Overcome 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.

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.

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.

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.

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.

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.

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.

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.

26S Proteasome Non-Atpase Subunit 1, PSMD1/Rpn2, Is a Potential Therapeutic Target in Multiple Myeloma

Background and Rationale Multiple myeloma (MM) is a cancer of the plasma cells that involves the production of a large amount of immunoglobulins, making MM cells sensitive to disturbances in their proteolytic pathways. Yet, they often develop resistance to conventional proteasome inhibitors (PIs). The 26S proteosome is made of the 20S core particle (CP), which degrades proteins and is targeted by PIs, and the 19S regulatory particle (RP), which recognizes ubiquitinated proteins and feeds them into the 20S CP. Here, we focused on identifying and validating novel therapeutic targets within the 19S RP to overcome the PIs resistance in MM. PSMD1/Rpn2, the non-ATPase regulatory subunit 1 is a key structural component of 19S RP. In the current study, we utilized our preclinical in vitro and in vivo models to show that targeting Rpn2 triggers anti-MM activity and overcomes PI-resistance. Results The largest non-ATPase subunit, Rpn2 which is a scaffolding protein and is responsible for the docking of other proteasome subunits within 19S RP. Our immunoblot analysis showed higher Rpn2 expression in MM versus normal peripheral blood mononuclear cells. Immunohistochemistry studies showed significantly higher Rpn2 expression in BM biopsies from MM patients than from healthy individuals. Knocking down Rpn2 using transiently transfected siRNA decreased the viability of various MM cell lines, as assessed by WST-1 assays, including cell lines that are PI-resistant (ANBL6.BR) or carry p53 alterations (JJN3, ARP1) (p< 0.01). Both stable inducible Rpn2-KD and inducible Rpn2-KO AMO1 cells had significantly reduced cell growth. Importantly, transfection with Rpn2-WT rescued cells from the growth-inhibitory activity of Rpn2 siRNA. Rpn2 blockade increased the level of k48-ubiquitinated proteins, reflecting inhibition of proteasome-mediated protein degradation, and caused cell death through cell cycle arrest, apoptosis associated with the activation of caspases, and endoplasmic reticulum stress response signaling. Finally, using Dox-inducible Rpn2-KD AMO1 MM cells in a xenograft mouse model, we found that Rpn2 depletion reduced tumor growth and prolonged mouse survival (p < 0.005). Conclusion Our preclinical data demonstrate the therapeutic potential of targeting Rpn2 and provide the preclinical basis for developing Rpn2 inhibitors to overcome PI resistance in MM.

P-100: Allosteric HSP70 inhibitors perturb mitochondrial proteostasis and overcome proteasome inhibitor resistance in multiple myeloma

P-100: Allosteric HSP70 inhibitors perturb mitochondrial proteostasis and overcome proteasome inhibitor resistance in multiple myeloma

Allosteric HSP70 inhibitors perturb mitochondrial proteostasis and overcome proteasome inhibitor resistance in multiple myeloma

Proteasome inhibitor (PI) resistance remains a central challenge in multiple myeloma. To identify pathways mediating resistance, we first mapped proteasome-associated genetic co-dependencies. We identified heat shock protein 70 (HSP70) chaperones as potential targets, consistent with proposed mechanisms of myeloma cells overcoming PI-induced stress. We therefore explored allosteric HSP70 inhibitors (JG compounds) as myeloma therapeutics. JG compounds exhibited increased efficacy against acquired and intrinsic PI-resistant myeloma models, unlike HSP90 inhibition. Shotgun and pulsed SILAC mass spectrometry demonstrated that JGs unexpectedly impact myeloma proteostasis by destabilizing the 55S mitoribosome. Our data suggest JGs have the most pronounced anti-myeloma effect not through inhibiting cytosolic HSP70 proteins but instead through mitochondrial-localized HSP70, HSPA9/mortalin. Analysis of myeloma patient data further supports strong effects of global proteostasis capacity, and particularly HSPA9 expression, on PI response. Our results characterize myeloma proteostasis networks under therapeutic pressure while motivating further investigation of HSPA9 as a specific vulnerability in PI-resistant disease.

Autophagy Blockade Disrupts Myeloma Cell Recovery from Proteasome Inhibition and Enhances Apoptosis

