Proteasome Inhibitor Resistance Research Articles

Different Strategies to Overcome Resistance to Proteasome Inhibitors-A Summary 20 Years after Their Introduction.

Proteasome inhibitors (PIs), bortezomib, carfilzomib, and ixazomib, are the first-line treatment for multiple myeloma (MM). They inhibit cytosolic protein degradation in cells, which leads to the accumulation of misfolded and malfunctioned proteins in the cytosol and endoplasmic reticulum, resulting in cell death. Despite being a breakthrough in MM therapy, malignant cells develop resistance to PIs via different mechanisms. Understanding these mechanisms drives research toward new anticancer agents to overcome PI resistance. In this review, we summarize the mechanism of action of PIs and how MM cells adapt to these drugs to develop resistance. Finally, we explore these mechanisms to present strategies to interfere with PI resistance. The strategies include new inhibitors of the ubiquitin-proteasome system, drug efflux inhibitors, autophagy disruption, targeting stress response mechanisms, affecting survival and cell cycle regulators, bone marrow microenvironment modulation, and immunotherapy. We list potential pharmacological targets examined in in vitro, in vivo, and clinical studies. Some of these strategies have already provided clinicians with new anti-MM medications, such as panobinostat and selinexor. We hope that further exploration of the subject will broaden the range of therapeutic options and improve patient outcomes.

Understanding the Transcription Factor NFE2L1/NRF1 from the Perspective of Hallmarks of Cancer.

Cancer cells subvert multiple properties of normal cells, including escaping strict cell cycle regulation, gaining resistance to cell death, and remodeling the tumor microenvironment. The hallmarks of cancer have recently been updated and summarized. Nuclear factor erythroid 2-related factor 1 (NFE2L1, also named NRF1) belongs to the cap'n'collar (CNC) basic-region leucine zipper (bZIP) family. It acts as a transcription factor and is indispensable for maintaining both cellular homoeostasis and organ integrity during development and growth, as well as adaptive responses to pathophysiological stressors. In addition, NFE2L1 mediates the proteasome bounce-back effect in the clinical proteasome inhibitor therapy of neuroblastoma, multiple myeloma, and triple-negative breast cancer, which quickly induces proteasome inhibitor resistance. Recent studies have shown that NFE2L1 mediates cell proliferation and metabolic reprogramming in various cancer cell lines. We combined the framework provided by "hallmarks of cancer" with recent research on NFE2L1 to summarize the role and mechanism of NFE2L1 in cancer. These ongoing efforts aim to contribute to the development of potential novel cancer therapies that target the NFE2L1 pathway and its activity.

MiR-34c-5p Inhibition Affects Bax/Bcl2 Expression and Reverses Bortezomib Resistance in Multiple Myeloma Cells.

Developing resistance to anticancer drugs complicates the clinical treatment of multiple myeloma patients. Previous studies revealed a link between the unfolded protein response (UPR) and miRNAs with acquired drug resistance. This study aimed to determine the expression profile of XBP1, hsa-miR-34c-5p, hsa-miR-214, and hsa-miR-30c-2* in resistant and sensitive multiple myeloma cell lines to a proteasome inhibitor, bortezomib. After establishing bortezomib-resistant cells, the expression level of XBP1, hsa-miR-214, hsa-miR-34c-5p, and hsa-miR-30c-2* in both cell lines were assessed by qRT-PCR. Hsa-miR-34c-5p was suppressed to study its effect on the expression profile of Bax/Bcl-2. Statistical analysis was done by t-test in two clinically resistant and sensitive cells to bortezomib. MTT assay confirmed the creation of the resistant cell line. The qRT-PCR screening showed a significant difference between XBP1 and miR-34c-5p levels in resistant and sensitive cells. Following hsa-miR-34c-5p blockage, while Bax was overexpressed, Bcl-2 expression was reduced in the resistant cell line, overcoming cells resistant to bortezomib. Our findings demonstrate miR-34c-5p is differentially expressed between bortezomib-sensitive and -resistant MM cells. Inhibiting miR-34c-5p re-sensitized resistant cells to bortezomib by modulating Bax/Bcl-2 expression, suggesting this miRNA regulates apoptosis and drug resistance and may be a promising therapeutic target for overcoming proteasome inhibitor resistance in MM.

