Apoptosis In Multiple Myeloma Cells Research Articles

Induction of m6A methylation in adipocyte exosomal LncRNAs mediates myeloma drug resistance

BackgroundTherapeutic resistance occurs in most patients with multiple myeloma (MM). One of the key mechanisms for MM drug resistance comes from the interaction between MM cells and adipocytes that inhibits drug-induced apoptosis in MM cells; MM cells reprogram adipocytes to morph into different characterizations, including exosomes, which are important for tumor-stroma cellular communication. However, the mechanism by which exosomes mediate the cellular machinery of the vicious cycle between MM cells and adipocytes remains unclear.MethodsAdipocytes were either isolated from bone marrow aspirates of healthy donors or MM patients or derived from mesenchymal stem cells. Co-culturing normal adipocytes with MM cells was used to generate MM-associated adipocytes. Exosomes were collected from the culture medium of adipocytes. Annexin V-binding and TUNEL assays were performed to assess MM cell apoptosis. Methyltransferase activity assay and dot blotting were used to access the m6A methylation activity of methyltransferase like 7A (METTL7A). RIP, MeRIP-seq, and RNA–protein pull down for assessing the interaction between long non-cording RNAs (LncRNAs) and RNA binding proteins were performed. Adipocyte-specific enhancer of zeste homolog 2 (EZH2) knockout mice and MM-xenografted mice were used for evaluating MM therapeutic response in vivo.ResultsExosomes collected from MM patient adipocytes protect MM cells from chemotherapy-induced apoptosis. Two LncRNAs in particular, LOC606724 and SNHG1, are significantly upregulated in MM cells after exposure to adipocyte exosomes. The raised LncRNA levels in MM cells are positively correlated to worse outcomes in patients, indicating their clinical relevancy in MM. The functional roles of adipocyte exosomal LOC606724 or SNHG1 in inhibition of MM cell apoptosis are determined by knockdown in adipocytes or overexpression in MM cells. We discovered the interactions between LncRNAs and RNA binding proteins and identified methyltransferase like 7A (METTL7A) as an RNA methyltransferase. MM cells promote LncRNA package into adipocyte exosomes through METTL7A-mediated LncRNA m6A methylation. Exposure of adipocytes to MM cells enhances METTL7A activity in m6A methylation through EZH2-mediated protein methylation.ConclusionThis study elucidates an unexplored mechanism of how adipocyte-rich microenvironment exacerbates MM therapeutic resistance and indicates a potential strategy to improve therapeutic efficacy by blocking this vicious exosome-mediated cycle.

Carfilzomib inhibits the proliferation and apoptosis of multiple myeloma cells by inhibiting STAT1/COX-2/iNOS signaling pathway.

BackgroundThe ubiquitin-proteasome pathway (UPP) plays a key role in the intracellular degradation of abnormal and misfolded proteins in eukaryotic cells. Multiple myeloma (MM) is a common hematologic malignancy caused by clonal expansion of malignant plasma cells. Proteasome-targeted drugs such as carfilzomib, which is a selective proteasome inhibitor (PI), could play an important role in the treatment of diseases such as MM.MethodsMM cells were treated with different concentrations of carfilzomib and apoptosis as well as the viability of MM cells were measured by flow cytometry analysis and MTT assay. We also measured the effect of carfilzomib on the proliferation of myeloma cells by DNA incorporation of the pyrimidine analog BrdU. The effect of carfilzomib on apoptosis was detected by immunofluorescence TUNEL staining and western blot. We also verified its effect on the STAT1/COX-2/iNOS pathway by western blot.ResultsCarfilzomib inhibited the growth of MM cells in a concentration-dependent manner, with the strongest inhibitory activity on RPMI-8226 cells. Carfilzomib also induced apoptosis of MM cells in a concentration-dependent manner, with the strongest effect on RPMI-8226. BrdU assay was then performed with RPMI-8226 cells, and the results showed that carfilzomib inhibited cell proliferation in a concentration-dependent manner. Immunofluorescence TUNEL staining and western blot assays showed that carfilzomib induced apoptosis in a dose-dependent manner, and promoted the expression of apoptosis-related proteins such as cleaved-caspase-3, cleaved-caspase-3, Bax and Bcl2. Western blot also verified that carfilzomib promoted STAT1 inhibition and subsequently inhibited COX-2 and iNOS.ConclusionsInhibition of the STAT1/COX-2/iNOS signaling pathway by carfilzomib not only inhibited MM cell proliferation, but was also an important mechanism of inducing MM cell apoptosis.

