anti-MM activity of RA190 against both bortezomib-sensitive and -resistant cells (Fig. 1c). Immunoblot analysis showed higher SOD1 expression in isogenic [37] bortezomib-resistant (ANBL6-BR) versus -sensitive (ANBL6-WT) MM cells (Fig. 1c, inset). These findings are in agreement with a previous study [25] showing upregulated SOD1 levels in bortezomib-resistant MM cells. Importantly, RA190 decreased SOD1 levels in both bortezomib-resistant and -sensitive MM cell lines (Fig. 1d).

To further examine the role of SOD1 in bortezomib resistance, ANBL6-BR cells were transfected with scr-siRNA or SOD1-siRNA, followed by IC50 measurement 24 h post transfection using WST assay. The results showed a significantly lower IC50 of bortezomib for SOD1-siRNA-versus scr-siRNA-transfected ANBL6-BR cells (Fig. 1e, p < 0.05). These data suggest that siRNA-mediated knockdown of SOD1 re-sensitized ANBL6-BR cells to bortezomib. Conversely, a forced overexpression of SOD1 using SOD1-WT plasmid in bortezomib-sensitive ANBL6-WT cells increases IC50 of bortezomib in these cells (data not shown). Taken together, our data show that: (1) blockade of SOD1 through either SOD1-siRNA or Rpn13 inhibitor RA190 overcomes bortezomib resistance in MM cells; and (2) RA190-induced downregulation of SOD1 contributes to the mechanism(s) whereby RA190 overcome bortezomib resistance in MM cells.

Clinical relevance of SOD1 in MM

We next examined SOD1 protein expression in MM cell lines, patient tumor cells and normal PBMCs. A markedly elevated SOD1 expression was noted in both MM cell lines and patient MM cells versus normal PBMCs (Fig. 2a). To assess its clinical relevance, we retrospectively analyzed prognostic significance of SOD1 baseline expression in MM bone marrow (BM) biopsy samples on survival of 170 newly diagnosed, uniformly treated, MM patients. We found a statistically significant inverse correlation between SOD1 levels and overall survival (Fig. 2b; p = 0.0005). Using publicly available GEP database (GSE6477), we next examined SOD1 gene expression in samples from normal healthy individuals (n = 15), individuals with monoclonal gammopathy of undetermined significance (MGUS) (n = 22), patients with smoldering (SMM) (n = 24) or active MM (n = 69), as well as patients with relapsed disease (n = 28). SOD1 levels are significantly higher in patients with active or relapsed MM versus normal healthy donors and individuals with MGUS or SMM (Fig. 2c; p < 0.0001). These data show that SOD1 expression correlates with the progression of MM, with maximal SOD1 levels noted in patients with relapsed disease.

Fig. 2. SOD1 expression and prognostic relevance in MM.

Fig. 2

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a Purified tumor cells from MM patients (n = 5), normal PBMCs (n = 5), and MM cell lines (n = 10) were analyzed for SOD1 expression level by immunoblotting with anti-SOD1 or anti-β-actin Abs. Blots shown are representative of three independent experiments. b Kaplan–Meier plots on the prognostic relevance of SOD1 expression on the overall survival of MM patients (patients: n = 175; p = 0.00054). The red line indicates patients with elevated SOD1 expression and shorter survival, whereas the blue line represents a group of patients with lower SOD1 and longer survival. c SOD1 gene expression data collected using Affymetrix Human Genome U133A [HG-U133A] Array platform based on normal plasma cells (n = 15), MGUS (n = 22), SMM (n = 24), newly diagnosed MM (n = 69), and relapsed MM patients (n = 28) samples (p < 0.0001; Data accession number GSE6477). The analysis of data was based on the information available on the following website: http://www.canevolve.org/AnalysisResults/AnalysisResults.html.

Validation of SOD1 inhibition as an anti-MM strategy using in vitro models of MM

We further determined the functional significance of SOD1 in MM.1S cells using RNA interference strategy. The specificity of the SOD1-siRNA was confirmed in an immunoblot showing reduction in SOD1 expression (Fig. 3a, immunoblot). Transfection of MM cells with SOD1-siRNA, but not scr-siRNA, induced a significant decrease in their viability (Fig. 3a; p < 0.001). These data indicate a role of SOD1 in MM cell survival. We next utilized a panel of MM cell lines and patient tumor cells to examine whether a biochemical inhibition of SOD1 similarly triggers anti-MM activity. An earlier study developed and characterized novel small molecule LCS-1 that targets SOD1 [38]. Here, we found that LCS-1 in a concentration-dependent manner triggers significant inhibition of SOD1 enzymatic activity in MM cells (Fig. 3b). Importantly, LCS-1 in a dosedependent manner reduces the viability of various MM cell lines, including those resistant to conventional therapies dexamethasone (MM.1R), doxorubicin (Dox40), or melphalan (LR5) (Fig. 3c). The variation in the IC50 of LCS-1 against the cell lines may be due to their distinct genetic backgrounds and/or drug-resistance characteristics. A prior report showing that antioxidants confer resistance to high dose melphalan [39], coupled with our data that blockade of antioxidant SOD1 overcomes melphalan resistance (LR5), further validates SOD1 as a potential therapeutic target in MM. In concert with our findings, SOD1 inhibition showed antitumor activity in other cancer models [38, 40-42]. Moreover, earlier studies have shown elevated SOD1 in PI resistant cells [24, 43].

Fig. 3. Anti-MM activity of SOD1 inhibitor LCS-1.

Fig. 3

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a MM.1S cells were transfected with scr-siRNA or SOD1-siRNA, followed by cell viability analysis 24 h post transfection using WST assay (mean ± s.d.; n = 3; p < 0.0001). Inset: immunoblot showing SOD1 expression in cells transfected with scr-siRNA or SOD1-siRNA. b MM.1S cells were treated for 4 h with DMSO control or LCS-1 at the indicated concentrations, followed by analysis of cytosolic extracts for SOD1 enzyme activity using colorimetric assay kit (mean ± s.d.; n = 3; p < 0.05). c MM cell lines were treated for 48 h with DMSO or LCS-1 at the indicated concentrations, followed by assessment for cell viability using WST assay (n = 3; p < 0.05). Cell viability data are presented in a heat map. d Purified CD138+ patient MM cells were treated for 48 h with LCS-1 at indicated concentrations, followed by assessment for cell viability using CellTiter-Glo assay (mean ± s.d. of triplicate cultures; p < 0.005 for all patient samples). e Bortezomib-sensitive (ANBL6-WT) and Bortezomib-resistant (ANBL6-BR) cells were treated with either bortezomib or LCS-1 for 48 h, followed by assessment for cell viability. The bar graph shows the IC50 ratio (ANBL6-BR/ANBL6-WT) of LCS-1 and bortezomib (mean ± s.d.; n = 3). f Normal PBMCs from healthy donors (PBMCs#1-PBMCs#5) were treated for 48 h with DMSO control or LCS-1 at indicated concentrations, followed by assessment for cell viability using CellTiter-Glo assay (mean ± s.d. of triplicate cultures). g MM.1S cells were cultured for 48 h with or without BMSCs in the presence or absence of indicated concentrations of LCS-1, followed by assessment for cell proliferation using CellTiter-Glo assay (mean ± s.d.; n = 3; p < 0.005). h MM.1S cells were cultured for 48 h with or without pDCs in the presence or absence of indicated concentrations of LCS-1, followed by assessment for cell proliferation using CellTiter-Glo assay (mean ± s.d.; n = 3; p < 0.0001).

