Proteasome Inhibitor Sensitivity Research Articles

Ex vivo drug response heterogeneity reveals personalized therapeutic strategies for patients with multiple myeloma

Multiple myeloma (MM) is a plasma cell malignancy defined by complex genetics and extensive patient heterogeneity. Despite a growing arsenal of approved therapies, MM remains incurable and in need of guidelines to identify effective personalized treatments. Here, we survey the ex vivo drug and immunotherapy sensitivities across 101 bone marrow samples from 70 patients with MM using multiplexed immunofluorescence, automated microscopy and deep-learning-based single-cell phenotyping. Combined with sample-matched genetics, proteotyping and cytokine profiling, we map the molecular regulatory network of drug sensitivity, implicating the DNA repair pathway and EYA3 expression in proteasome inhibitor sensitivity and major histocompatibility complex class II expression in the response to elotuzumab. Globally, ex vivo drug sensitivity associated with bone marrow microenvironmental signatures reflecting treatment stage, clonality and inflammation. Furthermore, ex vivo drug sensitivity significantly stratified clinical treatment responses, including to immunotherapy. Taken together, our study provides molecular and actionable insights into diverse treatment strategies for patients with MM.

MSC Senescence-Related Genes Are Associated with Myeloma Prognosis and Lipid Metabolism-Mediated Resistance to Proteasome Inhibitors.

Complex carcinogenic mechanisms and the existence of tumour heterogeneity in multiple myeloma (MM) prevent the most commonly used staging system from effectively interpreting the prognosis of patients. Since the microenvironment plays an important role in driving tumour development and MM occurs most often in middle-aged and elderly patients, we hypothesize that ageing of bone marrow mesenchymal stem cells (BM-MSCs) may be associated with the progression of MM. In this study, we collected the transcriptome data on MM from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) databases. Differentially expressed genes in both senescent MSCs and MM tumour cells were considered relevant damaged genes. GO and KEGG analyses were applied for functional evaluation. A PPI network was constructed to identify hub genes. Subsequently, we studied the damaged genes that affected the prognosis of MM. Least absolute shrinkage and selection operator (lasso) regression was used to identify the most important features, and a risk model was created. The reliability of the risk model was evaluated with the other 3 GEO validation cohorts. In addition, ROC analysis was used to evaluate the novel risk model. An analysis of immune checkpoint-related genes, tumour immune dysfunction and exclusion (TIDE), and immunophenotypic scoring (IPS) were performed to assess the immune status of risk groups. pRRophetic was utilized to predict the sensitivity to administration of chemotherapeutic agents. We identified that MAPK, PI3K, and p53 signalling pathways were activated in both senescent MSCs and tumour cells, and we also located hub genes. In addition, we constructed a 14-gene prognostic risk model, which was analysed with the ROC and validated in different datasets. Further analysis revealed significant differences in predicted risk values across the International Staging System (ISS) stage, sex, and 1q21 copy number. A high-risk group with higher immunogenicity was predicted to have low proteasome inhibitor sensitivity and respond poorly to immunotherapy. Lipid metabolism pathways were found to be significantly different between high-risk and low-risk groups. A nomogram was created by combining clinical data, and the optimization model was further improved. Finally, real-time qPCR was used to validate two bortezomib-resistant myeloma cell lines, and the test confirmed that 10 genes were detected to be expressed in resistant cell lines with the same trend as in the high-risk cohort compared to nonresistant cells. Fourteen genes related to ageing in BM-MSCs were associated with the prognosis of MM, and by combining this genotypic information with clinical factors, a promising clinical prognostic model was established.

The deubiquitinase OTUD1 regulates immunoglobulin production and proteasome inhibitor sensitivity in multiple myeloma

Serum monoclonal immunoglobulin (Ig) is the main diagnostic factor for patients with multiple myeloma (MM), however its prognostic potential remains unclear. On a large MM patient cohort (n = 4146), we observe no correlation between serum Ig levels and patient survival, while amount of intracellular Ig has a strong predictive effect. Focused CRISPR screen, transcriptional and proteomic analysis identify deubiquitinase OTUD1 as a critical mediator of Ig synthesis, proteasome inhibitor sensitivity and tumor burden in MM. Mechanistically, OTUD1 deubiquitinates peroxiredoxin 4 (PRDX4), protecting it from endoplasmic reticulum (ER)-associated degradation. In turn, PRDX4 facilitates Ig production which coincides with the accumulation of unfolded proteins and higher ER stress. The elevated load on proteasome ultimately potentiates myeloma response to proteasome inhibitors providing a window for a rational therapy. Collectively, our findings support the significance of the Ig production machinery as a biomarker and target in the combinatory treatment of MM patients.

Metformin Induces Resistance of Cancer Cells to the Proteasome Inhibitor Bortezomib.

The anti-diabetic drug metformin is currently tested for the treatment of hematological and solid cancers. Proteasome inhibitors, e.g., Bortezomib, are approved for the treatment of multiple myeloma and mantle cell lymphoma but are also studied for lung cancer therapy. We here analyzed the interaction of the two drugs in two cell lines, namely the mantle cell lymphoma Jeko-1 and the non-small-cell lung cancer (NSCLC) H1299 cells, using proliferation and survival assays, native-gel analysis for proteasome activity and assembly, and expression analysis of proteasome assembly factors. Our results demonstrate that metformin treatment induces resistance of cancer cells to the proteasome inhibitor Bortezomib by impairing the activity and assembly of the 26S proteasome complexes. These effects of metformin on proteasome inhibitor sensitivity in cancer cells are of potential relevance for patients that receive proteasome inhibitor therapy.