Trifluoperazine Synergistically Potentiates Bortezomib-Induced Anti-Cancer Effect in Multiple Myeloma via Inhibiting P38 MAPK/NUPR1.

Multiple myeloma (MM) is a common hematological malignancy. Bortezomib (BTZ) is a traditional medicine for MM treatment, but there are limitations for current treatment methods. Trifluoperazine (TFP) is a clinical drug for acute and chronic psychosis therapy. Lately, researchers have found that TFP can suppress tumor growth in many cancers. We attempted to study the effects of BTZ and TFP on MM in vivo and in vitro. We concentrated on the individual and combined impact of BTZ and TFP on the proliferation and apoptosis of MM cells via Cell Counting kit-8 assay, EdU assay, western blot, and flow cytometry. We found that combination therapy has a strong synergistic impact on MM cells. Combination therapy could induce cell arrest during G2/M phase and induce apoptosis in MM cells. Meanwhile, BTZ combined with TFP could play a better role in the anti-MM effect in vivo through MM.1s xenograft tumor models. Furthermore, we explored the mechanism of TFP-induced apoptosis in MM, and we noticed that TFP might induce MM apoptosis by inhibiting p-P38 MAPK/NUPR1. In summary, our findings suggest that TFP could synergistically enhance the BTZ-induced anti-cancer effect in multiple myeloma, which might be a promising therapeutic strategy for MM treatment.

Disruption of Lipid Raft Microdomains, Regulation of CD38, TP53, and MYC Signaling, and Induction of Apoptosis by Lomitapide in Multiple Myeloma Cells

Multiple myeloma (MM) is characterized by accumulation of a malignant clone of plasma cells in the bone marrow. Curative treatments are not yet available. Therefore, we undertook a drug repurposing approach to identify possible candidates from a chemical library of 1,230 FDA-approved drugs by virtual drug screening. As a target, we have chosen the non-receptor Bruton's tyrosine kinase (BTK) which is one of the main regulators of the MM biomarker CD38. In silico virtual screening was performed by using PyRx. Flow cytometry was applied for cell cycle and apoptosis analysis. Furthermore, protein and gene expression was determined by western blotting and microarray hybridization. Lipid raft staining was observed by confocal microscopy. The in silico identified lipid-lowering lomitapide presented with the strongest cytotoxicity among the top 10 drug candidates. This drug arrested the cell cycle in the G2/M phase and induced apoptosis in MM cells. Western blot analyses revealed that treatment with lomitapide induced cleavage of the apoptosis regulator PARP and reduced the expression of CD38, an integral part of lipid rafts. Using confocal microscopy, we further observed that lipid raft microdomain formation in MM cells was inhibited by lomitapide. In four MM cell lines (KMS-12-BM, NCI-H929, RPMI-8226, and MOLP-8) treated with lomitapide, microarray analyses showed not only that the expression of CD38 and BTK was down-regulated, but also that the tumor suppressor gene TP53 and the oncogene c-MYC were among the top deregulated genes. Further analysis of these data by Ingenuity pathway analysis (IPA) suggested that lomitapide interferes with the cross-talk of CD38 and BTK and apoptosis-regulating genes via TP53 and c-MYC. Lomitapide treatment led to disruption of lipid raft domains and induction of pro-apoptotic factors and might, therefore, be considered as a potential therapeutic agent in MM.

NBR2/miR-561-5p/DLC1 axis inhibited the development of multiple myeloma by activating the AMPK/mTOR pathway to repress glycolysis.

