CD56 Has a Critical Role in Regulating Multiple Myeloma Cell Growth and Response to Therapies

Multiple myeloma (MM) is a disease derived from genetically abnormal clonal plasma cells. MM cells aberrantly express several surface antigens compared with normal plasma cells. Among others, CD56/NCAM1 is present at variable levels in approximately 70% of MM patients. Very little is known about its role in MM; however, CD56 positivity in MM correlates with greater osteolytic burden and lower frequency of good prognostic features, such as the presence of t(11;14). We first analyzed 569 patients with MM diagnosed between 1/1/2005 and 12/31/2014 at the Ohio State University Wexner Medical Center, stratifying them based on the percentage of CD56-expressing clonal MM cells. The mean percentage of CD56-expressing clonal MM was 26.5%, with range from 0 to 100%; the Mean Fluorescent Intensity values varied, with a quarter of patients expressing CD56 at high intensity. We then evaluated patient outcomes based on the percentage of CD56-expressing clonal MM cells. We noticed that MM patients with more than 30 or 50 percent of CD56-expressing MM clonal cells have inferior clinical outcomes than patients with less than 30 or 50 percent of CD56-expressing MM clonal cells, with median overall survival of 9.61 versus 7.64 years (log-rank p-value: 0.004) or 9.30 versus 6.47 years (log-rank p-value: 0.0009), respectively. We then demonstrated by conventional and real-time PCR analyses that the predominately expressed CD56 isoform in MM has signaling potential with a transmembrane portion and cytosolic tail. Therefore, we evaluated the functional role of CD56 in MM. By gain-of function studies in U266 and MM.1S MM cell lines, we showed that overexpression of CD56 promotes MM growth and viability; the opposite effect occurred with CD56 silencing in H929, OPM-2, and RPMI-8226 MM cell lines, which leads to reduced MM growth and increased apoptotic cell death. Overexpression of CD56 resulted in the phosphorylation and hence activation of ribosomal protein S6 kinase A3 (RSK2) and of the transcription factor, cAMP responsive element binding protein 1 (CREB1). This induced CREB1 binding to DNA consensus CRE elements, and promoted transcription of CREB1 targets, the anti-apoptotic genes BCL2 and MCL1. CD56 silencing in H929 and OPM-2 MM cell lines had opposite effects, with reduction of phospho-RSK2, phospho-CREB1, MCL1, and BCL2 levels. We then used shRNAs targeting RSK2 and CREB1 or specific inhibitors (BI-D1870 that is a RSK2 inhibitor, and 666-15 that is a CREB1 inhibitor) at 0.1-1 microM concentration. We evaluated viability by MTT assay or Zombie dye staining on CD138 positive MM cells and apoptosis by Annexin V-PI staining. We demonstrated that CD56 positive MM cell lines (H929, OPM-2, and RPMI-8226) or patients with high CD56 expression (>30% of CD56-expressing clonal MM cells) are more sensitive to RSK2/CREB1 inhibition compared with CD56 negative MM cell lines (U266, L363, and MM.1S) or patients with low CD56 expression (<30% of CD56-expressing clonal MM cells). Using similar strategies, we also proved that CREB1 is essential to CD56-protumoral phenotype, since CREB1 inhibition reduces cellular growth and viability in CD56 overexpressing U266 cells. RSK2 and CREB1 inhibition mimic CD56 silencing with decrease of BCL2 and MCL1 mRNA and protein levels. Furthermore, we observed that CD56 signaling by CREB1 activation decreases CRBN expression, reducing responses to lenalidomide. Conversely, CREB1/RSK2 blockade rescued CRBN levels in CD56 positive MM cells and increased lenalidomide response. These results support the hypothesis that targeting CREB1 is hence a so far unexplored but potentially effective synthetic lethal strategy for CD56-expressing MM patients. In conclusion, our study defines an effective threshold for therapeutic intervention in CD56-expressing MM patients. Moreover, our data pioneer the use of CREB1/RSK2 inhibition in CD56-expressing MM cells, either as single agents or in combination with lenalidomide, suggesting that CD56 can be a prognostic and predictive factor of response in MM. Disclosures No relevant conflicts of interest to declare.