Mitochondrial Electron Transport Chain Inhibition Promotes Resistance to Proteasome Inhibitors in Multiple Myeloma

INTRODUCTIONProteasome inhibitors (PI) such as bortezomib (Velcade), carfilzomib (Kyprolis) and ixazomib (Ninlaro)) have been shown to be efficacious in multiple myeloma (MM) therapy. However, despite being an effective first line therapy, resistance to PI usually develops, leading to relapse and refractory disease. Previous studies have implicated a role of OXPHOS, glycolysis, antioxidants and serine metabolism in PI resistance. Mitochondrial metabolism plays a central role in malignant progression not only by generating ATP but also by providing precursors for synthesis of several biomolecules such as proteins, nucleotides, fatty acids and antioxidants that can influence the efficacy of MM therapies. We previously determined reduced mitochondrial electron transport chain (ETC) activity promotes sensitivity to the BCL-2 antagonist, venetoclax. However, the relationship between the metabolic state of the mitochondria and proteasome inhibitor (PI) sensitivity is not fully understood. Unexpectedly, we found ETC inhibition or reduced ETC activity to promote resistance to PIs. Here, we investigate the mechanistic basis for divergent effects of mitochondrial stress on sensitivity to PIs and venetoclax.METHODSWe have used RNA-Seq and carbon isotope tracing using labeled U 13C-glucose or U 13C-glutamine, flow cytometry and western blot analysis in MM cell lines treated with mitochondrial Complex I inhibitor, IACS-010759 (IACS) +/- Bortezomib; and immunostaining of MM patient samples and interrogation of Multiple Myeloma Research Foundation's CoMMpass Study (NCT01454297, Interim Analysis 15).RESULTSWe find that mitochondrial ETC (complex I-V) inhibition antagonizes bortezomib (BTZ) and carfilzomib (CFZ) induced cell death in MM in contrast to promoting sensitivity to venetoclax. Additionally, cell lines exhibiting intrinsically reduced ETC activity were more resistant to PI in comparison to cell lines with higher ETC activity. Evaluation of CoMMpass MM trial (NCT0145429, IA15) and serial samples from 50 patients before PI treatment and after relapse, show pathways related to OXPHOS and TCA cycle to be downregulated in poor survival patients, corroborating our in vitro observations on reduced ETC activity promoting resistance to PI. To elucidate the mechanistic basis of ETC-inhibition induced PI resistance we performed RNA-Seq and U 13C-glucose and glutamine tracing in L363 cells treated with the ETC Complex I inhibitor IACS +/- BTZ. RNA-Seq analysis and further confirmation by western blot analysis reveals integrated stress response (ISR) upregulation, ATF4 induction, and suppression of protein translation and global protein ubiquitination levels, likely responsible for resistance to proteasome inhibition in cells co-treated with IACS and BTZ compared to BTZ alone. Stable isotope tracing reveals an upregulation of reductive carboxylation; while RNA-Seq data and flow cytometry demonstrate increase in the cystine/glutamate transporter SLC7A11. The ensuing metabolic rewiring in mitochondrially suppressed MM induces several metabolic vulnerabilities including sensitivity to the SLC7A11 inhibitor, erastin. We also show that knockdown of ATF4 re-sensitizes ETC-inhibited cells to BTZ while ablating sensitivity to venetoclax (previously reported). Furthermore, examination of patient samples demonstrates inter-cellular heterogeneity in ATF4 expression. Our results thus, support the role ATF4 as key determinants of PI and BCL-2 antagonist efficacy.CONCLUSIONWe show mitochondrial ETC inhibition induces ISR mediated resistance to PI. These ETC-inhibited cells are however sensitive to BCL-2 antagonists and afford additional metabolic vulnerabilities that can be capitalized upon to target metabolic heterogeneity in MM. Our study underscores the need for implementing combinatorial regimens in MM cognizant of mitochondrial metabolic heterogeneity-mediated resistance. DisclosuresLonial: Janssen: Consultancy, Honoraria, Research Funding; BMS/Celgene: Consultancy, Honoraria, Research Funding; AMGEN: Consultancy, Honoraria; GlaxoSmithKline: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria. Boise: AstraZeneca: Consultancy, Research Funding; Abbvie: Consultancy. Jaye: Stemline Therapeutics: Honoraria. Nooka: Janssen Oncology: Consultancy, Research Funding; GlaxoSmithKline: Consultancy, Other: Travel expenses; Sanofi: Consultancy; Bristol-Myers Squibb: Consultancy; Takeda: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Oncopeptides: Consultancy; Adaptive technologies: Consultancy; Karyopharm Therapeutics: Consultancy.

Abstract LB110: Multiple myeloma cells depend on the NRF1-DDI2 proteasome stress response pathway for survival