Abstract 7191: Targeting IGF1R/INSR pathway with approved ALK inhibitors inhibits AKT signaling and overcomes proteasome inhibitor resistance in multiple myeloma

Abstract Background: Resistance to proteasome inhibitors (PI) bortezomib (BTZ) and carfilzomib (CFZ) is a major obstacle to the successful treatment of multiple myeloma (MM); thus, identification of novel therapeutic options is an unmet medical need. ALK inhibitors have been shown to exhibit anti-MM activity in monotherapy or in combination with CFZ. Our data showed that MM cells were negative for ALK; thus, we aimed to identify the mechanism of action of the ALK inhibitor ceritinib in MM cells and the mechanism of synergy between ceritinib and CFZ. Methods: A set of PI-naïve and PI-resistant MM cells was used in this study. Genome-wide CRISPR/Cas9-based screening using the Brunello library was performed in the AMO-1 cell line. Kinase inhibitor selectivity data were retrieved from ChEMBL, v30 database. RNA sequencing was used to determine expression changes after treatment, and RNA-seq data from patients included in the CoMMpass study were analyzed. Western blotting was performed to assess the levels of total and phosphorylated protein. Unbiased LC-MS/MS was performed to determine the effects of ceritinib and CFZ on the intracellular metabolites. An in vivo mouse model based on the orthotopic injection of AMO-BTZ cells into the femur of NSG mice was used to determine the effect of the drug combination in vivo. Results: Among approved ALK inhibitors, ceritinib has been identified as the most synergistic cytotoxic combination with CFZ in PI-naïve and PI-resistant MM cells. An in silico search of the ChEMBL database identified InsR and IGF1R as receptor tyrosine kinase that are inhibited by ceritinib. PI-naïve, PI-resistant cell lines, as well as MM patients were negative for ALK, but positive for InsR and IGF1R expression. CRISPR/Cas9-screening identified genes involved in the negative regulation of mTORC signaling (DDIT4, NPRL2/3, TSC1/2) and the transcription factor FOXO1 as the major resistance candidates to ceritinib. Subsequently, FOXO1 inhibition using a selective chemical inhibitor protected the cells from ceritinib-induced cytotoxicity. Next, ceritinib inhibited mTORC signaling and induced the expression of genes related to cell cycle arrest, which are downstream of FOXO1. Likewise, ceritinib, by targeting the InsR/IGF1R, impaired purine-pyrimidine and amino acid homeostasis, suggesting metabolic and proliferation shut-down and amino acid starvation due to downstream mTORC and Akt inhibition and FOXO1 induction. The combination of ceritinib and CFZ was superior to CFZ in PI-resistant MM in vivo and showed strong synergistic cytotoxicity in primary cells from MM patients progressing under or after PI-containing therapy. Conclusion: Ceritinib, an FDA-approved drug, overcomes PI resistance in MM by inhibiting InsR/IGF1R and downstream Akt signaling. Therefore, ceritinib is a promising treatment option for PI-resistant MM. Supported by the project National Institute for Cancer Research (Programme EXCELES, ID Project No. LX22NPO5102) - Funded by the European Union - Next Generation EU. Citation Format: Andrej Besse, Tiberiu Totu, Marianne Kraus, Anthonius P. Janssen, Jana Veprkova, Max Mendez Lopez, Ondrej Slaby, Marija Buljan, Mario van der Stelt, Christoph Driessen, Lenka Besse. Targeting IGF1R/INSR pathway with approved ALK inhibitors inhibits AKT signaling and overcomes proteasome inhibitor resistance in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7191.

MUC20 regulated by extrachromosomal circular DNA attenuates proteasome inhibitor resistance of multiple myeloma by modulating cuproptosis

BackgroundProteasome inhibitors (PIs) are one of the most important classes of drugs for the treatment of multiple myeloma (MM). However, almost all patients with MM develop PI resistance, resulting in therapeutic failure. Therefore, the mechanisms underlying PI resistance in MM require further investigation.MethodsWe used several MM cell lines to establish PI-resistant MM cell lines. We performed RNA microarray and EccDNA-seq in MM cell lines and collected human primary MM samples to explore gene profiles. We evaluated the effect of MUC20 on cuproptosis of PI-resistant MM cells using Co-immunoprecipitation (Co-IP), Seahorse bioenergetic profiling and in vivo assay.ResultsThis study revealed that the downregulation of Mucin 20 (MUC20) could predict PI sensitivity and outcomes in MM patients. Besides, MUC20 attenuated PI resistance in MM cells by inducing cuproptosis via the inhibition of cyclin-dependent kinase inhibitor 2 A expression (CDKN2A), which was achieved by hindering MET proto-oncogene, receptor tyrosine kinase (MET) activation. Moreover, MUC20 suppressed MET activation by repressing insulin-like growth factor receptor-1 (IGF-1R) lactylation in PI-resistant MM cells. This study is the first to perform extrachromosomal circular DNA (eccDNA) sequencing for MM, and it revealed that eccDNA induced PI resistance by amplifying kinesin family member 3 C (KIF3C) to reduce MUC20 expression in MM.ConclusionOur findings indicated that MUC20 regulated by eccDNA alleviates PI resistance of MM by modulating cuproptosis, which would provide novel strategies for the treatment of PI-resistant MM.