BackgroundExtensive protein synthesis in multiple myeloma (MM) cells renders them vulnerable to proteasome inhibitors (PI), a cornerstone of anti-myeloma therapy. However, relapse is inevitable and PI resistance remains a major barrier to improving outcomes. Adaptive responses to PI include upregulation of autophagy, another major degradation pathway in cells. We hypothesised that autophagy inhibition increases cell death and impedes recovery of MM cells exposed to Carfilzomib (K), a second generation PI effective in newly diagnosed and relapsed/refractory MM.Methods:Human myeloma cell lines (HMCL) and patient samples were exposed to a 1-hour pulse of K to mimic pharmacokinetics, ± autophagy inhibitors (AI) vacuolar protein sorting kinase 34 inhibitor (VPS34i) or unc-51 like autophagy initiating kinase inhibitor (ULKi). Apoptosis was assessed by Annexin V/Propidium iodide (AnV/PI) flow cytometry (FC) and autophagic flux by western blotting (WB) and FC for light chain 3B (LC3B) ± bafilomycin (B). Cell cycle was examined by FC using KI67/PI.Results:Basal autophagic flux was seen in all HMCL (MM1s, H929, KMS12BM) tested (±B). Autophagic flux increased in response to K: MM1s mean fluorescence intensity (MFI) control (CTL) 318±28 vs K 567±72, (mean±SEM),p=0.01, n=6). Autophagy was inhibited in all 3 HMCL with AI. Combined exposure to K and AI (VPS34i or ULKi) increased time dependent apoptosis in all HMCL tested: MM1s 31±8% (K) vs 58±9% (K+VPS34i), p=0.03, (n=3), and 13±0.4% (K) vs 63±16% (K+ULKi), p=0.03 (n=3), (K 10nM, AI 1uM) 48 hours(h) post K exposure.Basal autophagic flux was detected in all CD138+ primary MM patient samples tested by LC3B FC assay (MFI CTL B- 407±57 vs B+ 1081±125, p<0.0001, n=14). This increased in response to K: CTL B- 407±57 vs K B- 862±224, p=0.03, n=14. VPS34i alone reduced viable cell numbers in 26/33 primary samples (% viable cells relative to starting viable MM cell number at day 0: CTL 99±14% vs VPS34i 71±12%, p<0.0001 at 48h), and cell loss correlated with basal autophagic flux (r=0.9, Pearson's correlation coefficient, p=0.0003, n=10). Addition of VPS34i to K increased loss of viable CD138+ cells (n=33) (Fig 1). Both apoptosis with K and enhanced cell death with K+VPS34i correlated with basal autophagic flux (r=0.8, p=0.0005 and r=0.8, p<0.0001 respectively), but no correlation was seen with previous therapy, disease stage, or adverse risk by cytogenetics or ISS stage.K treated MM1s cells were examined for kinetics of ubiquitinated protein build up and autophagic induction by WB. Increased ubiquitinated proteins were detectable up to 24h post K pulse, whilst autophagic flux increased up to 72h but returned to baseline thereafter (n=3). MM1s cells were treated with K±VPS34i for up to 7 days and apoptosis, absolute cell number and cell cycle assessed. In K treated cells, apoptosis was maximal at 72h but declined thereafter. Viable cell numbers fell initially but increased after 96h, indicating cell recovery. However, in MM1s cells treated with K+VPS34i/ULKi, apoptosis was enhanced with a rapid loss of viable cells (Fig 2) and viable cell numbers remained low at all timepoints (Fig 3). Cell cycle assays confirmed persistent G0/G1 arrest in MM1s cells treated with K+VPS34i or ULKi at all timepoints, whilst cell proliferation recovered in K treated cells on day 3, (15±3% cells in S/G2M in K treated cells vs 2±0.4% with K+VPS34i, p=0.02, n=4, 11±3% in S/G2M with K vs 1± 0.7% with K+ULKi, p=0.04, n=3, day 4)(Fig 4). To confirm that effects of AI were mediated via autophagy, KMS28PE (ATG12 null HMCL) were treated with K±VPS34i. These autophagy depleted cells showed limited basal or K induced autophagic flux and addition of VPS34i to K had no effect on apoptosis or cell cycle.ConclusionMM cells displayed active autophagic flux that increased with K treatment. Inhibition of autophagy increased K induced apoptosis, prolonged K-induced cell cycle arrest and prevented MM cell recovery. Addition of AI to K increased cytotoxicity in patient samples and many patients showed sensitivity to AI alone; these effects correlated with basal autophagic flux. These data suggest that autophagy plays a vital role in cell recovery from proteasome inhibition independent of proteotoxic stress. Dual inhibition of the proteasome and autophagosome may therefore help overcome PI resistance, which remains an unmet need in MM. [Display omitted] DisclosuresAuner: Takeda, Karyopharm: Other: Advisory role; Amgen: Research Funding; Janssen: Speakers Bureau. Khwaja: Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astellas: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Yong: Sanofi: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Takeda: Honoraria; GSK: Honoraria; Amgen: Honoraria; BMS: Research Funding; Autolus: Research Funding.