Essential Drug Targets and Pathways in PI-Resistant MM Cells

Background Proteasome inhibitors (PI) have emerged as a powerful, cell biology-based treatment option for multiple myeloma (MM) and build a central backbone for MM treatment with three proteasome-inhibiting drugs currently approved: bortezomib (BTZ), carfilzomib (CFZ) and ixazomib. However, despite the high anti-MM activity of PI, MM cells adapt to the selective pressure of PI treatment in most cases to date and most MM patients relapse during or after treatment with PI, develop PI-refractory disease and ultimately die. Therefore, understanding and overcoming PI resistance is a key challenge for MM therapy. Our previous in vitro studies on PI-resistant MM suggest that PI-adapted, MM cells show very distinct features of general metabolism and cell biology that differentiate them from PI-sensitive MM, derived from the same cell line. We hypothesize that this highly specialized and adapted nature of PI-resistant MM offers novel areas of vulnerability, that differ from the therapeutic targets in PI-sensitive MM. The aim of our study was to identify essential drug targets and pathways in PI-resistant MM using genome-wide functional screening with the CRISPR/Cas9 system that could serve as novel therapeutic targets in PI-resistant MM. Methods We used genome-wide CRISPR/Cas9-based loss-of-function screening with Brunello library in L363-BTZ and RPMI-8226-BTZ cells, adapted to grow in the presence of 90 nM BTZ. The overlapping bortezomib genetic sensitivity candidates were further validated in the set of BTZ-resistant cells (L363-BTZ, RPMI-8226-BTZ, MM1S-BTZ and AMO-BTZ) cells using shRNA silencing or single-gene specific knockout or genetic overexpression using CCK8 viability assay. Subsequent functional analysis of the highest ranking BTZ sensitivity candidates in BTZ-adapted cells included apoptosis and cell cycle analysis, qPCR and western blotting, SILAC, proteasome activity determination using activity-based probes and FRAP analysis. Results CRISPR/Cas9-screening identified two candidate genes for BTZ sensitivity, ECPAS (KIAA0368; Ecm29 Proteasome Adaptor and Scaffold protein) and PSME1 (an 11S regulator complex subunit), as consistent screening hits in two independent BTZ-adapted MM cell lines. Both genes are related to proteasome, but do not build the proteasome core particle and do not have a proteolytic activity. Specific knock-down or knock-out of ECPAS sensitized PI-naïve cells to BTZ and CFZ, while significantly more sensitizing BTZ-adapted cells to both PI. Likewise, overexpression of PSMF1, an inhibitor of 11S regulator complex, sensitized BTZ-resistant as well as sensitive cells to BTZ. ECPAS-depleted BTZ-adapted cells showed accumulation of poly-ubiquitinated proteasome substrate proteins, induction of the unfolded protein response, cell cycle arrest and induction of apoptosis, together with changes in protein synthesis after the treatment with 50 nM bortezomib, in contrast to BTZ-adapted control cells. FRAP analysis of cells with GFP-tagged PSMD6 revealed that the intracellular mobility of proteasomes in ECPAS-depleted cells was reduced. Importantly, proteasome activity determined by activity-based probes was not impaired in ECPAS-depleted cells. Conclusion In conclusion, BTZ-resistant MM cells uniquely show a high dependency on the proteasome adaptor and scaffold protein ECPAS, which has been shown to be involved in coupling of proteasome in different compartments and promotes proteasome dissociation under oxidative stress. Specifically in PI-resistant MM, ECPAS is important to ensure functional proteasome, is involved in controlling the intracellular mobility of proteasomes, likely to ensure high proteasome turnover. ECPAS therefore represents a novel candidate that may be targeted to specifically re-sensitize PI-resistant MM cells to proteasome inhibitor treatment. Disclosures No relevant conflicts of interest to declare.

The Molecular Landscape and Essential Pathways Involved in Bone Marrow-Mediated Carfilzomib Resistance of Multiple Myeloma

Background Multiple myeloma (MM) remains an incurable malignancy, with most patients relapsing and dying from the disease. Anti-myeloma drugs, such as proteasome inhibitors (PIs) bortezomib and carfilzomib (CFZ), have considerably improved prognosis in myeloma. Despite these advances, disease heterogeneity, early relapse and treatment resistance still pose major challenges in MM treatment. Understanding the mechanisms that mediate PI resistance provide a key to targeting both, PI-resistant minimal residual disease that drives relapsed MM after prolonged PI-containing frontline therapy, as well as PI-refractory, aggressive advanced MM. While key mechanisms of the in vitro-generated PI resistance in MM have been revealed in cell line models, we lack understanding of PI resistance in vivo, where in particular clonal heterogeneity and the tumor microenvironment (TME) within the bone marrow (BM) add additional levels of complexity. Therefore, the aim of our study was to analyze the molecular landscape and changes occurring during MM progression under CFZ treatment in vivo and to identify key molecular processes contributing to CFZ-resistance of MM cells in the presence of stromal cells in vitro, to ultimately identify new molecular pathways and develop innovative treatment strategies in PI-resistant MM. Methods The NSG mice intrafemorally engrafted with human RPMI-8226 cells were either untreated or treated long-term with 4 mg/kg CFZ (intravenously) until they became drug resistant. At this point, CFZ naïve and CFZ-resistant cells were isolated and processed for single-cell RNA sequencing (scRNA-seq, 10x Genomics) with the aim to characterize a transcriptional CFZ-resistance signature in refractory cells. To investigate the role of the TME as well as the importance of cell-cell interactions in CFZ-resistance in vitro, we performed two independent genome-wide CRISPR/Cas9 library screenings. In the first one, Brunello library transduced RPMI-8226 cells were co-cultured with human stromal cells (HS5) and treated with CFZ to identify CFZ sensitivity/resistance candidate genes. In the second experiment, Brunello library and synthetic Notch (synNotch) receptor transduced HS5 cells were co-cultured with synNotch ligand transduced RPMI-8226 cells to identify genes that are essential for establishing cell-cell contacts between stromal and MM cells. Subsequent functional analysis of the highest-ranking CFZ sensitivity/resistance candidates in the RPMI-8226+HS5 co-culture included shRNA-silencing, single-gene knockouts, viability assays, cell cycle analysis and protein synthesis analysis using the SUnSET assay. Results ScRNA-seq analysis of CFZ-refractory RPMI-8226 cells growing in the BM of NSG mice showed a different transcriptional landscape, compared to CFZ-naïve cells isolated from the BM of untreated mice. The unsupervised clustering analysis, using UMAP, revealed that cells exposed to CFZ show distinct populations with a strong increase in the OXPHOS and protein folding capacity as well as down-regulation of several genes involved in proliferation and apoptosis, when compared to naïve cells. The CRISPR/Cas9 library screening where RPMI-8226 cells were co-cultured with HS5 cells and exposed to CFZ revealed several CFZ sensitivity candidates at the cut-off of false discovery rate (FDR) < 0.01 and fold change above 1.5-fold. Those genes are involved in cytokine signaling, cell growth, invasion, metastasis and quality control of translational elongation. At the same time, the CRISPR/Cas9 library screening, where synNotch receptor transduced HS5 cells were co-cultured with synNotch ligand transduced RPMI-8226 cells revealed gene candidates at the cut-off of FDR < 0.01 and fold change greater than 1.5-fold, which mediate stronger or weaker cell-cell interaction. Those genes are particularly involved in cytokine signaling and mitochondrial metabolism. Conclusion In conclusion, MM cells that acquired CFZ-resistance upon cell-cell contact with certain cell types within the TME, such as stromal cells, differ significantly from CFZ-naïve cells. CFZ-resistance, caused by cell-cell contact with stromal cells, is presumably mediated via decreased proliferative as well as protein synthesis capacity of MM cells. Therefore, stimulation of MM cells to proliferate and synthesize more proteins may be a key to targeting CFZ-resistance in vivo. Disclosures No relevant conflicts of interest to declare.

Functional Multi-Omics Reveals Genetic and Pharmacologic Regulation of Surface CD38 in Multiple Myeloma

Background: CD38 is a surface ectoenzyme expressed at high levels on myeloma plasma cells and is the target for the monoclonal antibodies (mAbs) daratumumab and isatuximab. These antibodies have multiple mechanisms of action, primarily involving recruiting and modulating components of the immune system, but they may also carry direct anti-tumor effects. CD38 density on tumor cells is an important determinant of mAb efficacy while CD38 is lost after mAb treatment. Several small molecules have been found to increase tumor surface CD38, with the goal of boosting mAb efficacy in a co-treatment strategy. However, we do not yet have a broad global sense of the transcriptional or post-transcriptional networks that most strongly impact CD38 expression. There may be alternate strategies to even more potently increase CD38 expression that have not yet been identified. Furthermore, prior clinical studies showed that CD38 downregulation after daratumumab treatment was accompanied by increases in the complement inhibitors CD55 and CD59. Are there other features of myeloma surface remodeling driven by CD38 downregulation? Here we sought to extend our currently limited insight into CD38 surface expression by using a multi-omics approach. Methods: Genome-wide CRISPR interference screening was performed in RPMI-8226 cells stably expressing the dCas9-KRAB fusion protein. Cells were grown for 14 days after library transduction, flow-sorted on the top and bottom 25% of CD38 surface expression, and sgRNA's deep sequenced. Antibody-dependent cellular cytoxicity assays were performed with NK92-CD16 cells. Cell surface proteomics was performed using N-glycoprotein cell surface capture in triplicate. Phosphoproteomics was performed used immobilized metal affinity chromatography in triplicate. Murine studies were performed in NSG mice under approved IACUC-approved institutional protocols. Results: A genome-wide CRISPR-interference screen in RPMI-8226 cells demonstrated that transcriptional and epigenetic factors played the most prominent role in surface CD38 regulation (Fig. 1A). One of the genes that when knocked down led to greatest surface CD38 increase was RARA. This finding supports the promise of all-trans retinoic acid (ATRA), which leads to RARA degradation, as a potent agent to induce CD38 upregulation. Validation of additional screen hits TLE3 and HEXIM1 also illustrated that these negative regulatory transcription factors suppress CD38 expression at baseline (not shown). We found the transcription factor SPI1 to be a prominent positive regulator of CD38. SPI1 knockdown led to daratumumab resistance both in vitro and in vivo, similar to the resistance observed after CD38 knockdown (not shown). Analysis of myeloma patient ATAC-seq data, assessing transcription factor motifs present at the CD38 locus, combined with a predictive machine learning model, further identified XBP1 as one of the most potent transcriptional regulators of CD38 (Fig. 1B). We next used "antigen escape profiling" - knockdown of CD38 followed by unbiased cell surface proteomics - to mimic surface alterations in the context of CD38 loss. We found minimal changes in other cell surface proteins beyond CD38 (Fig. 1C), indicating the CD38 loss alone is not sufficient to remodel the myeloma surfaceome. This finding also supports the hypothesis that complement or other immune system interactions are necessary to lead to other myeloma surface protein alterations. In a parallel analysis of pharmacologic regulation, we also used cell surface proteomics integrated with RNA-seq to demonstrate that ATRA leads to few other surface protein changes beyond CD38 (not shown). In contrast, other molecules, such as azacytidine and panobinostat, led to broader changes across many more surface proteins, showing a lack of specificity when driving CD38 upregulation. Finally, unbiased phosphoproteomics revealed partial inhibition of the MAP kinase pathway after daratumumab binding (Fig. 1D). This result may comprise a direct anti-proliferative effect of anti-CD38 therapeutic antibody engagement in myeloma. Conclusions: Our work provides a resource to design strategies to enhance efficacy of CD38-targeting immunotherapies in myeloma. Our approach also outlines a broad multi-omic strategy to evaluate surface and transcriptional regulation of other key immunotherapeutic targets in hematologic malignancies. Figure 1 Figure 1. Disclosures Choudhry: Genentech: Current Employment, Current equity holder in publicly-traded company. Ramkumar: Senti Biosciences: Current Employment, Current holder of individual stocks in a privately-held company.