Long non-coding RNA NBR2 exerts a tumor-suppressive effect in a variety of cancers, but its role in multiple myeloma (MM) is unclear. This article will elucidate the role of NBR2 in MM. The expressions of NBR2, miR-561-5p, and deleted in liver cancer 1 (DLC1) in MM cell lines were determined by quantitative real time polymerase chain reaction (qRT-PCR). The regulatory relationship of the NBR2/miR-561-5p/DLC1 axis was predicted by bioinformatics and confirmed via a dual-luciferase reporter assay. The effect of NBR2 on the biological behavior of MM cells was verified by loss- and gain-of-function experiments (cell counting kit-8, colony formation, flow cytometry, extracellular acidification rate, and lactate production measurement). The effects of the NBR2/miR-561-5p axis on the biological behavior of MM cells, the activation of the AMPK/mTOR signaling pathway (western blot), and DLC1 expression (western blot) were verified by rescue experiments. The upregulation of NBR2 in MM cell lines induced a decrease in the viability, proliferation capacity, glycolysis, and lactic acid production, and an increase in apoptosis of MM cells. NBR2 regulated the biological behavior of MM cells and the activation of the AMPK/mTOR signaling pathway by targeting miR-561-5p. DLC1 was the target gene of miR-561-5p and the protein expression of DLC1 was regulated by the NBR2/miR-561-5p axis. Collectively, NBR2/miR-561-5p/DLC1 axis inhibits the development of MM by activating the AMPK/mTOR pathway to repress glycolysis.

Circ_SEC61A1 contributes to the progression of multiple myeloma cells via regulating miR-660-5p/CDK6 axis

BackgroundCircular RNAs (circRNAs) have been reported to play critical roles in the malignant progression of diverse human cancers, including multiple myeloma (MM). This study aimed to explore the functional role and underlying mechanism of circ_SEC61A1 in MM progression. MethodsQuantitative real-time polymerase chain reaction (qRT-PCR) assay was performed to detect the expression levels of circ_SEC61A1, microRNA (miR)-660-5p and cyclin-dependent kinase 6 (CDK6) mRNA. The localization of circ_SEC61A1 in MM cells was tested by the subcellular fractionation location assay. Actinomycin D assay was conducted to determine the characteristics of circ_SEC61A1. Cell proliferation was evaluated by colony formation assay, 5-ethynyl-2’-deoxyuridine (EdU) incorporation assay and 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Western blot assay was exploited to examine the expression of proteins. Cell migration and invasion were tested via transwell assay, and cell apoptosis was measured by flow cytometry analysis. Dual-luciferase reporter assay was utilized to confirm the interaction between miR-660-5p and circ_SEC61A1 or CDK6. ResultsCirc_SEC61A1 level was increased in MM tissues and cells. Circ_SEC61A1 was a stable circRNA and mainly located in cytoplasm. Circ_SEC61A1 silence restrained the proliferation, metastasis and expedited the apoptosis in MM cells. CDK6 was identified as the target of miR-660-5p, and circ_SEC61A1 sponged miR-660-5p to positively regulate CDK6 expression. The inhibitory impacts of circ_SEC61A1 knockdown on the progression of MM cells were mitigated by miR-660-5p inhibition. MiR-660-5p overexpression blocked the malignant phenotypes of MM cells by targeting CDK6. ConclusionOur study manifested that circ_SEC61A1 could accelerate MM progression at least partially through modulating miR-660-5p/CDK6 axis.

Andrographolide, isolated from Andrographis paniculata, induces apoptosis in monocytic leukemia and multiple myeloma cells via augmentation of reactive oxygen species production

Background: Andrographolide (Andro) is a diterpenoid component of the plant Andrographis paniculata that is known for its anti-tumor activity against a variety of cancer cells. Methods: We studied the effects of Andro on the viability of the human leukemia monocytic cell line THP-1 and the human multiple myeloma cell line H929. Andro was compared with cytosine arabinoside (Ara-C) and vincristine (VCR), which are well-established therapeutics against hematopoietic tumors. The importance of reactive oxygen species (ROS) production for the toxicity of each agent was investigated by using an inhibitor of ROS production, N-acetyl-L-cysteine (NAC). Results: Andro reduced the viability of THP-1 and H929 in a dose-dependent manner. H929 viability was highly susceptible to Andro, although only slightly susceptible to Ara-C. The agents Andro, