Proteasome Inhibitor Resistance In Multiple Myeloma Research Articles

Ubiquitin receptor PSMD4/Rpn10 is a novel therapeutic target in multiple myeloma

AbstractPSMD4/Rpn10 is a subunit of the 19S proteasome unit that is involved with feeding target proteins into the catalytic machinery of the 26S proteasome. Because proteasome inhibition is a common therapeutic strategy in multiple myeloma (MM), we investigated Rpn10 and found that it is highly expressed in MM cells compared with normal plasma cells. Rpn10 levels inversely correlated with overall survival in patients with MM. Inducible knockout or knockdown of Rpn10 decreased MM cell viability both in vitro and in vivo by triggering the accumulation of polyubiquitinated proteins, cell cycle arrest, and apoptosis associated with the activation of caspases and unfolded protein response-related pathways. Proteomic analysis revealed that inhibiting Rpn10 increased autophagy, antigen presentation, and the activation of CD4+ T and natural killer cells. We developed an in vitro AlphaScreen binding assay for high-throughput screening and identified a novel Rpn10 inhibitor, SB699551 (SB). Treating MM cell lines, leukemic cell lines, and primary cells from patients with MM with SB decreased cell viability without affecting the viability of normal peripheral blood mononuclear cells. SB inhibited the proliferation of MM cells even in the presence of the tumor-promoting bone marrow milieu and overcame proteasome inhibitor (PI) resistance without blocking the 20S proteasome catalytic function or the 19S deubiquitinating activity. Rpn10 blockade by SB triggered MM cell death via similar pathways as the genetic strategy. In MM xenograft models, SB was well tolerated, inhibited tumor growth, and prolonged survival. Our data suggest that inhibiting Rpn10 will enhance cytotoxicity and overcome PI resistance in MM, providing the basis for further optimization studies of Rpn10 inhibitors for clinical application.

High Glutathione and NAD(P)H Levels, and Increased Protein Folding Capacity Are Involved in Mediating Proteasome Inhibitor-Resistance in Multiple Myeloma

Background. Proteasome inhibitors (PI) are a backbone for the treatment of multiple myeloma (MM). However, most MM patients ultimately acquire PI-resistance and die from MM. The biology of PI resistance is poorly understood and established treatment options for PI-refractory MM are lacking. We have recently generated a next-generation in vitro model for PI-refractory MM, based on single MM cell-derived subclones with specific (50-100x) resistance to either bortezomib (AMO-BTZ) or carfilzomib (AMO-CFZ). These cells consistently show downregulation of IRE1/XBP1, a hallmark of BTZ-refractory primary MM cells, and do or do not carry the PSMB5 A310G mutation. Our recent quantitative proteomic analysis suggested that the PI-resistant phenotype of AMO-BTZ and AMO-CFZ is in particular characterized by massive metabolic changes that render MM cells largely independent from proteasome activity. To verify such metabolic adaptation as a hallmark of proteasome inhibitor resistance of MM, and to identify therapeutic targets, we performed whole metabolome profiling of proteasome inhibitor-sensitive AMO-1 cells in comparison to BTZ or CFZ-resistant clones from the same cell line.Methods. Metabolomic profiling was performed using Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectroscopy (UPLC-MS/MS); data were quantified and normalized by Metabolon, Inc. Metaboanalyst v3.0 software was used for Metabolite Set Enrichment Analysis (MSEA). Reactive oxygen species (ROS) formation was determined by flow cytometry after staining the cells with H2DCFDA and exposure to 0.08% H2O2. Protein folding was determined by MERO-GFP construct, viability was determined by MTS assay.Results. Based on MSEA, PI-adapted cells showed substantial metabolic changes in glutathione metabolism, protein biosynthesis, malate/aspartate shuttle, metabolism of purines, pyrimidines, amino acids and tricarboxylic acid (TCA). Relative to AMO-1, in both AMO-BTZ and AMO-CFZ, glutathione (l-γ-Glutamyl-l-cysteinyl-glycine; GSH and GSSG) was consistently identified as the metabolite with the most significant quantitative increase (fold change (FC) relative to AMO-1 for GSH in AMO-BTZ=52.92; in AMO-CFZ=56.79; FC for GSSG in AMO-BTZ=36.92; in AMO-CFZ=54.17), while many other metabolites in glycine/serine and cysteine/methionine pathways that are associated with glutathione synthesis were decreased. Consistently, PI-adapted cells showed accumulation of nicotinamide adenine dinucleotide (phosphate) (NAD(P)+) and reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) (FC for NAD+ in AMO-BTZ=55.81; in AMO-CFZ=2.16; FC for NADH in AMO-BTZ=19.10; in AMO-CFZ=1.65), presumably to maintain balance between reduced and oxidized glutathione, together with changes in TCA and malate/aspartate shuttle fueling the NAD(P)H production and transport, respectively. PI-resistant cells in addition showed altered carbohydrate metabolism and a shift in availability of membrane lipids, from lysolipids to sphingomyelins, in comparison to non-adapted cells.The adaptive changes in these cells correspond well to the proteomic changes previously described, confirming that the metabolic adaptive changes of PI-refractory MM cells are in particular in the regulation of redox homeostasis and protein folding. Functional experiments confirmed that the PI-adapted cells show larger capacity to buffer ROS production and that protein re-folding after DTT challenge is more effective in PI-refractory MM, compared to PI-sensitive cells. Consequently, co-treatment of PI-adapted cells with respective proteasome inhibitors (bortezomib and carfilzomib) in the presence of the inhibitor of protein-disulfide isomerase (PDI), 16F16, re-sensitized cells to proteasome inhibition (FC of IC50 for bortezomib or carfilzomib alone vs bortezomib or carfilzomib + 16F16 in AMO-BTZ=1.26 or 1.88, respectively; in AMO-CFZ=1.25 or 2.59 respectively; in AMO-1=1.1 or 0.95, respectively.Conclusion. PI refractory MM cells show massive metabolic changes in particular in glutathione and NAD(P)H production. This is associated with increased protein folding capacity and likely results in decreased proteasome load. PI-refractory MM cells can be re-sensitized to PI treatment by reducing glutathione levels or by affecting protein folding. DisclosuresNo relevant conflicts of interest to declare.