Abstract Introduction: We previously showed that an imbalance between polyubiquitinated proteins awaiting proteasomal degradation and proteasome activity is a main determinant of proteasome inhibitor (PI) sensitivity in MM. While PIs have radically improved survival of MM patients, resistance is inevitable over time and understanding the molecular mechanisms underlying it is critically important to develop novel therapies to improve patient outcomes. The proteasome stress response (PSR) is an evolutionarily conserved homeostatic mechanism triggered by insufficient proteasome activity and aimed at restoring homeostasis via de novo proteasome biogenesis. The PSR master regulator NRF1 is constitutively degraded by the proteasome in homeostatic conditions, but in face of proteasomal insufficiency is cleaved by the aspartic protease DDI2, translocates to the nucleus, and induces proteasome subunit gene transcription. It is unknown whether blocking DDI2 or NRF1 could target an intrinsic vulnerability of MM. Materials and methods: We used human MM cell lines with distinct baseline sensitivity to PIs. NRF1 cleavage and nuclear localization were assessed via western blotting. We used CRISPR-Cas9 to knock out (KO) DDI2 and NRF1. Cell viability or tumor growth of DDI2/NRF1 KO cells versus cells edited with a non-targeting gRNA were compared in in vitro and in vivo growth competition studies, respectively. Addback studies were performed by stably expressing WT or aspartic protease dead DDI2 in DDI2 KO AMO1-VR monoclones. Chymotryptic-like proteasome activity was measured via cleavage of a fluorescent substrate upon incubation with protein lysates from DDI2 WT, KO, or addback cells. Results: We detected full-length and cleaved NRF1 in human MM cell lines, but not other hematologic cancer cell lines, suggesting constitutively active PSR in MM. Query of the dependency map (depmap.org) shows that MM is the cell line type most highly dependent on DDI2. Consistently, DDI2 or NRF1 KO is cytotoxic alone or in combination with PI across a panel of MM cells with distinct PI sensitivity, including de novo, PI-resistant KMS20. In vivo, DDI2 KO leads to reduced plasmacytoma formation in NOD SCID mice and results in prolongation of animal survival. While biallelic DDI2 or NRF1 KO monoclones could not be generated across 7 distinct MM cell lines, consistent with these genes being essential, we successfully established DDI2 KO monoclones in AMO1-VR, an isogenic AMO1 cell line variant adapted to grow in continuous bortezomib. Even in this cell line model of acquired PI resistance, DDI2 KO sensitized cells to PI carfilzomib. In this model, DDI2 KO blocks NRF1 cleavage and nuclear import, thereby impairing proteasome recovery after irreversible PI treatment and sensitizing to PI. Wild-type, but not catalytically-dead DDI2 addback rescues these phenotypes, confirming a causative link. Conclusions: Our data show that MM cells are intrinsically dependent on an intact DDI2/NRF1 axis to survive in baseline conditions and upon proteasomal inhibition. We propose that DDI2 is an unexplored, promising molecular target for MM treatment by disrupting the PSR and exacerbating proteotoxicity. This study provides the preclinical rationale for the development of novel therapeutics targeting the DDI2/NRF1 pathway to overcome acquired and de novo PI resistance and improve patient outcomes in MM. Citation Format: Tianzeng Chen, Matthew Ho, Jenna Briere, Maria Moscvin, Peter G. Czarnecki, Kenneth C. Anderson, T. Keith Blackwell, Giada Bianchi. Multiple myeloma cells depend on the NRF1-DDI2 proteasome stress response pathway for survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB110.

Proteasome Subunits Differentially Control Myeloma Cell Viability and Proteasome Inhibitor Sensitivity.

We generated eight multiple myeloma cell lines resistant to bortezomib; five acquired PSMB5 mutations. In 1,500 patients such mutations were rare clinically. To better understand disruption of proteasomes on multiple myeloma viability and drug sensitivity, we systematically deleted the major proteasome catalytic subunits. Multiple myeloma cells without PSMB5 were viable. Drug-resistant, PSMB5-mutated cell lines were resensitized to bortezomib by PSMB5 deletion, implying PSMB5 mutation is activating in its drug resistance function. In contrast, PSMB6 knockout was lethal to multiple myeloma cell lines. Depleting PSMB6 prevented splicing of the major catalytic subunits PSMB5, PSMB7, PSMB8, and PSMB10; however, PSMB6 engineered without splicing function or catalytic activity, also restored viability, inferring the contribution of PSMB6 to proteasome structure to be more important than functional activity. Supporting this, bortezomib sensitivity was restored in drug-resistant multiple myeloma cell lines by low level expression of mutated PSMB6 lacking splicing function. Loss of PSMB8 and PSMB9 was neither lethal nor restored bortezomib sensitivity. Significant codependency of PSMB5, PSMB6, and PSMB7 expression was observed. We demonstrated elevated levels of PSMB6 and 7, but not 8 and 9, in some, but not all, serial patient samples exposed to proteasome inhibitors. In summary, we show PSMB6 and PSMB7, but not PSMB5, to be essential for multiple myeloma cell survival, this dependency is structural and that upregulation or activating mutation of PSMB5, 6, and 7 confers proteasome inhibitor resistance, while depletion confers sensitivity. IMPLICATIONS: These findings support modulation of PSMB5, PSMB6, or PSMB7 expression as a new therapeutic strategy.

YWHAE/14-3-3ε expression impacts the protein load, contributing to proteasome inhibitor sensitivity in multiple myeloma

AbstractHigh protein load is a feature of multiple myeloma (MM), making the disease exquisitely sensitive to proteasome inhibitor (PIs). Despite the success of PIs in improving patient outcome, the majority of patients develop resistance leading to progressive disease; thus, the need to investigate the mechanisms driving the drug sensitivity vs resistance. With the well-recognized chaperone function of 14-3-3 proteins, we evaluated their role in affecting proteasome activity and sensitivity to PIs by correlating expression of individual 14-3-3 gene and their sensitivity to PIs (bortezomib and carfilzomib) across a large panel of MM cell lines. We observed a significant positive correlation between 14-3-3ε expression and PI response in addition to a role for 14-3-3ε in promoting translation initiation and protein synthesis in MM cells through binding and inhibition of the TSC1/TSC2 complex, as well as directly interacting with and promoting phosphorylation of mTORC1. 14-3-3ε depletion caused up to a 50% reduction in protein synthesis, including a decrease in the intracellular abundance and secretion of the light chains in MM cells, whereas 14-3-3ε overexpression or addback in knockout cells resulted in a marked upregulation of protein synthesis and protein load. Importantly, the correlation among 14-3-3ε expression, PI sensitivity, and protein load was observed in primary MM cells from 2 independent data sets, and its lower expression was associated with poor outcome in patients with MM receiving a bortezomib-based therapy. Altogether, these observations suggest that 14-3-3ε is a predictor of clinical outcome and may serve as a potential target to modulate PI sensitivity in MM.