To examine whether LCS-1 similarly alters viability of patient MM cells, we next examined CD138+ MM cells from two newly diagnosed (patients #1 and #2) and three patients with MM refractory to multiple therapies including dexamethasone, bortezomib, and lenalidomide (patient #2-patients #5) (Fig. 3d). MM was considered refractory when disease progressed on therapy or within 60 days of discontinuing therapy. A concentration-dependent reduction in the viability of all patient MM cells was observed after LCS-1 treatment (Fig. 3d), indicating that LCS-1 triggers cytotoxicity even in bortezomib-resistant patient MM cells. We similarly examined the activity of LCS-1 in bortezomib-sensitive (ANBL6-WT) and bortezomib-sensitive (ANL6-BR) isogenic MM cell lines [37]. The IC50 of these agents for ANBL6-WT and ANBL6-BR were 2.5 and 12 nM for bortezomib and 2.5 and 4.6 μM for LCS-1; the IC50 ratio (ANBL6-BR/ANBL6-WT) of LCS-1 is therefore significantly less than bortezomib, further demonstrating the ability of LCS-1 to overcome bortezomib resistance (Fig. 3e; p < 0.001). Finally, LCS-1 at the half-maximal inhibitory concentration for patient MM cells (100–400 nM) showed no significant toxicity against normal peripheral blood mononuclear cells (Fig. 3f), suggesting a favorable therapeutic index for SOD1 inhibition in MM.

SOD1 inhibition decreases tumor-promoting activity of MM bone marrow accessory cells

The MM-host BM milieu confers growth, survival, and drug resistance in MM cells [30, 44]. Specifically, the interaction of BMSCs with tumor cells triggers MM cell growth, as well as protects against drug-induced cytotoxicity. Moreover, BM accessory cells such as pDCs can also promote MM cell growth, survival, drug resistance, and immune suppression in the MM BM milieu [18, 27]. Therefore, we next assessed the effect of LCS-1 using our patient MM-BMSCs or MM-pDCs in vitro coculture assays [10, 26, 27]. Even in these cocultures of MM cells with BMSCs or pDCs, LCS-1 triggered a concentration-dependent decrease in the viability of MM cells (Fig. 3g, h, respectively). These data suggest that LCS-1 retains its ability to trigger MM cell death in the presence of the tumor-protective BM microenvironment.

Blockade of SOD1 triggers oxidative stress and proteasome disassembly

Our data show that Rpn13 inhibitor (RA190)-induced MM cell death is associated with a decrease in antioxidant enzyme SOD1; and that targeting of SOD1 using LCS-1 triggers anti-MM activity. We next examined whether blockade of SOD1 through either Rpn13 inhibition via RA190 or SOD1 inhibition by LCS-1 correlates with an increase in intracellular ROS levels, including superoxide anions (O2−). Treatment of MM.1S cells with LCS-1 or RA190 induced a significant increase in ROS levels and O2− levels (Fig. 4a, p < 0.001 and p < 0.006, respectively). Conversely, co-treatment of MM.1S cells with superoxide (O2−) scavenger N-acetyl-1-cysteine significantly reduced LCS-1- or RA190-induced cytotoxicity (Supplementary Fig. 1A). The similar results were observed using bortezomib-resistant ANBL6-BR cells (Supplementary Fig. 1B). These findings show the functional significance of SOD1/ROS/O2− signaling axis during LCS-1-induced apoptotic signaling in MM cells, including those that are bortezomib resistant.

Fig. 4. Blockade of SOD1 triggers oxidative stress, mitochondrial UPR signaling, and proteasome inhibition.