ZHX2 mediates proteasome inhibitor resistance via regulating nuclear translocation of NF-κB in multiple myeloma.

BackgroundMultiple myeloma (MM) is an incurable hematological malignancy. Although proteasome inhibitors and immunomodulators have significantly improved patient outcomes, some patients respond poorly to treatment and almost all patients will relapse. Mechanisms of proteasome inhibitor resistance in multiple myeloma have not been fully elucidated. ZHX2 is a transcription regulator degraded via proteasome and presents both oncogenic or tumor suppressive effect in different cancers, however, it is still unknown that the role of ZHX2 in myeloma. In this study, we aim to demonstrate the effect and mechanism of ZHX2 on proteasome inhibitor resistance in MM.Methods GSE24080 gene expression profile datasets from Gene Expression Omnibus (GEO) were analyzed to evaluate the relationship between ZHX2 expression level and survival in MM. Expression of ZHX2 in human MM cell lines at baseline and after bortezomib (BTZ) treatment was determined by Western blotting (WB). The proliferation and apoptosis rate of MM cells treated with BTZ after the knockdown of ZHX2 were analyzed by flow cytometry. Nuclear translocation of NF‐κB after the knockdown of ZHX2 was evaluated by WB and immunofluorescence, and the expression of NF‐κB target genes was measured by real‐time quantitative PCR. Co‐immunoprecipitation (Co‐IP) and WB were used to detect the interaction of ZHX2 with NF‐κB.ResultsWe found that higher ZHX2 expression was correlated with poorer clinical outcomes of patients. In addition, ZHX2 expression was relatively higher in RPMI‐8226 and MM.1S cell lines and the level of ZHX2 protein was upregulated after BTZ treatment. Knockdown of ZHX2 significantly enhanced the sensitivity of MM cells to BTZ, inhibited nuclear translocation of NF‐κB, and reduced mRNA expression of NF‐κB target genes. It was also revealed that ZHX2 directly binds to NF‐κB.ConclusionOur study showed that ZHX2 can promote proteasome inhibitor resistance in MM cells by regulating the nuclear translocation of NF‐κB.

In vitro and ex vivo gene expression profiling reveals differential kinetic response of HSPs and UPR genes is associated with PI resistance in multiple myeloma

Extensive inter-individual variation in response to chemotherapy (sensitive vs resistant tumors) is a serious cause of concern in the treatment of multiple myeloma (MM). In this study, we used human myeloma cell lines (HMCLs), and patient-derived CD138+ cells to compare kinetic changes in gene expression patterns between innate proteasome inhibitor (PI)-sensitive and PI-resistant HMCLs following test dosing with the second-generation PI Ixazomib. We found 1553 genes that changed significantly post treatment in PI-sensitive HMCLs compared with only seven in PI-resistant HMCLs (p < 0.05). Genes that were uniquely regulated in PI-resistant lines were RICTOR (activated), HNF4A, miR-16-5p (activated), MYCN (inhibited), and MYC (inhibited). Ingenuity pathway analysis (IPA) using top kinetic response genes identified the proteasome ubiquitination pathway (PUP), and nuclear factor erythroid 2-related factor 2 (NRF2)-mediated oxidative stress response as top canonical pathways in Ix-sensitive cell lines and patient-derived cells, whereas EIF2 signaling and mTOR signaling pathways were unique to PI resistance. Further, 10 genes were common between our in vitro and ex vivo post-treatment kinetic PI response profiles and Shaughnessy’s GEP80-postBz gene expression signature, including the high-risk PUP gene PSMD4. Notably, we found that heat shock proteins and PUP pathway genes showed significant higher upregulation in Ix-sensitive lines compared with the fold-change in Ix-resistant myelomas.

Blockade of deubiquitylating enzyme Rpn11 triggers apoptosis in multiple myeloma cells and overcomes bortezomib resistance.