CRIP1 involves the pathogenesis of multiple myeloma via dual-regulation of proteasome and autophagy

Multiple myeloma (MM) is an incurable hematological malignancy of the plasma cells. The maintenance of protein homeostasis is critical for MM cell survival. Elevated levels of paraproteins in MM cells are cleared by proteasomes or lysosomes, which are independent but inter-connected with each other. Proteasome inhibitors (PIs) work as a backbone agent and successfully improved the outcome of patients; however, the increasing activity of autophagy suppresses the sensitivity to PIs treatment. The transcription levels of CRIP1 were explored in plasma cells obtained from healthy donors, patients with newly diagnosed multiple myeloma (NDMM), and relapsed/refractory multiple myeloma (RRMM) using Gene expression omnibus datasets. Doxycycline-inducible CRIP1-shRNA and CRIP1 overexpressed MM cell lines were constructed to explore the role of CRIP1 in MM pathogenesis. Proliferation, invasion, migration, proteasome activity and autophagy were examined in MM cells with different CRIP1 levels. Co-immunoprecipitation (Co-IP) with Tandem affinity purification/Mass spectrum (TAP/MS) was performed to identify the binding proteins of CRIP1. The mouse xenograft model was used to determine the role of CRIP1 in the proliferation and drug-resistance of MM cells. High CRIP1 expression was associated with unfavorable clinical outcomes in patients with MM and served as a biomarker for RRMM with shorter overall survival. Invitro and invivo studies showed that CRIP1 plays a critical role in protein homeostasis via the dual regulation of the activities of proteasome and autophagy in MM cells. A combined analysis of RNA-seq, Co-IP and TAP/MS demonstrated that CRIP1 promotes proteasome inhibitors resistance in MM cells by simultaneously binding to de-ubiquitinase USP7 and proteasome coactivator PA200. CRIP1 promoted proteasome activity and autophagosome maturation by facilitating the dequbiquitination and stabilization of PA200. Our findings clarified the pivotal roles of the CRIP1/USP7/PA200 complex in ubiquitin-dependent proteasome degradation and autophagy maturation involved in the pathogenesis of MM. A full list of funding sources can be found in the acknowledgements section.

Decoding the secrets: how conformational and structural regulators inhibit the human 20S proteasome.

Acquired resistance to drugs that modulate specific protein functions, such as the human proteasome, presents a significant challenge in targeted therapies. This underscores the importance of devising new methodologies to predict drug binding and potential resistance due to specific protein mutations. In this work, we conducted an extensive computational analysis to ascertain the effects of selected mutations (Ala49Thr, Ala50Val, and Cys52Phe) within the active site of the human proteasome. Specifically, we sought to understand how these mutations might disrupt protein function either by altering protein stability or by impeding interactions with a clinical administered drug. Leveraging molecular dynamics simulations and molecular docking calculations, we assessed the effect of these mutations on protein stability and ligand affinity. Notably, our results indicate that the Cys52Phe mutation critically impacts protein-ligand binding, providing valuable insights into potential proteasome inhibitor resistance.

Abstract C084: Metabolomic profiling identifies molecular modifiers contributing to proteasome inhibitor resistance in multiple myeloma

Abstract Metabolic reprogramming plays a crucial role in the maintenance and differentiation of various cancers, including multiple myeloma (MM), the second-most common incurable hematological malignancy. Drug resistance poses a significant challenge to conventional proteasome inhibitor (PI) based therapy. A body of literature suggests that several metabolites are significantly deregulated in MM patients, however, their role in driving resistance is unclear. This study aims to identify metabolites modulated during acquired resistance to proteasome inhibitors in relapsed refractory (RR)MM. Two clinically relevant cell line models of PI-resistance (PIR) were developed by dose-escalation of the PI over one year. Cell cytotoxicity assays, immunoblotting, immunofluorescence, and flow cytometric analyses were used to assess the resistance phenotype. Mass spectrometry-based metabolomic profiling was performed to identify altered metabolites in resistant as compared to sensitive cell lines. PIR cells demonstrated pan-resistance to multiple PIs, including a >10-fold increase in IC50 for oprozomib compared to parental cells. PIR cells had higher bioenergetic and glycolytic activity (Seahorse) while gene expression profiling suggested downregulation of oxidative phosphorylation. Mass spectrometry-based metabolomic profiling identified distinct metabolites deregulated in drug-resistant cells. The metabolomic profiles of the drug-resistant cell lines showed that the lipid synthesis and beta-oxidation of fatty acid pathways were highly modulated. Several pathway components demonstrated a negative association with overall survival in multiple myeloma patient datasets (MMRF, GSE31161 & GSE9782). PI resistance led to increased phosphorylation of ACLY involved in fatty acid synthesis and increased expression of CPT1A indicative of activation of the beta-oxidation pathway. Use of ACLY and FASN tool compounds affected proliferation, survival, and response to treatment in PIR cells. Metabolites modulated during acquired resistance to proteasome inhibitor in relapsed refractory (RR)MM were identified and potential mechanisms driving resistance were studied. Citation Format: Snehal M Gaikwad, Vincent Keith Hughitt, Nana Gyabaah-Kessie, Mikaela Matera-Vatnick, Aleksandra A Michalowski, Anaisa Quintanilla-Arteaga, Thorkell Andresson, Beverly A Mock. Metabolomic profiling identifies molecular modifiers contributing to proteasome inhibitor resistance in multiple myeloma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C084.

CNOT4 Knockout Induces Proteasome Inhibitor Resistance in Multiple Myeloma Cells

Background: The outcomes of multiple myeloma (MM) patients have been significantly improved with novel therapies including proteasome inhibitors (PI). However, many patients develop PI resistance and becoming refractory to PI. Although several studies have identified the mechanisms of PI resistance, such as mutation to the proteasome β5 subunit, ER stress suppression, autophagy activation, and activation of efflux drug pumps, it remains unclear yet. To investigate new genes that may contribute to PI resistance, we performed a genome-wide CRISPR dropout screening in an MM cell line. Materials and Methods: To identify the genes responsible for PI resistance, we used a CRISPR library (Kosuke Yusa, V3) targeting 18,740 human genes. The knockout cells, in which an CRISPR library lentivirus was introduced into a Cas9-expressing AMO1 MM cell line, were treated with ixazomib (IXA), carfilzomib (CFZ), and DMSO for two weeks. Next, we used next generation sequencing (NGS) to count sgRNA copy numbers of the pre-drug control group and the drug-treated group. NGS analysis of the treated cells showed that inactivated genes which induced higher levels of cell proliferation in the PI group than in the DMSO group were considered candidate genes for PI resistance. Results: We identified 35 genes for PI resistance including the genes encoding subunits of the proteasome 19S complex, consistent with previous reports that shows reducing 19S subunits protects MM cells from bortezomib. Among 35 candidate genes, we chose the top hit gene, CNOT4, to further investigate. CNOT4 is a component of CCR4-NOT complex which removes the mRNA poly(A) tail for degradation of mRNA. In addition, CNOT4 has E3 ubiquitin ligase activity and is reported to control the proteasome complex formation. To further validate the effect of PI resistance with inactivated CNOT4, we generated CNOT4 KO AMO1 cells and performed an MTS assay. We observed a decrease in PI sensitivity compared with AMO1 wildtype (WT) (IXA IC 50: 24.8 nM (KO) vs 13.0 nM (WT), CFZ IC 50: 7.45 nM (KO) vs 2.76 nM (WT)). To investigate the influence of CNOT4 on proteasome complex formation and its activity, we first measured the expression of ubiquitinated proteins by Western blotting. In CNOT4 KO AMO1 cells, the expression of ubiquitinated proteins is increased with or without PI treatment, suggesting that inactivated CNOT4 can suppress the function of degrade ubiquitinated proteins in proteasome 19S complex. To elucidate the mechanism underlying the resistance effect, we next checked the expression of ER stress marker proteins. In CNOT4 KO AMO1 cells, the expression of PERK and IRE1α proteins are decreased with PI treatment, suggesting that inactivated CNOT4 can suppress the ER stress levels in spite of the increases of ubiquitinated proteins. Conclusion: Our CRISPR screen revealed that CNOT4 inactivation induced PI resistance in human MM cells. CNOT4 inactivation suppresses the function of 19S proteasome, which is reported the influence of PI resistance, and downregulates the ER stress signal leading to avoid PI induced cell death. Our findings demonstrate that CNOT4 plays an important role in PI sensitivity.

Targeting IGF1R/Insr Pathway with Approved ALK-Inhibitors Overcomes Proteasome Inhibitor Resistance in Multiple Myeloma

Background: The treatment of multiple myeloma (MM) has advanced rapidly with the discovery of proteasome inhibitors (PIs) bortezomib (BTZ) and carfilzomib (CFZ). PIs disrupt the equilibrium between production and disposal of excess and/or misfolded proteins in MM cells, leading to apoptosis. However, despite this success, essentially all patients with MM eventually develop resistance to PIs. Therefore, identifying strategies to overcome PIs resistance is clinically important. ALK inhibitors exhibit anti-MM activity ex vivo. Previously, the ALK inhibitor ceritinib has been shown to inhibit IGF1R/InsR signaling. Signaling through the IGF1/IGF1R axis contributes to acquired resistance to BTZ, and PI activity is reduced in the presence of IGF1, suggesting that IGF1R inhibitors may enhance the cytotoxic effect of PIs. To date, IGF1R/InsR inhibitors have not been successful as single agents in clinical trials and no formally approved drugs are available. Importantly, the drug repurposing approach offers a promising strategy for drug development, particularly for MM. Therefore, we aimed to identify i) whether ALK inhibitors are cytotoxic in MM by targeting the IGF1R/InsR pathway, and ii) whether they can be combined with PIs to overcome PI resistance in vitro and in vivo. Methods: A set of PI-naïve and PI-resistant cells was used in this study. The cytotoxicity of drugs was determined by CCK8 assay in cell lines and by CellTiter-Glo assay in primary cells. Genome-wide CRISPR/Cas9-based loss-of-function screening using the Brunello library was performed in the AMO-1 cell line to identify the mechanism of action of ceritinib. Kinase inhibitor selectivity data were retrieved from ChEMBL, v30. Western blotting was performed to assess the levels of total and phosphorylated proteins. RNA sequencing was used to determine the number of transcripts in PI-sensitive and PI-resistant cells, and RNA-seq data from patients included in the CoMMpass study were analyzed. Unbiased LC-MS/MS was performed to determine the effects of ceritinib and carfilzomib on intracellular metabolites. An in vivo mouse model based on orthotopic injection of AMO-BTZ cells into the femur of NSG mice was used to determine the effect of the drug combination in vivo. Results: Initially, seven ALK inhibitors were tested in PI-naïve and PI-adapted MM cell lines. Based on IC 50 values, the most effective ALK inhibitors to induce cytotoxicity in MM were ceritinib > brigatinib > entrectinib. The combination of ceritinib, brigatinib, and entrectinib showed synergistic cytotoxicity with PI BTZ and CFZ and overcame PI-resistance in four different sets of PI-adapted cells. The strongest synergistic cytotoxicity was observed between ceritinib and CFZ in CFZ-adapted cells. CRISPR/Cas9-screening identified genes involved in the negative regulation of mTORC signaling (DDIT4, NPRL2/3, TSC1/2) and transcription factor FOXO1 as the major resistance candidates to ceritinib. Subsequently, ceritinib treatment significantly inhibited mTORC signaling and impaired purine-pyrimidine and amino acid production pathways, suggesting a metabolic and proliferation shut-down and amino acid starvation due to targeting of the upstream receptor tyrosine kinases (RTK), which are essential for cell viability and thus not identified by CRISPR screening. Further search in the ChemBL database identified InsR and IGF1R, as RTK that was potently inhibited by ceritinib. PI-naïve and PI-resistant cell lines as well as MM patients were negative for ALK, but positive for InsR and IGF1R expression. Accordingly, co-treatment of PI-resistant cells with a combination of InsR/IGF1R inhibitors and CFZ induced strong synergistic cytotoxicity, resembling the synergistic cytotoxicity observed for ceritinib and CFZ, whereas InsR/IGF1R inhibitors did not show any synergistic cytotoxicity with ceritinib, suggesting targeting the same pathway. Importantly, the combination of ceritinib and CFZ was superior to CFZ in an orthotopic mouse model bearing PI-resistant MM cells and showed strong synergistic cytotoxicity in primary cells from MM patients progressing under or after PI-containing therapy. Conclusion: Ceritinib, an FDA-approved drug, overcomes PI resistance in MM by targeting InsR/IGF1R signaling, which is essential for PI resistance in MM. Therefore, ceritinib represents a promising, potential option for the treatment of PI-resistant MM.

PSMC2 Overexpression Enhances Proteasome Activity, Mitigates ER Stress, and Correleates with Reduced Overall Survival in Multiple Myeloma

Background: Multiple myeloma (MM) remains a highly treatable but generally incurable plasma cell dyscrasia. These plasma cells synthesize massive amounts of paraprotein relying heavily on the ubiquitin proteasome system to maintain homeostasis and are exquisitely sensitive to proteasome inhibition to avoid terminal ER stress. We postulated that increased expression of clinically relevant proteasome subunits would correlate with reduced overall survival in MM patients treated with proteasome inhibitors. Objective: To correlate proteasome gene expression levels to clinicals outcomes in both the phase III APEX trial and the MMRF COMMPASS Trial. To model the effects of these hits on MMCL UPS function. Results: We identified that 19S regulatory ATPases PSMC2 and PSMC6 correlated with poorer OS, outperforming the proteasome inhibitor (PI) target, PSMB5, in this analysis. We focused on modeling the more frequently upregulated PSMC2. We created PSMC2 overexpressing multiple myeloma cell lines, noting PSMC2 is readily incorporated in the higher order proteasome complexes. PSMC2 overexpressing cells exhibit higher levels of 20S proteasome activity (1.6 fold increase in ARH77 and 3 fold increase in U266 cell line compared to control, p< 0.01 in each case) and are more resistant to PI challenge (Bortezomib LD 50 of 30 nM v 15 nM in U266 overexpressed and control cells respectively, Bortezomib LD 50 of 18 nM v 10 nM in ARH77 overexpressed v control cells respectively). PIs are known to increase ER stress, and PSMC2 overexpressing cells are better able to mitigate PI mediated ER stress with PSMC2 overexpressing cells exhibiting less induction of ER stress sensors p-IRE1α and ATF4 compared to PSMC2 knockdown cells on bortezomib or tunicamycin challenge as quantitated by western blotting and utilizing an fluorescent ER stress sensor that models the conversion of XBP1u to XBP1s by pIRE1α. Mechanistically, PSMC subunits play a key role in the recognition and degradation of misfolded ER proteins, and increased 19S subunits may allow more effective ER associated protein degradation (ERAD) Conclusions: Taken together, our results suggest PSMC2 overexpression correlates with reduced OS in MM patients treated with bortezomib and that PSMC2 may represent an actionable therapeutic target to prevent or overcome PI resistance. PSMC2 overexpression increases proteasome activity in MM cells, mitigates ER stress on PI challenge and promotes PI resistance. mitigates ER stress upon PI challenge, and portending poorer clinical prognosis and more drug resistance, suggesting that 19S targeted agents may help overcome drug resistance in MM. Figure 1 A. PSMC2 overexpression increases chymotrypsin-like proteasome activity at baseline in U266 and ARH77 cells with a similar effect of residual proteasome activity after challenge with bortezomib or carfilzomib. B. ARH77 and U266 FLAG and vector cells were challenged with bortezomib for 18 hrs and then assayed by annexin PI staining. LD50 increased from 10 nm to 15 nm in ARH77 cells and from 12 to 25 in U266 Cells.

CRIP1 Involves the Pathogenesis of Multiple Myeloma Via Dual-Regulation of Proteasome and Autophagy

Multiple myeloma (MM) is an incurable hematological malignancy of plasma cells. The maintenance of protein homeostasis is critical for the survival of MM cells. Elevated paraprotein in MM cells would be cleared by proteasomes or lysosomes, which are independent but inter-connected with each other. Proteasome inhibitors (PIs) work as the backbone agent and successfully improved the outcome of patients, but the increasing activity of autophagy suppresses the sensitivity to PIs treatment. In the present study, we elucidated that CRIP1 was a critical player in protein homeostasis via the dual regulation of proteasome and autophagy in MM. A combination analysis of RNA-seq, Co-IP and Tandem affinity purification/mass spectrometry identified that high-level CRIP1 represents a biomarker for relapsed/refractory MM with shorter overall-survival of patients. CRIP1 plays a critical role in MM progression and PIs resistance by binding to deubiquitinase USP7 and proteasome coactivator PA200. CRIP1 promotes proteasome activity and autophagosome maturation in MM by stabilizing PA200. Therefore, our findings clarified the pivotal roles of CRIP1/USP7/PA200 complex formation in ubiquitin-dependent proteasome degradation and autophagy maturation involved in the progression of MM.

Ribosomal protein S3 mediates drug resistance of proteasome inhibitor: potential therapeutic application in multiple myeloma.

Multiple myeloma (MM) remains incurable due to drug resistance. Ribosomal protein S3 (RPS3) has been identified as a non-Rel subunit of NF-κB. However, the detailed biological roles of RPS3 remain unclear. Here, we report for the first time that RPS3 is necessary for MM survival and drug resistance. RPS3 was highly expressed in MM, and knockout of RPS3 in MM inhibited cell growth and induced cell apoptosis both in vitro and in vivo. Overexpression of RPS3 mediated the proteasome inhibitor resistance of MM and shortened the survival of MM tumor-bearing animals. Moreover, our present study found an interaction between RPS3 and the thyroid hormone receptor interactor 13 (TRIP13), an oncogene related to MM tumorigenesis and drug resistance. We demonstrated that the phosphorylation of RPS3 was mediated by TRIP13 via PKCδ, which played an important role in activating the canonical NF-κB signaling and inducing cell survival and drug resistance in MM. Notably, the inhibition of NF-κB signaling by the small-molecule inhibitor targeting TRIP13, DCZ0415, was capable of triggering synergistic cytotoxicity when combined with bortezomib in drug-resistant MM. This study identifies RPS3 as a novel biomarker and therapeutic target in MM.

Ancistrocladinium A Induces Apoptosis in Proteasome Inhibitor-Resistant Multiple Myeloma Cells: A Promising Therapeutic Agent Candidate.

The N,C-coupled naphthylisoquinoline alkaloid ancistrocladinium A belongs to a novel class of natural products with potent antiprotozoal activity. Its effects on tumor cells, however, have not yet been explored. We demonstrate the antitumor activity of ancistrocladinium A in multiple myeloma (MM), a yet incurable blood cancer that represents a model disease for adaptation to proteotoxic stress. Viability assays showed a potent apoptosis-inducing effect of ancistrocladinium A in MM cell lines, including those with proteasome inhibitor (PI) resistance, and in primary MM cells, but not in non-malignant blood cells. Concomitant treatment with the PI carfilzomib or the histone deacetylase inhibitor panobinostat strongly enhanced the ancistrocladinium A-induced apoptosis. Mass spectrometry with biotinylated ancistrocladinium A revealed significant enrichment of RNA-splicing-associated proteins. Affected RNA-splicing-associated pathways included genes involved in proteotoxic stress response, such as PSMB5-associated genes and the heat shock proteins HSP90 and HSP70. Furthermore, we found strong induction of ATF4 and the ATM/H2AX pathway, both of which are critically involved in the integrated cellular response following proteotoxic and oxidative stress. Taken together, our data indicate that ancistrocladinium A targets cellular stress regulation in MM and improves the therapeutic response to PIs or overcomes PI resistance, and thus may represent a promising potential therapeutic agent.

CRIP1 INDUCES PIS RESISTANCE BY FORMING A COMPLEX WITH USP7 /PA200 AND ENHANCING PROTEASOME ACTIVITY AND AUTOPHAGY IN MULTIPLE MYELOMA

Introduction: Multiple myeloma (MM) is a plasma malignancy characterized by an accumulation of misfolded immunoglobulins in the cell. Although proteasome inhibitor (PI) can treat MM by inhibiting ubiquitin-proteasome system (UPS), autophagy as a compensatory protein clearance mechanism, it still leads to drug resistance. The discovery of new targets that can simultaneously inhibit UPS and autophagy will provide great potential for the treatment of MM. Methods: The proliferation, invasion, migration, proteasome activity and autophagy were detected in CRIP1- knockdown /overexpression and control MM cells. Co-IP (Co-Immunoprecipitation) with TAP/MS (Tandem affinity purification/Mass spectrum) was performed to detect the binding of USP7, CRIP1 and PA200. Myeloma xenograft model was used to determine the role of CRIP1 to promote proliferation of MM cells and induce BTZ resistance in vivo. Results: The levels of CRIP1 were significantly increased in plasma cells from NDMM (newly diagnosed multiple myeloma) compared to healthy donors, and further increased in patients with RRMM (relapsed/refractory multiple myeloma). Moreover, the high expression of CRIP1 is significantly related to the poor prognosis of MM patients, especially those treated with BTZ. Our results in vitro and in vivo showed that CRIP1 knockdown noteworthy inhibited the cell growth, invasion, migration. MM cells were more sensitive to PIs-induced cell growth arrest when CRIP1 knockdown. The proteasome activity and the protein level of LC3B-Ⅱ of the MM cells were significantly reduced with CRIP1 knockdown. Co-IP analysis showed that CRIP1 formed a complex with USP7/PA200. The protein level of CRIP1 decreased and the ubiquitination CRIP1 increased with USP7 inhibition. These data suggested that CRIP1 is the substrate of USP7.CRIP1 promotes the de-ubiquitination and stabilization of PA200 mediated by USP7 by interacting with USP7 and PA200, which in turn promotes MM cell survival and drug resistant. Functional inhibition of CRIP1 by knocking down of USP7 or PSME4 in CRIP1 OE MM cell line led to a decrease of the activity of proteasome and autophagy, thus enhancing the sensitivity to PIs in intro and in vivo. Conclusion: CRIP1 plays a critical role in MM progression and PIs resistance by formation of CRIP1/USP7/PA200 complex. High level of CRIP1 is a biomarker for high-risk MM patients. CRIP1 is a potential target for MM therapy, can simultaneously inhibit proteasome activity and autophagy. Encore Abstract - previously submitted to EHA 2023 The research was funded by the following foundations:(a) CAMS Innovation Fund for Medical Sciences (CIFMS) (2021-I2M-1-040 and 2022-I2M-1-022), (b) National NaturalScience Foundation of China (82170194, 81920108006, 82270175) and Natural Science Foundation of Fujian Province of China (2021J02040). Keywords: Diagnostic and Prognostic Biomarkers, Multiple Myeloma, Tumor Biology and Heterogeneity No conflicts of interests pertinent to the abstract.

CD317 maintains proteostasis and cell survival in response to proteasome inhibitors by targeting calnexin for RACK1-mediated autophagic degradation

Unbalanced protein homeostasis (proteostasis) networks are frequently linked to tumorigenesis, making cancer cells more susceptible to treatments that target proteostasis regulators. Proteasome inhibition is the first licensed proteostasis-targeting therapeutic strategy, and has been proven effective in hematological malignancy patients. However, drug resistance almost inevitably develops, pressing for a better understanding of the mechanisms that preserve proteostasis in tumor cells. Here we report that CD317, a tumor-targeting antigen with a unique topology, was upregulated in hematological malignancies and preserved proteostasis and cell viability in response to proteasome inhibitors (PIs). Knocking down CD317 lowered Ca2+ levels in the endoplasmic reticulum (ER), promoting PIs-induced proteostasis failure and cell death. Mechanistically, CD317 interacted with calnexin (CNX), an ER chaperone protein that limits calcium refilling via the Ca2+ pump SERCA, thereby subjecting CNX to RACK1-mediated autophagic degradation. As a result, CD317 decreased the level of CNX protein, coordinating Ca2+ uptake and thus favoring protein folding and quality control in the ER lumen. Our findings reveal a previously unrecognized role of CD317 in proteostasis control and imply that CD317 could be a promising target for resolving PIs resistance in the clinic.