Elongator Complex Regulates MCL1 Dependency Via IRE1-XBP1 Axis of the ER Stress Response Pathway in Multiple Myeloma

Evasion of apoptosis is a hallmark of cancer wherein overexpression and amplification of pro-survival BCL2-family genes like MCL1 is a common observation. MCL1 is frequently amplified in many hematological cancers like Multiple Myeloma (MM) that depend on it for survival. BH3 mimetic drugs, like the BCL2-specific inhibitor Venetoclax, have been successfully used in the clinic to treat certain cancers, and MCL1-selective inhibitors are currently in clinical development. While inhibition of MCL1 displays promising preclinical activity, many cancer models display acquired or intrinsic resistance to MCL1 inhibitors (MCL1i). As MCL1-targeted therapies progress clinically, understanding mechanisms that lead to resistance will be important to not only identify therapeutically-exploitable targets to combat resistance, but to also determine if these biomarkers could stratify patients most likely to respond to an MCL1i. Here, we used a genome-wide CRISPR knock-out screen to identify mechanisms of resistance to MCL1i AZD5991 in two MM cell lines, KMS11 and KMS34. We used a sgRNA library consisting of about 118,000 sgRNAs (~6 sgRNAs/gene), and treated the cells with DMSO or 1uM AZD5991 for 16 days (5 doublings). We identified 316 genes in KMS11 and 184 genes in KMS34 with >4-fold enrichment of sgRNAs; and 221 genes with >2-fold enrichment of sgRNAs in both cell lines. The sgRNAs targeting BAK and BAX were the most enriched overlapping hits. Using GSEA analysis of the 221 common genes with enriched sgRNAs, we discovered that the tRNA wobble uridine modification as the most enriched pathway. The tRNA U34 mcm5s2 modification is catalyzed by the elongator complex ELP1-6 and cytosolic thiouridylase CTU1/2. Each subunit of the elongator complex is essential for its function and loss of any subunit results in destabilization of the complex. By knocking out ELP4 in five MM cell lines (KMS11, KMS34, KMS12-PE, MM.1S, and RPMI-8226), we first validated the destabilization of the complex by showing a robust decrease in the protein levels of ELP1 and ELP3 via western blot. As the elongator complex has additional functions, we also knocked-out tRNA U34 modification pathway specific CTU1 in KMS11, KMS34, and KMS12-PE cells. We showed that genetic knock-out of ELP4 and CTU1 results in increased resistance to MCL1i in all cell lines tested. We observed the highest increase in MCL1i resistance upon ELP4-KO in KMS11 and RPMI-8226 cell lines. To understand the mechanism behind elongator complex mediated regulation of MCL1 dependency, we performed RNAseq and global proteomics in KMS11 cells (Parental, non-targeting control [NTC], ELP4-KO and CTU1-KO) and RPMI-8226 cells (Parental, NTC, and ELP4-KO). We show that the elongator complex is a regulator of IRE1-XBP1 axis of the ER stress response pathway; and knockout of IRE1 also results in MCL1i-resistance in KMS11 and RPMI8226 cell lines. Mechanistically, we show that loss of elongator complex-mediated downregulation of IRE1-XBP1 axis leads to stabilization of MCL1 and upregulation of BCL-XL and NOXA expression. We further show that upon treatment with MCL1i, KMS11-ELP4-KO cells have less disruption of MCL1:Bim complex and an increase in BCL-XL:Bim complex as compared with KMS11-NTC cells. The net increase in pro-survival MCL1 and BCL-XL proteins in ELP4-KO cells resulting in lower levels of unsequestered BIM upon AZD5991 treatment suggests a reduction in apoptotic priming. The mechanistic link between the elongator complex and ER stress response pathway led us to test ER stress inducing drugs in these cell lines. We observed that ELP4-KO results in increased resistance to proteasome inhibitor Bortezomib and other ER stress inducers like Tunicamycin, Thapsigargin, and BrefeldinA as a monotherapy or in combination with AZD5991. These data are consistent with our hypothesis that ELP4-KO cells have reduced apoptotic priming and are thus multi-drug resistant. As bortezomib is used in the clinic to treat MM patients, we asked if an elongator gene signature could be used to predict response to current therapies. We show that the elongator complex components could be used as a gene signature to stratify overall survival in MM patients (MMRF CoMMpass dataset). Moreover, ER stress response gene signature has been shown to be repressed in drug-resistant MM. Taken together, an integrated elongator and IRE-XBP1 gene signature could be a strong predictor of therapy response in MM . Disclosures Aryal: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Sundarrajan: AstraZeneca: Ended employment in the past 24 months. Boiko: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Jenkins: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Liu: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Ahdesmaki: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Bornot: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Jarnuczak: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Miele: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. McDermott: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Fawell: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Drew: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Boise: AbbVie/Genentech: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Honoraria, Research Funding. Cidado: AstraZeneca: Current Employment, Current equity holder in publicly-traded company.