Proteasome inhibition is an effective therapy for multiple myeloma (MM) patients; however, the emergence of drug resistance is common. Novel therapeutic strategies to overcome proteasome inhibitor resistance are needed. In this study, we examined whether targeting deubiquitylating (DUB) enzymes upstream of 20S proteasome overcomes proteasome inhibitor resistance. Gene expression analysis, immunohistochemical studies of MM patient bone marrow, reverse transcription-PCR and protein analysis show that Rpn11/POH1, a DUB enzyme upstream of 20S proteasome, is more highly expressed in patient MM cells than in normal plasma cells. Importantly, Rpn11 expression directly correlates with poor patient survival. Loss-of-function studies show that Rpn11-siRNA knockdown decreases MM cell viability. Pharmacological inhibition of Rpn11 with O-phenanthroline (OPA) blocks cellular proteasome function, induces apoptosis in MM cells and overcomes resistance to proteasome inhibitor bortezomib. Mechanistically, Rpn11 inhibition in MM cells activates caspase cascade and endoplasmic stress response signaling. Human MM xenograft model studies demonstrate that OPA treatment reduces progression of tumor growth and prolongs survival in mice. Finally, blockade of Rpn11 increases the cytotoxic activity of anti-MM agents lenalidomide, pomalidomide or dexamethasone. Overall, our preclinical data provide the rationale for targeting DUB enzyme Rpn11 upstream of 20S proteasome to enhance cytotoxicity and overcome proteasome inhibitor resistance in MM.

The Rationale for Combined Proteasome and Autophagy Inhibition in Multiple Myeloma Established Using Novel Translational Platforms

Multiple myeloma (MM) is selectively sensitive to perturbations of protein catabolism. Genetic evidence has identified a link between plasma cell differentiation and upregulation of the unfolded protein response. The effective, selective, cytotoxic effect of proteasome inhibition observed in MM therapy further validates pathways of protein degradation as therapeutic targets in this incurable disease. Upon proteasome inhibition, cancer cells utilize compensatory pathways such as aggresome formation and macroautophagy. Despite a remarkable increase in the granularity of knowledge surrounding autophagy in mammalian cell biology, the role of autophagy in MM cell survival has not been studied. Lysosome-dependent macroautophagy (autophagy) pathway is known to be up-regulated by chemical inhibition of the proteasome. Therefore we theorized that autophagy is a pathway of proteasome inhibitor resistance in MM, and that simultaneous inhibition of both the ubiquitin-proteasome system (UPS) and the autophagy pathway would result in synergistic toxicity in MM cells. We chose to explore this hypothesis utilizing a chemical genetic approach, with the goal of repurposing off-patent autophagy inhibitors for immediate translation in the context of human clinical investigation. We selected for study the combination of the only FDA-approved proteasome inhibitor, bortezomib, and the 4-aminoquinoline autophagy inhibitor, hydroxychloroquine (HCQ). Indeed, HCQ exhibits a dose-dependent cytotoxic effect on cultured human MM cells in vitro. Using high-throughput, high-content epifluorescence microscopy, we have demonstrated a dose-dependent increase in autophagosome number and average vesicle area in MM cells treated with HCQ consistent with inhibition of late autophagy. These findings were confirmed by electron microscopy. Autophagy inhibition by HCQ was verified by LC3 immunoblot with and without bafilomycin clamp. Gold-standard pulse-chase studies of HCQ verified inhibition of long-lived protein degradation in MM cells. Of note, in vivo studies of HCQ in a bioluminescent orthotopic xenograft model of MM failed to demonstrate a statistically evident disease response or prolongation of survival. Drawing on the aforementioned biological and clinical insights, we predicted that combination autophagy and proteasome inhibition would result in synergistic cytotoxicity. To explore multiple combinations of these classes of therapeutic agents, we devised a high-throughput translational platform for assessing synergy between therapeutic agents. In this model, library plates of compounds are established which may be “crossed” with each other by alternating plate orientation and delivering compounds to experimental assay microtiter plates by robotic pin transfer. Experimental measurements (i.e. cytotoxicity, protein content, post-translational modifications) are then processed in a semi-automated manner employing the median effect principle and illustrative graphical outputs. This platform was validated by modeling known, effective combination therapies in current clinical use or study in MM. We observe potent, synergistic toxicity from the combination of bortezomib and HCQ in both MM1.S and RPMI-8826 MM cell lines. Of note, the combination index observed is <1 across the pharmacologically-achievable range of both compounds. As the bone marrow microenvironment is known to confer chemoresistance to MM cells, a co-culture assay was developed using high-content imaging to study microenvironmental interactions and MM biology with greater biological precision than established methods (i.e. thymidine uptake). Combined proteasome and autophagy inhibition exhibits synergistic cytotoxicity in this microenvironmental model, notably increasing activated caspase-3 in MM cells indicative of an apoptotic death. Based on these data we have initiated a Phase I clinical study at the University of Pennsylvania (D.V.), aimed at establishing the tolerability and pharmacodynamic effect of this combination in patients with proteasome-refractory MM.