The Contribution of Proteasome Subunits to Myeloma Cell Viability and Proteasome Inhibitor Sensitivity

Background Bortezomib (BTZ) is highly effective in the treatment of multiple myeloma; however, emergent drug resistance is common. The underlying mechanisms of such proteasome inhibitor resistance are still incompletely understood. Methods To further understand its resistant mechanism, we generated eight multiple myeloma (MM) cell lines resistant to bortezomib (BTZ) by exposure to increasing drug concentration: five of them acquired novel PSMB5 mutations. Given the rarity of similar mutations in over 1,500 analyzed MM patients, we explored in depth the role of the proteasome on MM cell viability and BTZ sensitivity by systematically deleting the major proteasome targets of BTZ by CRISPR. Results We demonstrated that MM cell lines without PSMB5 were surprisingly viable (mutation corresponding yeast gene pre2 is lethal). PSMB5 mutated, BTZ resistant, MM cell lines were re-sensitized to BTZ when PSMB5 was experimentally deleted, implying that this mutation is activating in its drug resistance function. In contrast PSMB6 knockout was lethal to MM cell lines, which were efficiently rescued by re-introduction of wild type PSMB6. Interestingly, reduction in PSMB6 levels also prevented the splicing of the major catalytic subunits PSMB5, PSMB7, PSMB8 and PSMB10. PSMB6 engineered with no splicing function or catalytic activity, also restored viability, inferring that the contribution of PSMB6 to proteasome structure is more important than functional activity. Supporting this observation, BTZ sensitivity was restored in resistant MM cells line by introducing low level expression of mutated PSMB6 lacking splicing function. As with PSMB6, PSMB7 knockout was lethal to MM cell lines. In contrast, loss of immunoproteasome subunits PSMB8 and PSMB9 was neither lethal nor restored sensitivity to BTZ. Our results demonstrate that expression of the three constitutive proteasome subunits PSMB5, PSMB6 and PSMB7 is highly co-dependent. This dependence is relying on the structure, but not the function, of PSMB5 and PSMB6. Conclusions In summary, PSMB5 and PSMB6, but not PSMB8 and PSMB9, are highly relevant for BTZ sensitivity in MM. Absence of PSMB6 or PSMB7, but not PSMB5, was lethal in MM cell lines. Expression of PSMB5, PSMB6 and PSMB7 was highly co-dependent. Together these findings suggest that the modulation of expression rather than function of PSMB5, PSMB6 or PSMB7 may be a new therapeutic strategy. Disclosures No relevant conflicts of interest to declare.

Targeting Glycolysis in Multiple Myeloma: Novel Strategies in the Treatment of Proteasome Inhibitor Resistant in Hypoxic Conditions

Introduction: Multiple myeloma (MM) is one of the hematological malignancy and characterized by the clonal expansion of plasma cells in the bone marrow. The treatment of MM patients has been dramatically changed by new agents such as proteasome inhibitors and immunomodulatory drugs, however, many patients will relapse even if new agents provide therapeutic advantages. Therefore, a new strategy is still needed to increase MM patient survival. Hypoxia is an important component of the bone marrow microenvironment. Hypoxia may increase myeloma cell survival. Because cells shift primarily to a glycolytic mode for generation of energy in hypoxic conditions, glycolytic activities can be targeted therapeutically in MM patients. The 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB) is responsible for maintaining the cellular levels of fructose-2,6-bisphosphate which is a regulator of glycolysis. Materials and Methods: In this study, we investigated whether PFKFB was involved in myeloma cells in hypoxia condition. We also investigated whether PFKFB inhibitors could suppress myeloma cells and enhance the sensitivity of myeloma cells to proteasome inhibition. Results: We first investigated the expression of PFKBP in the myeloma cell lines in hypoxia condition. PFKFB family contains four tissue-specific isoenzymes encoded by four different genes. We found expression of PFKBP3 and PFKBP4 were increased in hypoxia condition. We found gene expression of PFKBP3 and PFKBP4 were involved in myeloma cell lines and myeloma patient samples in hypoxia condition from the public microarray datasets (GSE80140 and GSE80545). In hypoxia condition, expression of hypoxia-inducible factor 1α (HIF1α) was increased and phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) was activated in myeloma cell lines. Expression of PFKBP3 and PFKBP4 were inhibited by HIF1α inhibitor and p38 MAPK inhibitor treatment. In the hypoxia condition, activity of proteasome inhibitors were reduced compared to normoxia condition. We next investigated whether PFKBP3 inhibitor, PFK158 and PFKBP4 inhibitor, 5MPN could inhibit the proliferation of myeloma cells. We found PFK158 and 5MPN treatment inhibited the growth of myeloma cells in a dose dependent manner in hypoxia condition. Combined treatment of myeloma cells with carfilzomib and PFK158 or 5MPN caused more cytotoxicity than each drug alone. Caspase 3/7 activity and cellular cytotoxicity was also increased. We found proteasomal activity was also reduced by carfilzomib and PFK158 or 5MPN treatment. Adenosine triphosphate (ATP) is the most important source of energy for intracellular reactions. Intracellular ATP levels drastically decreased after carfilzomib and PFK158 or 5MPN treatment. Because mitochondria generate ATP and participate in signal transduction and cellular pathology and cell death. The quantitative analysis of JC-1 stained cells changed mitochondrial membrane potential in cell death, which were induced by carfilzomib and PFK158 or 5MPN on myeloma cells. In the hypoxia condition and inhibitor treatment, glycolytic activities (e.g. glucose and lactate) were changed in myeloma cells. Conclusion: The PFKBP3 and PFKBP4 are enhanced in hypoxia condition and involved in proteasome inhibitor sensitivity. Our data also suggested that administration of PFKBP3 and PFKBP4 inhibitors may be a powerful strategy against myeloma cells and enhance cytotoxic effects of proteasome inhibitors in hypoxia condition. Disclosures No relevant conflicts of interest to declare.