Genomic Characterization of in Vitro Acquired-Resistance to Proteasome Inhibitors

Multiple myeloma (MM) is the second most common hematological malignancy characterized by an abnormal clonal proliferation of malignant plasma cells. Despite the introduction of novels agents that have significantly improved clinical outcomes, MM patients invariably relapse. A better understanding of the drug resistance mechanisms and development of biomarkers remain of major interest to improve the treatment of patients.In order to further investigate the mechanisms involved in resistance to proteasome inhibitors (PI), we have derived and characterized PI-resistant human myeloma cells lines (HMCLs) from different molecular subgroups including XG2BR t(12;14), XG7BR t(4;14), XG19BR t(14;16), and XG1BR t(11;14). These cell lines were cultured continuously with escalating concentrations of Bortezomib (Btz) during 12 months and showed a significant resistance to Bortezomib compared to their parental cell lines (mean IC50: Btz-resistant HMCLs =5.5nM vs parental HMCLs=2.5nM, p<0.05). Of interest, we demonstrated that Btz-resistant HMCLs are also significantly more resistant to Carfilzomib (Cfz) and Ixazomib (Ixa) PIs (mean IC50: Btz-resistant HMCLs =6nM for Cfz and =70nM for Ixa vs parental HMCLs=3nM, for Cfz, p<0.05 ; and =21nM for Ixa, p<0.05. No significant cross-resistance was observed with other therapeutic agents including melphalan, dexamethasone and IMIDs indicating that the observed drug resistance mechanisms are specifically related to PIs.In order to understand the PIs resistance mechanisms acquired by MM cells, we used a combination of genomic approaches including whole genome sequencing, and comparative transcriptomic analysis.Among the 40 mutations identified in Btz-resistant HMCLs compared to the parental ones, a mutation residing in the Btz-binding pocket in the proteasome beta5-subunit (PSMB5) gene was identified. This mutation has already been found in other models of PIs-resistant MM cell lines and in relapsed MM patients, and is associated to PIs- resistance by reducing the PI binding capacity and impairing the chemotrypsin-like catalytic activity of the 20S proteasome.When we compared the gene expression profiling of Btz-resistant HMCLs with the parental ones, we identified a gene expression signature significantly enriched in nucleotide excision repair (NER) pathway with an increased expression of ERCC1, ERCC5, LIG1, POLD1 in the Btz-resistant HMCLs (FC >1.5). Since, protein ubiquitination is essential in regulation and coordination of various pathways of DNA damage recognition, signaling and repair, proteasome inhibitors affect DNA repair, overexpression of DNA repair pathways may participate in drug resistance mechanisms.Using gene expression profiling data, we also identified a significant downregulation of 8 solute carrier protein (SLC) intake transporters (SLC6A6, SLC16A1, SLC16A14, SLC16A10, SLC25A13, SLC5A6, SLCO3A1, SLCO4A1) together with a significant upregulation of xenobiotic receptors (RXRA, RXRB) in Btz-resistant HMCLs compared to parental HMCLs (FC> or < 1.5; p value <0.05). In addition, several genes involved in antioxidant response (NQO1), and in glutathione regulation (MGST1, MGST2, GSTO1) were also overexpressed in Btz-resistant HMCLs (FC>1.5 p value <0.05).Investigating the deregulated genes involved on energy metabolism that is often associated with resistance, we identified an upregulation of glycolytic enzymes directly involved in glycolytic metabolism (ALDOC, ENO3, HK1, PDK1, PDK3, PFKB3, PFKB4, PFKL, SLC2A1 (FC>1.5 p value <0.05) in the Btz-resistant HMCLs.Altogether our data underline a significant deregulation of genes involved in cell metabolism and drug clearance system that allow the PI resistant-MM cells to maintain metabolic homeostasis and survival in stringent redox conditions. This is in accordance with described mechanisms linking drug resistance and glycolytic metabolism in cancers cells. Metabolomic analyzes are currently ongoing for functional validation.Altogether, drug-resistant cell lines represent an attractive preclinical model to test molecules targeting these pathways in order to identify new therapeutic strategies to overcome PI resistance in MM. DisclosuresDe Boussac: Diag2Tec: Current Employment. Kassambara: Diag2Tec: Consultancy. Machura: Diag2Tec: Current Employment. Chemlal: Diag2Tec: Current Employment. Vincent: Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees. Herbaux: Abbvie: Honoraria, Research Funding; Roche: Honoraria; Janssen: Honoraria; Takeda: Honoraria, Research Funding. Bruyer: Diag2Tec: Current Employment. Moreaux: Diag2Tec: Consultancy.

P-093: Ubiquitin receptor PSMD4/Rpn10 as therapeutic target in Multiple Myeloma

<h3>Background</h3> Our prior studies showed that targeting Ubiquitin Receptor (UbR) Rpn13 upstream of the 20S proteasome signaling cascade overcomes proteasome-inhibitor (PI)-resistance (Yan et al. Leukemia 2016; Yan et al, 2021 BCJ–please complete reference details). Besides Rpn13, 19S-associated UbR PSMD4/Rpn10 also plays a key role in chaperoning ubiquitinated substrate proteins for downstream 20S proteasomal degradation. In the present study, we show that inhibition of PSMD4 triggers potent anti-MM activity using both in in vitro and in vivo models of MM, including against PI-resistant MM cells. <h3>Methods</h3> Cell viability, and apoptosis assays were performed using WST/ CellTiter-Glo assay, and Annexin V staining, respectively. PSMD4 knockout 293 cell line was generated using CRISPR/Cas9. Doxcycycline (Dox)-inducible PSMD4-knockdown (KD) MM cell line was generated using short hairpin RNA (shRNA). A xenograft human MM model was used to characterize the role of PSMD4 on tumor progression. Statistical significance was assessed with Student's t test. <h3>Results</h3> 1) MM patient gene expression profiling database showed that PSMD4 expression inversely correlates with overall survival (n=175; p=0.00064). 2) RT-PCR, immunoblotting, and immunohistochemistry of MM patient BM showed higher PSMD4 levels in patient MM cells and MM cell lines versus normal cells. 3) Transient transfection of MM cells including, bortezomib-resistant MM cells with PSMD4-siRNA decreased their viability; conversely, transfection with PSMD4-WT rescued cells from growth-inhibitory activity of PSMD4-siRNA. Western blotting confirmed knockdown of PSMD4 by PSMD4-siRNA versus scrambled (scr)-siRNA, and restoration of PSMD4 levels in cells transfected with PSMD4-WT versus PSMD4-siRNA. 4) CRISPR/Cas9-PSMD4-KO showed reduced cell growth. 5) Both PSMD4-KO and PSMD4-KD cells showed elevated levels of polyubiquitylated proteins, indicating blockade of proteasome-mediated protein degradation; 6) PSMD4 blockade triggered apoptosis, cell-cycle arrest, activation of caspases, and endoplasmic reticulum stress response signaling in MM cells; and finally, 7) Transplantation of Dox-inducible PSMD4-KD MM.1S cells in mice correlated with significantly reduced tumor growth. <h3>Conclusion</h3> Our preclinical data validates targeting UbR PSMD4/Rpn10 to enhance cytotoxicity and overcome PI resistance in MM, as well as provides the basis for the development and clinical evaluation of PSMD4 inhibitors to improve patient outcome.

P-092: Multiple Myeloma cells depend on the DDI2/NRF1-mediated proteasome stress response for survival

<h3>Background</h3> Multiple myeloma (MM) cells depend on the ubiquitin-proteasome system for survival. Proteasome inhibitors (PIs) are FDA-approved in MM and have radically improved patient survival. However, acquisition of resistance is inevitable. The proteasome stress response (PSR) contributes to proteostasis 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, it is deglycosylated by NGLY1, cleaved by the aspartic protease DDI2, and translocates to the nucleus, to induce proteasome subunit gene transcription. Pharmacological targeting of NGLY1 was recently shown to sensitize cells to PIs. <h3>Hypothesis</h3> We hypothesize that blocking DDI2 or NRF1 could target an intrinsic vulnerability of MM and represent an innovative therapeutic strategy to overcome PI resistance. <h3>Methods</h3> We used MM cell lines with distinct baseline sensitivity to PIs, including AMO1-VR, an isogenic AMO1 cell line adapted to grow in continuous bortezomib. NRF1 cleavage and nuclear localization were assessed via western blotting. We used CRISPR-Cas9 to knock out (KO) DDI2/NRF1. Cell viability or tumor growth of DDI2/NRF1 KO versus non-targeting gRNA-edited cells 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. Proteasome subunit PSMA7, PSMB5, PSMB6 and PSMD11 transcription was evaluated via real time PCR. <h3>Results</h3> Full length and cleaved NRF1 is detectable in MM cell lines and positively correlates with polyubiquitinated proteins, suggesting constitutively active PSR in MM. DDI2/NRF1 KO is cytotoxic alone or in combination with PI carfilzomib in MM cells with distinct PI sensitivity, including de-novo PI-resistant KMS20. In vivo, DDI2 KO leads to reduced plasmacytoma formation in NSG mice and results in prolongation of animal survival. DDI2 KO in AMO1-VR sensitizes to carfilzomib. Further, DDI2 KO blocks NRF1 cleavage and nuclear import, thereby impairing proteasome subunit transcription and CT-L proteasome activity in baseline conditions and following carfilzomib treatment, resulting in increased sensitivity to PI. Wild-type, but not catalytically-dead DDI2 addback rescues these phenotypes, confirming a causative link. <h3>Conclusions</h3> MM cells exhibit baseline activation of NRF1 and are dependent upon DDI2 for survival. DDI2 KO blocks NRF1 cleavage and nuclear translocation, causing impaired proteasome subunit biogenesis and recovery of CT-L proteasome activity, thereby increasing sensitivity to PI. Add-back of wild-type, but not of catalytically-dead DDI2 fully rescues these phenotypes. Our study provides the preclinical rationale for development of novel therapeutics targeting DDI2/NRF1 in MM.

Inhibition of p62-ZZ Domain-Mediated Signaling Overcomes Bortezomib Resistance in Multiple Myeloma Cells Independent of Their p53 Status

Multiple myeloma (MM) cells depend on autophagy as well as proteasome activity for their survival. In MM cells, autophagy is essential to reduce the extraordinary ubiquitinated protein system stress derived from high levels of immunoglobulin synthesis. Thus, proteasome inhibitors (PIs) decrease tumor burden in the majority of MM patients but eventually a PI resistant MM clone(s) arises. Bortezomib (BTZ) resistance has been reported to correlate with p53 status of MM cells. Previous studies showed that MM patients harboring p53 genetic abnormalities are commonly resistant to standard therapies, increasing the need for new druggable targets.A possible mechanism of BTZ resistance involves the cargo receptor for ubiquitin-mediated autophagy SQSTM1/p62, that enhances MM cells survival by increasing their autophagic flux. We found that MM cells with wild type p53, are more sensitive to PI treatment, while MM cells harbouring mutant p53 or lacking p53 are more resistant to BTZ-mediated cytotoxicity. BTZ treatment induced a modest level of apoptosis in MM1.S cells (p53 wild type) at high doses (>5nM) by triggering the cleavage of caspase 3 and PARP-1. As expected BTZ treatment induced a significant compensatory increase in SQSTM1/p62 at the mRNA and protein levels in these cells. Conversely, BTZ treatment of p53 mutant MM cells failed to induce apoptosis, exhibiting only a mild effect on viability. This occurred in spite of a similar increase in SQSTM1/p62 expression as seen in MM.1S cells.To determine the role of p62 in the BTZ resistance of p53 mutant MM cells, we tested a novel small-molecule p62-ZZ domain inhibitor, XRK3F2 we recently identified. XRK3F2 treatment of mice with established MM, dramatically increased bone formation in MM-bearing bones, although it did not affect tumour burden in vivo . In vitro studies showed that high concentrations (10μM) of XRK3F2 were required to decrease the viability of primary and MM cell lines and induce a time-dependent activation of caspases 3, 7 and 9. XRK3F2 treatment dose-dependently increased levels of the lipidated form of LC3, LC3II, suggesting activation of pro-survival autophagy. Surprisingly, the expression levels of SQSTM1/p62 were either unchanged or increased following XRK3F2 treatment despite the increase of autophagosome formation, indicating that XRK3F2 inhibited the autophagic flux in MM cells. Defective autophagy occurs in tumor cells and leads to accumulation of p62, further promoting tumorigenesis by altering NFκB regulation and gene expression. We found that XRK3F2 treatment also significantly inhibited TNFα-induced NFκB activation, by inhibiting p65 nuclear translocation. These data suggested that XRK3F2 may sensitize MM cells to BTZ-induced cytotoxicity and possibly overcome BTZ resistance.To test this hypothesis, human MM cell lines MM1.S (p53 wild type), U266 (p53 mutant) and KMS-11 (p53 null) were exposed to different doses of a BTZ-XRK3F2 combination comprised of varying concentrations of BTZ combined with XRK3F2 at 5μM. We found treatment with the BTZ-XRK3F2 combination significantly reduced cell viability compared with either drug alone at the same doses, and increased MM cells sensitivity to BTZ, independent of their p53 status (IC50 BTZ vs BTZ-XRK3F2 for MM1.S 3 vs 1.5nM of BTZ; for U266 10 vs 1.7nM of BTZ; for KMS-11 7 vs 1nM BTZ).Mechanistic studies revealed that in p53 wild type MM cells, the combination (Bortezomib 3nM - XRK3F2 5 μM) significantly increased annexin V positive cells after 6h of treatment. This was followed by a reduction in expression of the anti-apoptotic Bcl-2 protein (mRNA and protein levels) and extensive activation of caspase 3 and cleaved PARP1 after 24h, demonstrating apoptosis-mediated cell death. Importantly, p62 expression was significantly reduced by the BTZ-XRK3F2 combination. The decrease in p62 level may result as a consequence of its caspase 3-dependent degradation. In p53 mutant cells, the combination significantly induced PARP1 cleavage, although only mildly increased caspase 3 activation. These results imply that the BTZ-XRK3F2 combination activates multiple parallel death pathways to enhance both apoptosis and necrosis.Taken together, these data suggest that p62-ZZ antagonists, alone or in combination with PIs, may have both anti-tumor and bone anabolic effects, and may overcome PI resistance in MM cells, making p62 an attractive target for the treatment of MM. DisclosuresXie:Oxis Biotech: Consultancy. Roodman:Amgen Denosumab Trial: Membership on an entity's Board of Directors or advisory committees.

Nelfinavir Overcomes Proteasome Inhibitor Resistance in Multiple Myeloma By Modulating Membrane Lipid Bilayer Composition and Fluidity