Anti-Myeloma Effects of Panobinostat in Combination with Duvelisib

Introduction: Panobinostat (Secura Bio) is a non-selective histone deacetylase inhibitor (HDACi) that has shown anti-tumor activity in preclinical studies in both solid and hematological malignancies. Duvelisib (Secura Bio) is an FDA-approved oral drug and a potent small molecule inhibitor of the delta (δ) and gamma (γ) isoforms of phosphoinositide-3 kinase (PI3K) with potential immunomodulating and antineoplastic activities. There is no data regarding in combination of an HDACi with duvelisib for multiple myeloma (MM) treatment. We evaluated the anti-myeloma effects of the panobinostat in combination with duvelisib. Methods: The human MM cell lines U266, RPMI 8226 and MM1s were obtained from ATCC. Primary MM tumor cells were isolated from MM patients' bone marrow (BM) aspirates and mononuclear cells (MCs) were isolated. The cells were seeded at 10 5 cells/well in 96-well plates and incubated for 24 h in the presence of vehicle, panobinostat or duvelisib alone, or the two drugs together for 48 h. Cell viability was quantified using the MTS cell proliferation assay. For the apoptosis assay, U266, RPMI 8226, MM1s, or primary MM tumor cells were cultured at 1 x 10 6 cells per well for 48 hours at 37°C in 5% CO2 with or without the addition of panobinostat or duvelisib. Next, the cells were washed twice with PBS, re-suspended in binding buffer, and stained with FITC-conjugated annexin V and fluorescent dye propidium iodide (PrI) following annexin V assay standard protocol (BioVision, USA). For each drug treatment, 1 x 10 5 gated events were recorded. Results and Discussion: The MTS cell proliferation assay showed that panobinostat alone inhibited cell proliferation of the MM cell lines U266 and MM1s but not RPMI8226. Panobinostat alone induced concentration-dependent inhibition of BMMCs. Panobinostat (20nM) in combination with duvelisib markedly increased inhibition of primary MM cell proliferation. Panobinostat alone induced MM cell apoptosis in all three MM cell lines. Panobinostat in combination with duvelisib enhanced MM cell apoptosis in MM1s but not U266 and RPMI 8226 cells. We further examined apoptosis of primary MM cell treated with panobinostat alone and in combination with duvelisib. The results showed panobinostat alone induced primary MM cell apoptosis and in combination with duvelisib markedly increased the induction of apoptosis. This study illustrates that the combination of panobinostat and duvelisib shows potent anti-MM effects in vitro and we are currently evaluating the anti-MM effects of this combination in vivo using our human MM xenograft models. *The study was partially funded by Secura Bio, Inc Disclosures No relevant conflicts of interest to declare.

Disrupting the Reverse Warburg Effect As a Therapeutic Strategy in Multiple Myeloma

Introduction: Altered cellular metabolism is a hallmark of every cancer cell. Aerobic glycolysis ("The Warburg Effect") is one of the earliest recognized metabolic abnormalities in cancer cells whereby extracellular glucose is preferentially metabolized and eventually processed to generate lactate and energy in the form of ATP before the former is released extracellularly, irrespective of oxygen availability. While extracellular lactate produced and released from cancer cells has traditionally been considered a waste metabolic by-product, recent understanding of cell metabolism suggests that it can also serve as a primary metabolic fuel for cancer cells via uptake by monocarboxylate transporters (MCTs). Our goal was to evaluate this "Reverse Warburg Effect" phenomenon in multiple myeloma (MM) cells and determine if it can be exploited for therapeutic purposes. Methods: All HMCLs, MM1S, RPMI-8226 and U266, were grown in RPMI-1640 cell culture medium containing 11 mM glucose and supplemented with 10% dialyzed fetal bovine serum (FBS) and 2 mM Glutamine. Primary MM cells were extracted using magnetic bead CD138 positive selection from MM patient bone marrow aspirates. For 13C-labeling experiments, HMCLs and primary MM cells were suspended in RPMI-1640 cell culture media containing 13C-labeled isotopes. Isotopomer analysis of glycolytic and tricarboxylic acid (TCA) cycle metabolites from HMCL and primary MM cell pellets was performed using Agilent Technologies 5975C gas chromatography-mass spectrometry. Small molecule inhibitors, AZD3965 and syrosingopine, were purchased from Selleck Chemicals and Sigma respectively. Cellular viability and proliferation were measured using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrasodium bromide (MTT) and CCK-8 assays respectively. MCT-1 and MCT-4 antibodies for western blotting were utilized to evaluate their cell membrane expression on HMCLs. Results: The HMCLs, MM1S and RPMI-8226 as well as primary CD138+ cells from MM patient bone marrow were cultured in cell culture media containing physiological levels (1 mM) of U-13C-Lactate. The incorporation of extracellular 13C into the intracellular glycolytic and TCA cycle metabolite pool was observed (Fig 1) based on the expected isotopomeric patterns, demonstrating the Reverse Warburg Effect in MM cells. The relative contribution of carbon substrate by extracellular lactate compared to extracellular glucose was assessed in the following HMCLs: MM1S, RPMI-8226 and U266 cells by culturing in cell culture media containing 3-13C-Lactate and U-13C-Glucose. Extracellular lactate (yellow bar) contribution to the formation of TCA metabolites equaled that of glucose (red bar) based on the expected isotopomer patterns, suggesting the relative importance of extracellular lactate as an essential nutrient like glucose (Fig 2). Since MCT-1 and MCT-4 are key bidirectional cell membrane transporters of lactate in and out of cells, we explored the clinical significance of their gene expression level on clinical outcomes using the COMMPASS dataset provided by the Multiple Myeloma Research Foundation (MMRF). When MM patients were dichotomized at above or below the median of the expression levels of fragments per kilobase of transcript per million (FPKM), MCT-1 and MCT-4 overexpression conferred a worse progression free survival and overall survival (Fig 3). The MCT-1/MCT-4 protein expression was detectable across the various HMCLs: MM1S, U266 and RPMI-8226 (Fig 4). Inhibition of MCT-1 by specific inhibitor AZD3965 was able to reduce proliferation but not affect viability of HMCLs at 48 hours (Fig 5). However, dual inhibition of MCT-1/MCT-4 using syrosingopine was able to significantly reduce proliferation and decrease viability of HMCLs in a dose dependent fashion (Fig 6). Finally, dual inhibition of MCT-1/MCT-4 using syrosingopine reduced the utilization of extracellular lactate into the TCA cycle pool by HMCLs in media containing 3-13C-Lactate (Fig 7). Conclusion: Utilization of extracellular lactate via Reverse Warburg Effect phenomenon appears highly active in MM cells. Disrupting the utilization of extracellular lactate by dual inhibition of both MCT-1 and MCT-4 appears therapeutic. In the future, dual inhibition of MCT-1/MCT-4 in combination with other anti-MM therapies should be evaluated to determine synergistic therapeutic potential. Figure 1 Figure 1. Disclosures Kumar: Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; KITE: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Carsgen: Research Funding; Sanofi: Research Funding; Novartis: Research Funding; Antengene: Consultancy, Honoraria; Beigene: Consultancy; Bluebird Bio: Consultancy; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tenebio: Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides: Consultancy; Astra-Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding; Roche-Genentech: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Research Funding.

Pyrroline-5-Carboxylate Reductase 1: A Novel Target for Sensitizing Myeloma to Cytotoxic Agents By Inhibition of PRAS40-Mediated Protein Synthesis