Fig. 4

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a MM.1S cells were treated for 16 h with DMSO control, LCS-1 (1.25 μM), or RA190 (0.3 μM); cells were then stained with membrane permeable DCFDA or DHE for the last 30 min, followed by measurement of ROS and superoxide anion levels using flow cytometry analysis. Superoxide anions oxidize HE to fluorescent ethidium, permitting analysis by flow cytometry (mean ± s.d.; n = 3). b MM.1S cells were treated for 16 h with DMSO control, LCS-1 (1.25 μM), or RA190 (0.3 μM), followed by assessment for Glutathione (GSH)/Glutathione disulfide (GSSG) levels using GSH/GSSG-Glo Assay kit (mean ± s.d.; n = 3; p < 0.005). c (upper panel) MM.1S cells were treated with DMSO control or LCS-1 (1.25 μM) for 16 h; cytosolic extracts were subjected to immunoblot analysis using anticytochrome-c or anti-β-actin Abs. c (lower panel) MM.1S cells were treated with DMSO control, LCS-1 (1.25 μM), or RA190 (0.3 μM) for 24 h; mitochondrial extracts were subjected to immunoblot analysis with anti-HSP60, anti-CLPP or anti-Cox IV Abs. d MM.1S cells were treated for 5 h with LCS-1 (1.25 μM), RA190 (0.3 μM) or bortezomib (2.5 nM); cell lysates were subjected to a native gel analysis, followed by incubation of gel at 37 °C for 30 min with fluorogenic proteasome substrate Suc-LLVY-AMC. 26S proteasomes (RP2CP and RP1CP) were visualized in-gel under UV light (360 nM). e MM.1S cells were treated with LCS-1 (1.25 μM) for indicated time periods; protein lysates were subjected to immunoblot analysis using antipolyubiquitin or anti-β-actin Abs. f Immunoblot showing the levels of Ub-GFP accumulation in a GFPu-1 reporter cell line treated for 16 h with indicated concentrations of LCS-1. g MM.1S cells were treated for 3 h with DMSO control, LCS-1 or bortezomib at indicated concentration; protein lysates were analyzed for proteasome activities (CT-L chymotrypsin-like; T-L trypsin-like; C-L caspase-like). The percentage of proteasome activity was normalized to DMSO control (mean ± s.d.; n = 3). Blots shown are representative of three independent experiments.

In addition, reduced glutathione (GSH) is a scavenger of ROS, and its ratio with oxidized glutathione (GSSG) serves as a marker of oxidative stress [45]. Examination of LCS-1 or RA190-treated MM cells showed a significant decrease in GSH/GSSG ratio, further confirming the induction of oxidative stress (Fig. 4b, p < 0.005). We and others have shown that oxidative stress causes defects in mitochondrial signaling and MM cell death [46-50]. In accord with these reports, LCS-1-induced O2− in MM cells is associated with the release of mitochondrial cytochrome-c into the cytosol (Fig. 4c), as well as induction of proteins mediating mtUPR signaling [49, 50], such as HSP60 and CLPP (Fig. 4c). Moreover, RA190, like LCS-1, also triggered mtUPR in MM cells (Fig. 4c).

Recent studies have linked mitochondrial stress to proteasome functioning, and proteasome disassembly [51]. The 26S proteasome holoenzyme consists of 20S proteolytic core particle (CP) and a 19S regulatory particle (RP) that recognizes and translocate ubiquitinated substrates in 20S CP for degradation via proteasomal catalytic activities [2]. 26S proteasome is classified into two forms RP1CP and RP2CP, depending upon whether one or two 19S RP attach to a single 20S CP [52]. Increased oxidant levels, such as O2−, trigger oxidation of 26S proteasome and decrease their levels [51, 53]. Consistent with these observations, we found that LCS-1-induced O2− triggered a marked decrease in both RP1CP and RP2CP forms of 26S proteasome (Fig. 4d). The similar results were noted in RA190- and bortezomib-treated cells (Fig. 4d). We confirmed inhibition of proteasome-mediated protein degradation in two ways: first, LCS-1 induced a marked increase in accumulation of polyubiquitinated proteins, reflecting inhibition of cellular protein degradation via the proteasome; and second, LCS-1 triggered accumulation of Ub-GFP, indicating impaired proteasome degradation (Fig. 4e, f, respectively). As previously reported for RA190 [10], LCS-1 blocked cellular proteasome function (Fig. 4e, f), without inhibiting 20S proteasome proteolytic activities (Fig. 4g).