INTRODUCTION Nelfinavir is a highly lipophilic, first generation HIV-protease inhibitor (HIV-PI) approved for HIV treatment. It has largely been replaced by next-generation HIV-PI with increased specificity and efficacy for HIV therapy, partly reflecting the significant rate of the off-target activity of nelfinavir. Increasing preclinical and clinical evidence shows that nelfinavir has broad anti-cancer activity as a single agent and in combination, potentially related to its off-target activity in mammalian cells. Nelfinavir is particularly effective in the treatment of proteasome inhibitor-refractory multiple myeloma (MM), where the combination of nelfinavir+bortezomib+dexamethasone yielded an overall response rate (ORR, PR or better) &amp;gt; 65% in a Phase II clinical trial. The targets and molecular mechanism of action of nelfinavir in MM are unknown. This hampers both, a rational clinical repositioning and development of nelfinavir as antineoplastic drug, as well as the design, synthesis and testing of next generation nelfinavir-like compounds with optimized antineoplastic activity and improved specificity or pharmacologic properties. We therefore aimed to take an unbiased target-identification approach to identify molecular targets of nelfinavir in human malignant cells and link them to cell biological processes and mechanisms that mediate sensitivity or resistance to nelfinavir treatment. METHODS Proteome-wide affinity-purification of targets binding the nelfinavir active site was combined with genome-wide CRISPR/Cas9-based screening to identify protein partners interacting with nelfinavir and candidate genetic contributors affecting nelfinavir cytotoxicity. Multiple intracellular reporter systems including RUSH system, ATP/ADP constructs; FRAP microscopy, Seahorse measurements, flow cytometry, qPCR, metabolic labelling, lipidomics and viability assays were used to dissect functional alterations in pathways related to nelfinavir targets. RESULTS We identified a common set of proteins interacting specifically with the active site of nelfinavir. These proteins are embedded in intracellular, lipid-rich membranes of mitochondria (VDAC1,2,3, ANT2), endoplasmic reticulum (BCAP31, CANX, SRPRB) and nuclear envelope (PGRMC2) and are consistent across multiple cancer cell types. ADIPOR2, a key regulator gene of membrane lipid fluidity, was identified as a key nelfinavir resistance gene, while genes involved in fatty acids (FAs) and cholesterol metabolism, vesicular trafficking and mitochondria biogenesis are candidate sensitivity genes. We further show that via binding to proteins in lipid-rich membranes nelfinavir affects membrane composition and reduces membrane fluidity, leading to induction of FAs synthesis and the unfolded protein response (UPR). Via its structural interference with membrane fluidity, nelfinavir impairs the function and mobility of a diverse set of membrane-associated proteins and processes, such as glucose flux and processing, mitochondria respiration, energy supply, transmembrane vesicular transport and ABCB1-mediated drug efflux, as we show in different reporter systems in live MM cells. These functional effects are prevented by addition of metabolically inert lipids to be incorporated in membranes, supporting a direct structural activity of nelfinavir. The adaptive biology of proteasome inhibitor (PI)-resistant myeloma relies on metabolic reprogramming and changes in lipid composition, drug export and down-modulation of the UPR. Modulation of membrane fluidity and depletion of FAs/cholesterol is synergistic with proteasome inhibitors in PI-resistant MM. Thus, the mechanism of action of nelfinavir perfectly matches with the biology of PI-resistant MM, serving as a molecular rational for its significant clinical activity. CONCLUSION We here demonstrate in vitro that the activity of nelfinavir against MM cells is triggered through changes in lipid metabolism and the fluidity of lipid-rich membranes. Pharmacologic targeting of membrane fluidity is a novel, potent mechanism to achieve anti-cancer activity, in particular against PI-refractory MM. This mechanism explains the clinical activity of nelfinavir in MM treatment as well as the key side effects of nelfinavir during antiretroviral therapy. Disclosures No relevant conflicts of interest to declare.

Identification of Novel Regulatory Pathway for Immunoglobulin Production Provides Rational Treatment for Bortezomib-Resistant Multiple Myeloma Patients