Introduction Multiple myeloma (MM) remains an incurable cancer despite advances in therapy. Therefore, the search for new targets is still essential to uncover potential treatment strategies. Metabolic changes, induced by the hypoxic bone marrow, contribute to both cancer cell survival and drug resistance. In this study, we aimed to identify which metabolic changes and downstream pathways are involved in myeloma cell growth and persistence. Methods Correlation of pyrroline-5-carboxylate reductase 1 and 2 (PYCR1 and PYCR2) with overall survival was investigated in the gene-expression data of MM patients (MMRF CoMMpass trial). To perform a tracer study, RPMI-8226 cells were supplemented with 13C-glutamine for 48h in both normoxia and hypoxia (<1% O 2, by chamber). For further in vitro investigation, 2 human MM cell lines (OPM-2 and RPMI-8226) were used. Proline concentrations in cell lysates were measured by ELISA-based proline assay kit. We used siRNA to establish a knockdown of PYCR1 and/or PYCR2. Levels of apoptosis were measured using AnnexinV and 7-AAD positivity on flow cytometry. Differential protein expression was evaluated with western blot. Proliferation was measured by assessing BrdU incorporation through flow cytometry. Pargyline was used as a PYCR1 inhibitor. All in vitro experiments were performed in hypoxic conditions. For the in vivo murine experiment, C57BL/KalwRij mice were inoculated with 1 million of eGFP+ 5TGM1 cells, and treated with vehicle, bortezomib (0.6 mpk, 2x/week, starting day 14), pargyline (100 mpk, 5x/week, starting day 1) or combination of both. Tumor burden was measured by flow cytometry when vehicle mice reached end-stage. Results Pyrroline-5-carboxylate reductase 1 and 2 (PYCR1 and PYCR2) are 2 mitochondrial enzymes that facilitate the last step in the enzymatic conversion of glutamine to proline. High expression of both enzymes correlated with a lower overall survival in the CoMMpass trial. Moreover, MM cells from relapse/refractory patients expressed significant higher levels of PYCR1. We performed a tracer study with RPMI-8226 cells, revealing an increased conversion of 13C-glutamine to proline in hypoxia compared to normoxia. We confirmed these results by increased proline production after 48h of hypoxic culture. SiRNA-mediated knockdown of PYCR1 or both PYCR1/2 combined with bortezomib increased apoptotic cell death in OPM-2 and RPMI-8226, which we confirmed by detecting upregulation of cleaved PARP and cleaved CASPASE 3 levels. In contrast, PYCR2 knockdown combined with bortezomib did not significantly alter apoptosis. Further investigation revealed that PYCR1 knockdown reduced proliferation, and led to a decrease in p-AKT, p-p42/44 MAPK and c-MYC levels. Mechanistically, we found that PYCR1 silencing affected protein synthesis, as shown by a downregulation of p-PRAS40, p-MTOR, p-p70, p-S6, p-4EBP1 and p-EIF4e levels. Next, we evaluated whether the clinically relevant anti-hypertensive agent and PYCR1 inhibitor, pargyline, was capable of inducing myeloma cell death. In vitro, pargyline reduced proline production, MM viability and increased apoptotic cell death. Pargyline was also capable of reducing viability in CD138+ cells of primary patient samples . Finally, in vivo combination of pargyline with bortezomib significantly reduced tumor burden in the 5TGM1 model. On protein level, we also observed a significant decrease in p-4EBP1 and p-EIF4e in the freshly isolated 5TGM1 cells for the combination therapy. Conclusion Hypoxia increased glutamine-to-proline conversion in myeloma cells by stimulating PYCR activity. Knockdown of PYCR1 and PYCR1/2 increased bortezomib efficacy and inhibited proliferation. Mechanistically, PYCR1 interference reduced PRAS40-mediated protein synthesis. Pargyline, a PYCR1 inhibitor, also reduced MM viability and increased apoptosis. In vivo, pargyline combined with bortezomib significantly reduced tumor burden in the 5TGM1 model compared to both single agents. In conclusion, this study identifies PYCR1 as a novel target in MM therapy. Disclosures De Veirman: Active Biotech AB: Research Funding. OffLabel Disclosure: Pargyline is a antihypertensive agent and irreversible MAO B inhibitor that also inhibits PYCR1. Pargyline is not approved by the FDA as a PYCR1 inhibitor.

PSMB4 and PSMD4 Are Correlated with 1q21 Amplification in CD138 + Plasma Cells: New Potential Druggable Targets in Myeloma Patients