Myeloma Cells Addicted to Glutamine for Biomass Production Are Sensitive to Lenalidomide

Introduction We have shown that in myeloma (MM) the immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide, in a CRBN dependent fashion, inhibit thioredoxin reductase and thus increase intracellular oxidative stress as a consequence of peroxide accumulation (Sebastian et al. 2017). Accordingly, we have also shown that CRBN expression alone does not fully correlate with lenalidomide sensitivity, as the cells ability to decompose H2O2 is important - MM cells with lower H2O2 decomposition capacity are more sensitive to IMiDs. We hypothezised that the specific metabolic pathways used by cells for biomass production would influence IMiD sensitivity since they also influence this cellular oxidative capacity. Cellular proteins are key elements of this biomass, and required for the proliferation of MM cells and production of immunoglobulin proteins. Methods We cultured MM cells under various nutrients conditions to understand its effect on cell proliferation and drug responses. Cell viability was assessed by MTT assay, the Luminescent Assay, and direct cell counting. MM cells glycolytic rate was measured using the Seahorse XF analyzer. Western blots were performed to quantify various protein expression levels in MM cells cultured under different nutrients conditions. CRBN CRISPR/Cas9 KO plasmid was used to generate CRBN knockout cells and a lentivirus system was used to over-express CRBN in MM cells. Results MM cells resistant to lenalidomide, but still expressing functional CRBN, show a higher glycolytic pathway use than sensitive cells. We found that the lenalidomide sensitive cell line MM1.S is highly dependent on extracellular glutamine for cell proliferation. In contrast, JJN3, a lenalidomide resistant cells line with wild type CRBN, preferentially consumes glucose. We found that glutamine depletion from culture media completely abolished lenalidomide sensitivity in MM1.S cell line. Western blot analysis revealed that antibody production by MM1.S was primarily dependent on glutamine availability, and that JJN3 antibody production was dependent upon glucose availability. While glutamine depletion from culture medium completely abolished lenalidomide sensitivity in MM1.S cell line, IKZF1, and IKZF3 degradation was unchanged. We further studied the role of CRBN in glutamine dependent cellular biomass protein production using isogenic cell lines (+/- wtCRBN). We found that wtCRBN expressing cells proliferate more and show higher antibody production in the presence of glutamine, over CRBN negative isogenic cell lines. Lenalidomide treatment further increased glutamine dependent antibody production in wtCRBN expressing cells. One possibile explanation is that activation of glutamine catabolism can facilitate protein demethylation via the supplementation α-ketoglutarate (α-KG). Therefore, we tested whether lenalidomide treatment could induce protein demethylation in sensitive cell lines. Western blot probed with mono-methyl lysine antibody showed lenalidomide induced protein demethylation. We further confirmed that protein demethylation, and lenalidomide sensitivity is a consequence of elevated α-KG by treating cells with cell-permeable 5-octyl- α-ketoglutarate which act as a substrate of the lysine demethylases. 5-Octyl-α-ketoglutarate treatment inhibited cell proliferation preferentially in wtCRBN expressing cells, and also enhanced lenalidomide induced sensitivity. Protein demethylation is associated with protein ubiquitination and proteasomal degradation. We thus hypothesize that lenalidomide induced protein demethylation also likely increases proteasome inhibitors sensitivity in MM. Conclusion MM cells with preferential glutamine consumption are likely to be more sensitive to lenalidomide, and extracellular glutamine depletion can induce lenalidomide resistance. MM cells expressing CRBN, but not dependent on glutamine for protein biomass production are more likely to be resistant to lenalidomide. Our study postulates that CRBN protein requirement in lenalidomide sensitivity cells is restricted to high glutamine dependency, and that it is quite likely CRBN has a role in glutamine metabolism. Moreover, MM cells that consume more glutamine are under higher oxidative stress and exhibit less H2O2 decomposition capacity and therefore increasing sensitivity to IMiDs. References Sebastian , S., et al., Blood 2017 129:991-1007 Disclosures Stewart: Ono: Consultancy; Roche: Consultancy; Seattle Genetics: Consultancy; Takeda: Consultancy; Amgen: Consultancy, Research Funding; Bristol Myers-Squibb: Consultancy; Celgene: Consultancy, Research Funding; Ionis: Consultancy; Janssen: Consultancy, Research Funding; Oncopeptides: Consultancy. Bergsagel:Janssen Pharmaceuticals: Consultancy; Ionis Pharmaceuticals: Consultancy; Celgene: Consultancy. Fonseca:AbbVie, Amgen, Bayer, Celgene, Kite, Janssen, Juno, Merck, Pharmacylics, Sanofi, Takeda: Other: Consultant/Advisor; Prognosticatin of MM based on Genetic Categorization by FISH: Patents & Royalties; Adaptive Biotechnologies: Other: Scientific Advisory Board.

The contribution of proteasome subunits to Myeloma cell viability and proteasome inhibitor sensitivity

We generated eight multiple myeloma (MM) cell lines resistant to bortezomib (BTZ) by exposure to increasing drug concentration: five of them acquired novel PSMB5 mutations. Given the rarity of similar mutations in over 1,500 analyzed MM patients, we explored in depth the role of the proteasome on MM cell viability and BTZ sensitivity by systematically deleting the major proteasome targets of BTZ. We demonstrated that MM cell lines without PSMB5 were surprisingly viable. PSMB5 mutated, BTZ resistant, MM cell lines were re-sensitized to BTZ when PSMB5 was experimentally deleted, implying that this mutation is activating in its drug resistance function.

YWHAE/14-3-3ε expression impacts the proteasome load contributing to proteasome inhibitor sensitivity in multiple myeloma

High protein load is a feature of multiple myeloma (MM), which makes this disease exquisitely sensitive to proteasome inhibition (PI). 14-3-3 are chaperone and scaffold proteins that exert a widespread influence on cellular processes through binding to serine/threonine-phosphorylated residues on target proteins. Here we report that loss of YWHAE, the coding gene for the 14-3-3ε protein, causes an imbalance in the protein load in MM cells potently impacting their sensitivity to PI.

Mitochondrial metabolism promotes adaptation to proteotoxic stress.

The mechanisms by which cells adapt to proteotoxic stress are largely unknown, but key to understanding how tumor cells, particularly in vivo, are largely resistant to proteasome inhibitors. Analysis of cancer cell lines, mouse xenografts and patient-derived tumor samples all showed an association between mitochondrial metabolism and proteasome inhibitor sensitivity. When cells were forced to use oxidative phosphorylation rather than glycolysis, they became proteasome inhibitor-resistant. This mitochondrial state, however, creates a unique vulnerability: sensitivity to the small-molecule compound elesclomol. Genome-wide CRISPR/Cas9 screening showed that a single gene, encoding the mitochondrial reductase FDX1, could rescue elesclomol-induced cell death. Enzymatic function and NMR-based analyses further showed that FDX1 is the direct target of elesclomol, which promotes a unique form of copper-dependent cell death. These studies elucidate a fundamental mechanism by which cells adapt to proteotoxic stress and suggests strategies to mitigate proteasome inhibitor-resistance.

Targeting Insulin-like Growth Factor in Multiple Myeloma: Novel Strategies in the Treatment of Proteasome Inhibitor Resistant Cells

Introduction: Multiple myeloma (MM) is one of the hematological malignancy that affects the plasma cells in bone marrow. The treatment of MM patients has been dramatically changed by new agents such as proteasome inhibitors and immunomodulatory drugs. However, most patients will relapse even if new agents provide therapeutic advantages. Therefore, a new strategy is still needed to increase MM patient survival. Insulin-like growth factor (IGF) cause intracellular signaling that ultimately results in cellular growth and proliferation. Because IGF signaling pathways have crucial functions in hematological malignancies and solid tumors, IGF pathways might regulate myeloma cell survival.Materials and Methods: In this study, we investigated whether IGF1R inhibitor could suppress myeloma cells. We also investigated whether IGF1 was involved in proteasome sensitivity against myeloma cells and cytokines were induced by IGF1 in human umbilical vein endothelial cells (HUVEC).Results: We first investigated the IGF signaling pathways by microarray gene expression data from the online Gene Expression Omnibus (GEO). IGF is tightly regulated by six related IGF-binding proteins (IGFBPs). Gene expression of IGF1, IGF1R, IGFBP2, IGFBP5, IGFBP7 and IGF2 are involved in myeloma and normal control samples from the public microarray datasets of GSE39754. We next investigated the proteasome inhibitor, bortezomib or carfilzomib activity. Proteasome inhibitors exhibit cell growth inhibition against myeloma cells. However, in the presence of IGF1, proteasome inhibitors activity was reduced. Linsitinib is an orally bioavailable small molecule inhibitor of the IGF1R. We investigated whether linsitinib could inhibit the proliferation of myeloma cells. We found linsitinib treatment inhibited the growth of myeloma cells in a dose dependent manner. Activity of Caspase 3/7 and cytotoxicity were also increased. Combined treatment of myeloma cells with carfilzomib and linsitinib caused more cytotoxicity than each drug alone. Caspase 3/7 activity and cellular cytotoxicity was also increased. We examined the intracellular signaling. Activity of caspase 3 and poly (ADP-ribose) polymerase (PARP) was increased after carfilzomib and linsitinib treatment. We next blocked IGF1R function by small interfering RNA (siRNA). We found drug sensitivity of the cells to the carfilzomib was increased compared to mock-transfected cells. Caspase 3/7 activity and cytotoxicity were increased after carfilzomib treatment in siRNA transfected cells.We next investigated the in vitro effects of IGF1 on cultured HUVEC. Because the entire bone marrow cavity is hypoxic condition, we investigated the IGF signaling pathways in the hypoxic condition by microarray gene expression data from GEO. Gene expression of IGF1R and IGF2 are increased in the hypoxia from the public microarray datasets of GSE36837. Gene amplification was confirmed by RT-PCR analysis. In the immunoblot analysis, p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) phosphorylation was increased by IGF1 stimulation in the time dependent manner. We also found that gene expression of monocyte chemotactic protein-1 (MCP-1) was enhanced by IGF1 stimulation.Conclusion: The IGF1 signaling pathway is involved in proteasome inhibitor sensitivity and plays a key role in chemokine production of the HUVEC. Our data also suggested that administration of IGF1R inhibitor, linsitinib, might be a powerful strategy against myeloma cells and enhance cytotoxic effects of proteasome inhibitors in those residual MM cells. DisclosuresOhyashiki:MSD,: Honoraria, Research Funding; Bristol Meyer Squibb KK,: Honoraria, Research Funding; Kyowakko Kirin KK,: Research Funding; Celegene KK,: Honoraria, Research Funding; Pfizer KK,: Honoraria, Research Funding; Novartis KK,: Honoraria, Research Funding; Taiho Pharmaceutical KK: Honoraria, Research Funding; Takeda Pharmaceutical KK,: Honoraria, Research Funding; Jansen Pharma KK,: Research Funding; Chugai KK,: Honoraria, Research Funding; Nihon-Seiyaku,: Research Funding; Ono Pharmaceutical KK,: Honoraria, Research Funding; Eizai,: Research Funding; Nippon-shinyaku,: Honoraria, Research Funding; Asahikase: Research Funding; Asteras KK,: Research Funding; Dainippon Sumitomo KK,: Honoraria, Research Funding.

Exosome-Transmitted PSMA3 and PSMA3-AS1 Promotes Proteasome Inhibitors Resistance in Multiple Myeloma

PIs resistance is a major challenge for multiple myeloma (MM). The bone marrow microenvironment facilitates crucial interactions between the myeloma cells and mesenchymal stem cells (MSCs) that permit MM to survive and proliferate progression. Exosomes are involved in intercellular communication, and in this study we investigated how the transfer of exosomic PMSA3 (encodes proteasome subunit α7) and lncPSMA3-AS1 from MSCs to MM cells affected proteasome inhibitors resistance (Figure 1).We firstly underscored that exosomes derived from r-MSCs (MSCs derived from bortezomib-resistant patients), but not from s-MSCs (MSCs derived from bortezomib-resistant patients) reduced the proteasome inhibitors sensitivity in MM cells (Figure 2).To further elucidate mechanisms of Proteasome inhibitors (PIs) resistance, we retrieved a database containing gene expression profile of 169 myeloma cases with clinical response and disease prognosis (GSE9782). The analysis of this dataset showed that the mRNA levels of PSMA3 and PSMA3-AS1 in CD138+ cells are upregulated in bortezomib-resistant patients (Figure 3A-3D). Moreover, Kaplan-Meier analysis showed that high PSMA3 levels in CD138+ MM cells were correlated with reduced progression-free survival (PFS) (p = 0.0307) and overall survival (OS) (p = 0.0328) (Figure 3E). Cox proportional hazards regression analysis further demonstrated that high PSMA3 was an independent prognostic factor for MM patients with bortezomib therapy in a multivariate analysis (p = 0.0013, HR = 1.3104, 95%CI = 1.1113-1.545). Further analysis of Oncomine data showed that the PSMA3 levels appeared a progressive increase in MGUS, SM, MM and PCL (Figure 3F-3H). Similarly, our PIs resistant models (U266BR, U266CR, U266IR, MM.1SBR, MM.1SCR, MM.1SIR) consistently displayed up-regulation of PSMA3 and PSMA3-AS1 expression (Figure 3J). Consistent with this previously published study, our clinical data showed that the mRNA levels of PSMA3 and PSMA3-AS1 are upregulated in CD138+ MM cells derived from bortezomib resistant patients relative to those from bortezomib sensitive patients (Figure 3I). In addition, r-MSCs had increased expression of PSMA3 and PSMA3-AS1 compared to s-MSCs (Figure 3K). Moreover, the expression of PSMA3 and PSMA3-AS1 in MSCs were positively correlated with that in CD138+ myeloma cells (Figure 3L). These data suggested that high levels of PSMA3 and PSMA3-AS1 were correlated with proteasome inhibitors resistance in MM.We further identified that PSMA3 and PSMA3-AS1 in MSCs could be incorporated into exosomes and transmitted to myeloma cells, thus promoting PIs resistance (Figure not shown).PSMA3-AS1 was capable of forming an RNA duplex with PSMA3 pre-mRNA at overlapping regions and this duplex transcriptionally promoted PSMA3 expression by increasing its stability, conferring bortezomib resistance to myeloma cells (Figure not shown).To evaluate the therapeutic potential of PSMA3-AS1 in MM in vivo, bioluminescent MM models (U266-luc), which recapitulates the clinical sequelae, anatomic distribution of MM lesions, and hallmark bone pathophysiology observed in MM patients were established. Intravenously administered siPSMA3-AS1 was found to be effective in increasing bortezomib sensitive (Figure 4).Moreover, circulating exosomal PSMA3 and PSMA3-AS1 derived from the plasma of MM patients were significantly associated with both progression-free survival (PFS) and overall survival (OS) in the univariate analysis, and were still statistically significant after adjusting for the international staging system (ISS) and several other clinical variables in the multivariate analysis (Figure not shown).In summary, our results indicated a unique role of exosomic lncPSMA3-AS1 in transferring proteasome inhibitors resistance from MSCs to MM cells, through a novel exosomic lncPSMA3-AS1/PSMA3 signaling pathway. Exosomic PSMA3 and PSMA3-AS1 may serve as a potential therapeutic target for proteasome inhibitors resistance and a prognostic predictor for clinical response. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