Introduction Monoclonal immunoglobulin (Ig) is a valuable diagnostic marker in patients with multiple myeloma (MM). An inevitable consequence of extensive Ig synthesis is overload of misfolded proteins that saturate proteasome capacity making the myeloma cells highly sensitive to proteasome inhibitors (PI). Even though PI are regularly used in the clinic, resistance often emerges leaving clinicians with limited treatment options. Therefore, there is a need for a robust marker selecting MM patients for precise PI-based combination therapy. Methods We performed a multiple database search for genes associated with Ig production and MM patients' survival. Additionally, we compared gene expression profiles (RNAseq) of primary MM cells with low and high Ig levels. Next, we validated the identified hits by shRNA knockdown and overexpression studies using myeloma cell lines, primary MM samples, and mouse models. We also applied mass spectrometry-based proteomic analysis, advanced biochemical approaches, and genetic models to reveal the Ig production pathway components and function. Finally, we performed a limited rational drug screening to select suitable compounds for combination treatment. Results RNAseq and database mining revealed a strong association between the expression of plasma cell-specific deubiquitinase OTUD1, Ig production, and MM patient survival. Suppression of OTUD1 with shRNAs in RPMI8226 and MM1.S cell lines reduced Ig levels, increased proliferation, and induced bortezomib resistance. Conversely, inducible OTUD1 overexpression enhanced Ig production, slowed down proliferation, and increased bortezomib sensitivity. In the xenografts mouse models cells with high OTUD1 levels synthesized more Ig and developed smaller tumors. Intriguingly, the transcription of Ig genes was not influenced by OTUD1 expression suggesting that OTUD1 functions as a posttranslational regulator of Ig assembly. To gain mechanistic insight into the Ig pathway regulation by OTUD1, we utilized the biotin proximity labeling method (Turbo-ID) combined with mass spectrometry analysis. We found several novel OTUD1 interaction partners including the E3 ubiquitin ligase KEAP1 and endoplasmic reticulum (ER) redox protein PRDX4. We demonstrated that KEAP1 acts upstream of OTUD1 by regulating OTUD1 ubiquitination and stability. Consistently, survival analysis revealed that MM patients with high KEAP1 expression (low OTUD1) had a worse prognosis than patients with low levels of KEAP1 (high OTUD1). PRDX4 regulates disulfite bonds formation during protein folding and is uniquely expressed in fully differentiated plasma cells. Here, we revealed that OTUD1 specifically deubiquitinates and thus stabilizes PRDX4 inside the ER. Additionally, we performed rescue genetic experiments and found a direct link between the OTUD1-PRDX4 axis and Ig production. The increase in OTUD1 expression (high Ig) led to a dramatic increase in the total pool of ubiquitinated proteins formed mainly by misfolded Ig, while OTUD1 knockdown (low Ig) had an opposite effect. We showed that changes in the level of ubiquitinated proteins correlated with PI sensitivity. Of note, OTUD1 did not affect the expression of proteasome subunits, either their enzymatic activity. Our mechanistic findings prompted us to propose a novel therapeutic opportunity in PI resistant MM patients. We hypothesize that the resensitization of Ig low MM cells to PI could be achieved by enhancing ER stress leading to an increase in misfolded proteins that would ultimately saturate proteasomes. Indeed, from clinically relevant drugs tested so far, the HSP-90 inhibitor (17-AAG) reverted the PI resistance in OTUD1 low (Ig low) myeloma cells. An in vivo validation of the combination treatment and testing of Ig involvement in PI sensitivity and proliferation of MM cells is ongoing. Conclusion Here we present the discovery of a novel regulatory mechanism for Ig production in plasma cells. Based on our results and previously published studies, we conclude that Ig synthesis is a clinically significant factor related to PI response and MM patient survival. Our findings suggest that the intracellular Ig level is an important biomarker to identify patients benefiting the most from PI-based therapies. Finally, we provide a rational solution for selective, combination therapy to overcome PI resistance in MM patients with a decreased capacity to synthesize Ig. Figure Disclosures Hajek: Janssen: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Consultancy, Honoraria; PharmaMar: Consultancy, Honoraria; Oncopeptides: Consultancy.

Targeting the p62-ZZ/N-End Rule Pathway in Multiple Myeloma Overcomes Proteasome Inhibitor-Resistance Via Induction of Necroptosis and Enhances the Bone Anabolic Effects of Proteasome Inhibitors

Multiple myeloma (MM) is incurable and 80% of MM patients develop MM bone disease (MMBD). MMBD lesions do not heal due to the persistent suppression of bone formation, which markedly increases mortality and contributes to MM drug resistance. Current treatments for MMBD, such as bisphosphonates and denosumab, target bone destruction but do not result in new bone formation. Although proteasome inhibitors (PIs) have greatly improved survival of MM patients, they only have transient bone anabolic effects. Further, development of drug resistance to PIs remains a major clinical problem. We previously showed that the ZZ domain of p62 (sequestosome-1),plays an important role in both MM growth and suppression of osteoblast (OB) differentiation in the MM microenvironment, by regulating multiple signaling pathways and acting as a cargo-receptor for autophagy. Recently, our collaborators showed that the p62-ZZ is a high-affinity N-recognin of the N-end rule pathway (NERP). p62-ZZ also serves as the molecular switch for necroptotic versus apoptotic cell death pathways. We previously reported that MM cells or TNFα prevent OB differentiation by inducing persistent epigenetic repression of the Runx2-P1 promoter in MM patient bone marrow stromal cells (BMSCs), via the transcriptional repressor Gfi1. We found that blocking p62-ZZ by saturating it with a novel synthetic p62-ZZ/NERP competing ligand, XRK3F2, prevented and reversed MM-induced Gfi1 occupancy at the Runx2-P1 promoter, allowing BMSCs to increase OB marker gene expression and to mineralize. These results suggest that targeting the p62-ZZ/N-end rule pathway would enhance the bone anabolic effects of PIs. To test this hypothesis, we first exposed normal OBs to different doses of bortezomib (Btz) or XRK3F2 or their combination. The combination significantly increased OB differentiation markers Runx2 (60%), Osterix (20%) and ATF4 (60%), and induced mineral deposition compared with either drug alone. The combination also blocked TNFα up-regulation of Gfi1 and suppression of OB differentiation. Interestingly, none of the concentrations tested decreased OB viability. Studies with MM patient-BMSCs showed that XRK3F2 reversed suppression of OB differentiation induced by MM cells, allowing them to mineralize. Importantly, our preliminary in vivo data showed that administration of XRK3F2 to mice with established MM induced dramatic cortical bone formation in MM-containing bones but had no effect on tumor burden. We and others previously showed that MM cells subjected to sustained proteasomal inhibition, rely on p62-mediated autophagic degradation to reduce the proteotoxic load caused by excessive immunoglobulin synthesis. We recently found that targeting the p62-ZZ domain in human MM cells, increases Btz-induced MM cell death, independently of their p53 status. The combination also significantly reduced cell viability in Btz resistant cells although no caspase 3 activation was observed, suggesting a caspase-3 independent cell death. To determine the mechanism(s) responsible for MM cell death induced by the combination, we pretreated MM cell lines and primary CD138+ MM cells with Z-DEVD (20μM), bafilomycin (Baf, 40nM), or necrostatin1 (NEC1, 50μM). The anti-MM effects of XRK3F2 or Btz+XRK3F2 were fully blocked by NEC1, an inhibitor of necroptosis, but not by inhibitors of caspase-dependent apoptosis (Z-DEVD) or autophagy (Baf), supporting that p62-ZZ regulates necroptosis in MM cells. RNAseq analysis of the additive effect of Btz+XRK3F2 on gene expression showed a total of 583 differentially regulated genes, including 374 significantly down-regulated and 209 significantly upregulated. GO term analysis of up-regulated DEGs identified an enrichment in the endoplasmic reticulum (ER) stress and ER unfolded protein response, and regulation of transcription in response to stress and autophagy. In summary, our results demonstrate that targeting the p62-ZZ/N-end rule pathway in combination with PIs in MM significantly reduces MM cell viability by activating multiple death pathway and overcomes PI-resistance of MM cells. In addition, targeting the p62-ZZ in OBs potentiates the bone anabolic action of PIs and reverses the persistent OB suppression induced by MM cells to allow bone formation. Thus, p62-ZZ plays a critical role in MM and bone cells and identifies p62-ZZ as an important molecular target for the treatment of MMBD. Disclosures Xie: Oxis Biotech: Consultancy; ID4Pharma: Other: Founder. Roodman:Amgen: Membership on an entity's Board of Directors or advisory committees.

Abstract 285: Ixazomib combined with the nuclear export inhibitors selinexor or eltanexor for the treatment of multiple myeloma

Abstract Introduction In the past decade, response and survival rates in multiple myeloma (MM) patients have been greatly increased by newer therapies. Nevertheless, most patients with MM eventually die from relapsed/refractory disease. Previous studies in our lab and in others have shown that exportin 1 inhibitors (XPO1i) are potent anti-MM drugs, whether used as single agents or in combination with proteasome inhibitors (PIs), immunomodulator drugs (IMiDs), anthracyclines, or alkylating agents. Ixazomib (IXA), an orally available PI, has been shown to be effective when combined with IMiDs, dexamethasone, alkylating agents, and/or prednisone for the treatment of patients with MM. We investigated whether the clinical XPO1i selinexor (SEL) or eltanexor (ELT) combined with IXA could overcome PI resistance in MM cell lines and in ex vivo bone marrow aspirates from PI-refractory patients. Materials and Methods PI-resistant MM cell lines 8226B25 and U226PSR and their respective parental cell lines, 8226 and U266, were treated in vitro and in vivo (mice) with SEL/IXA or ELT/IXA combinations or with each drug as a single agent. Bone marrow aspirates from newly diagnosed and PI-refractory MM patients were treated ex vivo with SEL/IXA or ELT/IXA and assayed for apoptosis. Mechanistic studies included NFkB and IkBα protein assays, immunofluorescence microscopy, ImageStream flow-cytometry, and proximity-ligation assay. IkBα knockdown and NFkB transcriptional activity were measured in SEL/IXA- or ELT/IXA-treated MM cells. Results SEL and ELT each sensitized parental and, to a lesser extent, PI-resistant MM cells to IXA, as measured by apoptosis. XPO1i/IXA treatments inhibited U266 and 8226 MM tumor growth and increased survival in mice; however, PI-resistant tumors were not sensitized by XPO1i/IXA treatment. Myeloma cells isolated from both newly diagnosed and PI-refractory MM patients were sensitized by XPO1i to IXA, as compared to untreated and single agent-treated MM cells, without affecting non-MM cells. Immunofluorescence microscopy, Western blot, and ImageStream analyses of MM cells showed increased nuclear IkBα with the XPO1i/PI treatments. Proximity ligation found increased IkBα-NFkB complexes in treated MM cells. IkBα knockdown abrogated XPO1i/PI-induced cytotoxicity in MM cells. XPO1i/PI treatment decreased NFkB transcriptional activity. Conclusions In previous studies, we found that SEL sensitized PI-resistant cell lines to carfilzomib and bortezomib; however, IXA was less effective in these same MM cells. SEL or ELT used in combination with IXA have the potential to overcome PI drug resistance in MM patients. Sensitization may be due to inactivation of the NFkB pathway by IkBα. Further investigation is underway to examine these mechanisms. The results presented in this study support combinatorial clinical trials in relapsed and refractory MM patients who use PI therapies. Citation Format: Joel G. Turner, Jana Dawson, Alexis Bauer, Juan Gomez, Erkan Baloglu, Yosef Landesman, Daniel M. Sullivan. Ixazomib combined with the nuclear export inhibitors selinexor or eltanexor for the treatment of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 285.