The amplification of the 1q21 (amp1q21) region is one of the most acquired cytogenetic abnormalities (CA) in multiple myeloma (MM) associated with a worse patient outcome and disease progression. Moreover, different studies have demonstrated that the number of copies (CN) 1q21 (gain1q21: three copies or amp1q21: ≥ four copies) have a different impact in the response to anti-MM therapies. Particularly, it has been proposed that in MM patients, additional copies of 1q21 may be associated with the resistance to proteasome inhibitor (PI) treatment as bortezomib. A recent study showed that newly diagnosed MM (MMD) patients carrying amp1q21 but not gain1q21 receiving carfilzomib-based treatment have an early disease progression with shorter overall survival. Previous studies underlined that the amplification of 1q21 can lead to the overexpression and/or dysregulation of several candidate genes associated with cell proliferation, apoptosis, and drug resistance. Here we aim to identify 1q21 target genes possibly correlated to the response to PI therapy. We evaluated a total cohort of 29 primary plasma cells (PCs) purified from bone marrow (BM) blood aspirates from 11 smoldering MM (SMM) and 18 MMD. The median age of our cohort was 70 years (range: 38-86). Fluorescence in situ hybridization (FISH) analysis was performed to access the presence or absence of copy number alteration (CNA) in the 1q21 region in all patients. 14 out of 29 patients carried 1q21 CNA (5 with gain1q21 and 9 with amp1q21). A score reflecting the number of 1q21 copies was calculated based on the hybridization pattern. The transcriptional profiles of the 29 BM PCs samples were generated on GeneChip ClariomD Arrays (Affymetrix Inc., Santa Clara, CA, USA). The samr package was used in R for call genes as differentially expressed between 1q21 CN-altered and wild-type samples. The correlation between the 1q21 copy number score and the gene expression levels was performed. Moreover, we have evaluated by FISH the 1q21 CNA in a panel of human myeloma cell lines (HMCLs): OCY-MY5, JJN3, RPMI-8226, NCI-H929, and OPM2. JJN3 were transfected with a control vector and PSMB4 and PSMD4 short hairpin RNA (shRNA) lentivectors. The gene and protein expression levels of PSMB4 and PSMD4 in MM cell lines were analyzed by qRT-PCR and Western Blot, respectively. Cell viability and proliferation were assessed using MTT assay and flow cytometry. Our bioinformatic analyses highlighted the overexpression of different genes (IL6R, ILF2, BCL9, MCL1, CSk1B, ADAR1, ARNT, ANP32E) in the 1q21 CNA samples with respect to the controls, as already reported in the literature. Our analysis showed a significantly higher expression of two proteasome subunits (PSMB4 and PSMD4) in patients with 1q21 CNA when compared with patients without (PSMB4 p=0.0006; PSMD4 p=<0.0001). Patients with amp1q21 showed a higher expression of PSMB4 when compared to the patients with gain1q21 (p=0,007). In our cohort, gene expression profile analysis also showed a strong positive correlation between gene expression levels and 1q21 CN for the proteasome subunits PSMB4 (p=<0.0001, r=0.5631) and PSMD4 (p=<0.0001, r=0.6391). Interestingly, we found that the PSMB4 and PSMD4 expression level was independent of the disease stage (SMM vs MM) and was only driven by 1q21 CN. We have evaluated PSMB4 and PSMD4 mRNA and protein expression levels in a 1q21 wild-type cell line (OCY-MY5) and in a panel of MM cell lines carrying different degrees of 1q21 CN (in order: JJN3, U266, RPMI-8226, OPM2, and NCI-H929). The mRNA expression level of PSMB4 and PSMD4 was higher in cell lines carrying 1q21 amp, following a 1q21 copy number fashion. Similar results were obtained when protein levels of MM cell lines were analyzed by Western Blot. To further determine the potential role of both proteasome subunits in the pathogenesis of amp1q21, we generated a PSMB4-shRNA and PSMD4-shRNA knockdown stable MM cell lines. Functional studies showed that blockade of PSMB4 and PSMD4 decreased MM cell viability. In conclusion, our study identified proteasome subunits PSMB4 and PSMD4 to be significantly upregulated in MM patients carrying amp1q21, correlated with 1q21 copy number but not with disease stage. In addition, knockdown of both, PSMB4 and PSMD4 decreased MM cell proliferation. Therefore, targeting PSMB4 and PSMD4 could be a strategy to treat MM patients with ampq21 Disclosures Giuliani: Celgene: Membership on an entity's Board of Directors or advisory committees, Other: congress, Research Funding; Bristol Mayers Squibb: Other: congress; GSK: Other: clinical studies; Takeda: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: Clinical studies, congress, Research Funding; Millenium Pharmaceutical: Other: clincial studies.

The Mitochondria-Independent Cytotoxic Effect of Leflunomide on RPMI-8226 Multiple Myeloma Cell Line

Leflunomide, an anti-inflammatory agent, has been shown to be effective in multiple myeloma (MM) treatment; however, the mechanism of this phenomenon has not been fully elucidated. The aim of the study was to assess the role of mitochondria and dihydroorotate dehydrogenase (DHODH) inhibition in the cytotoxicity of leflunomide in relation to the MM cell line RPMI 8226. The cytotoxic effect of teriflunomide—an active metabolite of leflunomide—was determined using MTT assay, apoptosis detection, and cell cycle analysis. To evaluate DHODH-dependent toxicity, the cultures treated with teriflunomide were supplemented with uridine. Additionally, the level of cellular thiols as oxidative stress symptom was measured as well as mitochondrial membrane potential and protein tyrosine kinases (PTK) activity. The localization of the compound in cell compartments was examined using HPLC method. Teriflunomide cytotoxicity was not abolished in uridine presence. Observed apoptosis occurred in a mitochondria-independent manner, there was also no decrease in cellular thiols level. Teriflunomide arrested cell cycle in the G2/M phase which is not typical for DHODH deficiency. PTK activity was decreased only at the highest drug concentration. Interestingly, teriflunomide was not detected in the mitochondria. The aforementioned results indicate DHODH- and mitochondria-independent mechanism of leflunomide toxicity against RPMI 8226 cell line.