SOD1-blockade activates caspases, p53/p21 signaling, and endoplasmic reticulum stress response

Treatment of MM.1S cells with LCS-1 triggers an increase in early- (Annexin V+/PI−) and late-stage (Annexin V+/PI+) apoptosis, accompanied by proteolytic cleavage of Poly (ADP) ribose polymerase (PARP) as well as activation of caspase-3, caspase-8, and caspase-9 (Fig. 5a, b). The similar results were obtained using bortezomib-resistant ANBL6-BR cells (data not shown). Treatment of MM.1S and ANBL6-BR with LCS-1 cells induces growth arrest, evidenced by a marked decrease in cell-cycle regulatory proteins (cyclin-B1, CDC25C, and CDC2) (Fig. 5c). Importantly, LCS-1 treatment in a concentration-dependent manner also upregulates p53/p21 signaling, as well as downregulates survival pathway proteins MCL-1, BclxL, or c-Myc in MM cells (Fig. 5d).

Fig. 5. Mechanisms of LCS-1-induced MM cell death.

Fig. 5

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a MM.1S cells were treated for 16 h with DMSO control or LCS (2 μM), and then analyzed for apoptosis using Annexin V/PI double staining assay (mean ± s.d.; n = 3; p < 0.001). b MM.1S and ANBL6-BR cells were treated for 16 h with DMSO control or LCS-1 (2 μM for MM.1S; 5 μM for ANBL6-BR); protein lysates were then subjected to immunoblotting using antibodies against PARP, caspase-3, caspase-8, caspase-9, and β-actin. FL full length, CF cleaved fragment. c MM.1S cells were treated for 16 h with DMSO control or LCS-1 (2 μM); protein lysates were then subjected to immunoblotting using specific antibodies against CDC2, CDC25C, Cyclin-B1, and β-actin. d MM.1S cells were treated with DMSO control or LCS-1 (2 μM); protein lysates were subjected to immunoblotting using specific antibodies against p53, p21, MCL-1, BclxL, c-Myc, and β-actin. e MM.1S cells were treated for indicated time periods with DMSO control or LCS-1 (2 μM); protein lysates were subjected to immunoblotting using specific antibodies against BIP, PERK, p-eIF2α, calnexin, and β-actin. f ANBL6-BR cells were treated for indicated time periods with DMSO control or LCS-1 (5 μM); protein lysates were subjected to immunoblotting using specific antibodies against p53, BIP, PERK, p-eIF2α, and β-actin. Blots shown are representative of three independent experiments.

We next examined whether LCS-1-induced accumulation of polyubiquitinated proteins in MM.1S cells increases endoplasmic reticulum (ER) stress and triggers associated unfolded protein response (UPR) signaling. We found a rapid and robust induction of UPR proteins (BIP, PERK, phosphorylated eIF2α, or a lectin protein calnexin) in LCS-1-treated MM.1S cells (Fig. 5e). The similar results were observed in LCS-1-treated bortezomib-resistant ANBL6-BR cells (Fig. 5f). Overall, these findings show that LCS-1-induced apoptosis in MM cells is associated with activation of the caspase-cascade, p53/p21 signaling, and ER stress response signaling, as well as downregulation of antiapoptotic pathways. Importantly, we show that LCS-1, like RA190 [10], triggers apoptosis even in bortezomib-resistant ANBL6-BR cells.

In vivo anti-MM activity of LCS-1 in distinct therapeutically relevant animal models