Dual-pharmacophore artezomibs hijack the Plasmodium ubiquitin-proteasome system to kill malaria parasites while overcoming drug resistance

Artemisinins (ART) are critical anti-malarials and despite their use in combination therapy, ART-resistant Plasmodium falciparum is spreading globally. To counter ART resistance, we designed artezomibs (ATZs), molecules that link an ART with a proteasome inhibitor (PI) via a non-labile amide bond and hijack parasite's own ubiquitin-proteasome system to create novel anti-malarials in situ. Upon activation of the ART moiety, ATZs covalently attach to and damage multiple parasite proteins, marking them for proteasomal degradation. When damaged proteins enter the proteasome, their attached PIs inhibit protease function, potentiating the parasiticidal action of ART and overcoming ART resistance. Binding of the PI moiety to the proteasome active site is enhanced by distal interactions of the extended attached peptides, providing a mechanism to overcome PI resistance. ATZs have an extra mode of action beyond that of each component, thereby overcoming resistance to both components, while avoiding transient monotherapy seen when individual agents have disparate pharmacokinetic profiles.

Metabolic Alterations in Multiple Myeloma: From Oncogenesis to Proteasome Inhibitor Resistance

Despite significant improvements in treatment strategies over the past couple of decades, multiple myeloma (MM) remains an incurable disease due to the development of drug resistance. Metabolic reprogramming is a key feature of cancer cells, including MM, and acts to fuel increased proliferation, create a permissive tumour microenvironment, and promote drug resistance. This review presents an overview of the key metabolic adaptations that occur in MM pathogenesis and in the development of resistance to proteasome inhibitors, the backbone of current MM therapy, and considers the potential for therapeutic targeting of key metabolic pathways to improve outcomes.

SUMOylation inhibition overcomes proteasome inhibitor resistance in multiple myeloma.

Proteasome inhibition is a highly effective treatment for multiple myeloma (MM). However, virtually all patients develop proteasome inhibitor resistance, which is associated with a poor prognosis. Hyperactive small ubiquitin-like modifier (SUMO) signaling is involved in both cancer pathogenesis and cancer progression. A state of increased SUMOylation has been associated with aggressive cancer biology. We found that relapsed/refractory MM is characterized by a SUMO-high state, and high expression of the SUMO E1-activating enzyme (SAE1/UBA2) is associated with poor overall survival. Consistently, continuous treatment of MM cell lines with carfilzomib (CFZ) enhanced SUMO pathway activity. Treatment of MM celllines with the SUMO E1-activating enzyme inhibitor subasumstat (TAK-981) showed synergy with CFZ in both CFZ-sensitive and CFZ-resistant MM cell lines, irrespective of the TP53 state. Combination therapy was effective in primary MM cells and in 2 murine MM xenograft models. Mechanistically, combination treatment with subasumstat and CFZ enhanced genotoxic and proteotoxic stress, and induced apoptosis was associated with activity of the prolyl isomerase PIN1. In summary, our findings reveal activated SUMOylation as a therapeutic target in MM and point to combined SUMO/proteasome inhibition as a novel and potent strategy for the treatment of proteasome inhibitor-resistant MM.

EMD originates from hyaluronan-induced homophilic interactions of CD44 variant-expressing MM cells under shear stress.

Extramedullary disease (EMD) is known to be associated with chemoresistance and poor prognosis in multiple myeloma (MM); however, the mechanisms of its development are not fully understood. Elucidating the mechanism of EMD development and its therapeutic targeting would greatly contribute to further improvement of treatment outcome in patients with MM. Here, we show that bone marrow stroma cell-derived hyaluronan (HA) elicits homophilic interactions of MM cells by binding to surface CD44, especially long-stretch variants, under physiological shear stress and generates cell clusters that might develop into EMD. We recapitulated the development of EMD via administration of HA in a syngeneic murine MM model in a CD44-dependent manner. HA-induced MM cell clusters exhibited the specific resistance to proteasome inhibitors (PIs) invitro and in murine models via γ-secretase-mediated cleavage of the intracellular domains of CD44, which in turn transactivated PI resistance-inducible genes. Treatment of HA-injected mice with anti-CD44 antibody or γ-secretase inhibitors readily suppressed the development of EMD from transplanted MM cells and significantly prolonged the survival of recipients by overcoming PI resistance. The HA-CD44 axis represents a novel pathway to trigger EMD development and could be a target of the prediction, prevention, and treatment of EMD in patients with MM.