(Immuno)proteasomes as therapeutic target in acute leukemia

The clinical efficacy of proteasome inhibitors in the treatment of multiple myeloma has encouraged application of proteasome inhibitor containing therapeutic interventions in (pediatric) acute leukemia. Here, we summarize the positioning of bortezomib, as first-generation proteasome inhibitor, and second-generation proteasome inhibitors in leukemia treatment from a preclinical and clinical perspective. Potential markers for proteasome inhibitor sensitivity and/or resistance emerging from leukemia cell line models and clinical sample studies will be discussed focusing on the role of immunoproteasome and constitutive proteasome (subunit) expression, PSMB5 mutations, and alternative mechanisms of overcoming proteolytic stress.

Increased expression of the tight junction protein TJP1/ZO-1 is associated with upregulation of TAZ-TEAD activity and an adult tissue stem cell signature in carfilzomib-resistant multiple myeloma cells and high-risk multiple myeloma patients.

Tight junction protein 1 (TJP1) has recently been proposed as a biomarker to identify multiple myeloma (MM) patients most likely to respond to bortezomib- and carfilzomib-based proteasome inhibitor regimens. Herein we report increased expression of TJP1 during the adaptive response mediating carfilzomib resistance in the LP-1/Cfz MM cell line. Moreover, increased TJP1 expression delineated a subset of relapsed/refractory MM patients on bortezomib-based therapy sharing an LP-1/Cfz-like phenotype characterized by activation of interacting transcriptional effectors of the Hippo signaling cascade (TAZ and TEAD1) and an adult tissue stem cell signature. siRNA-mediated knockdown of TJP1 or TAZ/TEAD1 partially sensitized LP-1/Cfz cells to carfilzomib. Connectivity Map analysis identified translation inhibitors as candidate therapeutic agents targeting this molecular phenotype. We confirmed this prediction by showing that homoharringtonine (omacetaxine mepesuccinate) — the first translation inhibitor to be approved by the U.S. Food and Drug Administration — displayed potent cytotoxic activity on LP-1/Cfz cells. Homoharringtonine treatment reduced the levels of TAZ and TEAD1 as well as the MM-protective proteins Nrf2 and MCL1. Thus, our data suggest the importance of further studies evaluating translation inhibitors in relapsed/refractory MM. On the other hand, use of TJP1 as a MM biomarker for proteasome inhibitor sensitivity requires careful consideration.

Glutaminase Inhibitor CB-839 Enhances Proteasome Inhibitor Sensitivity in Multiple Myeloma Cells

Multiple myeloma (MM) remains largely incurable due to the emergence of therapeutic resistance. We therefore set out in this study to identify druggable molecular mechanisms that convey resistance to proteasome inhibitors (PIs; e.g., bortezomib/VELCADE, carfilzomib/KYPROLIS), which are cornerstone agents in the treatment of MM. In comparing isogenic pairs of PI sensitive and resistant cells, we observed stark differences in cellular bioenergetics between the divergent phenotypes. While glycolysis rates between cell lines were similar, PI resistant cells exhibited increased mitochondrial respiration characterized by higher basal oxygen consumption rates (OCR) and overall respiratory capacity. Additionally, PI resistant cells were found to have lower activation of the AMP kinase (AMPK) energy stress pathway and increased levels of NAD(P)H, which serve as electron carriers in the mitochondrial process of oxidative phosphorylation. We determined that glutamine was the principle source of fuel driving mitochondrial respiration as removal of glutamine completely inhibited OCR as well as cell proliferation, whereas glucose and pyruvate were dispensable. Given the propensity for mitochondrial respiration in PI resistant cells and the dependence on glutamine for this process, we hypothesized that targeting glutamine utilization by PI resistant cells would restore their sensitivity to the cytotoxic effects of PIs. To test this possibility, we inhibited glutamine metabolism using the small molecule GLS1 inhibitor CB-839. CB-839 repressed basal OCR and total respiratory capacity and reduced cell viability to varying degrees in a panel of PI sensitive and resistant MM cell lines. Most notably, we found that CB-839 synergistically enhanced the cytotoxic activity of multiple PIs, including bortezomib, carfilzomib, ixazomib, and oprozomib, in a genetically diverse panel of 15 PI sensitive and resistant MM cell lines. The effects of CB-839 were the most apparent in combination with carfilzomib (Crflz), where it enhanced Crflz-induced death by >4-fold. CB-839 enhanced Crflz-induced apoptosis as measured by the activation of caspase 3, 7, 8 and the cleavage of the caspase-3 substrate PARP. Mechanistically, the combination of CB-839 and Crflz induces a strong and synergistic ER stress response, characterized by the induction of ATF4 and CHOP. Our findings suggest that the acquisition of PI resistance may involve adaptations in cellular bioenergetics that may be exploited therapeutically by targeting glutamine metabolism. Furthermore, our results support the combination of clinical stage compound CB-839 with PIs, particularly Crflz, for the treatment of refractory MM. DisclosuresNo relevant conflicts of interest to declare.

Tight Junction Protein 1: New Insights into Proteasome Inhibitor Resistance and Myeloma Pathophysiology

In this issue of Cancer Cell, Zhang etal. report that TJP1 suppresses EGFR/JAK1/STAT3-mediated signaling and increases the proteasome inhibitor sensitivity of myeloma cells by altering the cellular proteasome capacity versus proteasome load of undegraded intracellular proteins.