Having defined the anti-MM activity of LCS-1 in vitro, we next examined whether LCS-1 similarly affects MM cell growth in vivo using our human plasmacytoma xenograft model [7, 28]. This model has been useful in validating novel anti-MM therapies bortezomib, carfilzomib, ixazomib, lenalidomide, and pomalidomide, which have translated to clinical trials and FDA approval. Treatment of MM.1S-bearing mice with intraperitoneal injections of LCS-1 (20 mg/kg) inhibits MM growth and prolongs host survival (Fig. 6a, b, respectively). Importantly, LCS-1 was well tolerated, with no significant weight loss in LCS-1-treated mice (data not shown). We next examined whether LCS-1 retains its anti-MM activity against bortezomib-resistant MM cells in vivo. For these studies, we utilized the SCID-hu model [27, 28], which recapitulates the human BM milieu in vivo. Bortezomib-resistant (ANBL6-BR) MM cells were injected directly into human bone chips implanted subcutaneously in SCID mice, and MM cell growth was assessed by serial measurements of circulating levels of shIL-6R in mouse serum. LCS-1 treatment markedly inhibited MM cell growth in this SCID-hu model (Fig. 6c). These findings suggest that: (1) the anti-MM activity of LCS-1 is retained in the presence of the MM-promoting BM microenvironment; and (2) LCS-1 overcomes bortezomib resistance in vivo. Together, our data show potent in vivo anti-MM activity of SOD1 inhibitor LCS-1 and suggest a favorable therapeutic index.

Fig. 6. LCS-1 inhibits xenografted human MM cell growth and prolongs host survival.

Fig. 6

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a Subcutaneous model: mice bearing human MM.1S MM tumors were treated with vehicle control or LCS-1 (20 mg/kg, intraperitoneally) every other day for 14 days. Tumor volume (mean tumor volume ± s.d. in mm3, nine mice/group) versus time is shown. b Kaplan–Meier plots shows survival of mice utilized in (a). Mice were euthanized when tumor volume reached 2000 mm3 (p < 0.05). c SCID-hu model: SCID-hu mice bearing human bortezomib-resistant ANBL6-BR MM tumors (seven mice/ group) were treated with either vehicle control or LCS-1 (10 mg/kg), and mouse serum samples were analyzed for shIL-6R using ELISA.

In summary, while our preclinical studies [10, 18] suggest translational utility of targeting Rpn13 in MM, there are presently no clinical agents available. We here extend our prior study to identify downstream signaling pathways triggered by Rpn13 inhibition in MM cells. Our proteomic analysis of Rpn13 inhibitor (RA190)-treated MM cells identified redox homeostasis-associated SOD1 as a mediator of Rpn13 signaling. Using both in vitro and in vivo models of MM including MM patient tumor cells and MM xenograft models, we validate SOD1 inhibition, either genetically or biochemically, as a potential therapeutic strategy to overcome bortezomib resistance in MM. Moreover, we delineated mechanisms whereby SOD1 inhibition can overcome PI resistance. Importantly, recent clinical trials in patients with amyotrophic lateral sclerosis already showed safety and tolerability of clinical drugs targeting SOD1 (ClinicalTrials.gov Identifier: NCT02623699; NCT00706147), which suggests the possibility for clinical trials evaluating these agents in MM. Collectively, our studies provide the rationale for the development of SOD1 inhibition-based therapies to overcome drug resistance, enhance MM cytotoxicity, and improve patient outcome in MM.

Supplementary Material

Figure 1
Supplementary File

Acknowledgements

The grant support for this investigation was provided by Dr Miriam and Sheldon Adelson Medical Research Foundation, as well as by the National Institutes of Health Specialized Programs of Research Excellence (SPORE) grant P50100707, R01CA207237, and RO1 CA050947. KCA is an American Cancer Society Clinical Research Professor. We are thankful to Krishan Chauhan (Undergraduate Summer Intern, WIT/DFCI) for the literature research and helpful discussion on free radical-mediated signaling cascades.

Footnotes

Supplementary information The online version of this article (https://doi.org/10.1038/s41375-020-0865-2) contains supplementary material, which is available to authorized users.

Conflict of interest KCA is on Advisory Board of Millenium-Takeda, Gilead, Janssen, Bristol Myers Squibb, and Sanofi Aventis, and is a Scientific Founder of Oncopep and C4 Therapeutics. DC is consultant to Stemline Therapeutic, Inc., and Equity owner in C4 Therapeutics. The remaining authors declare no conflict of interest.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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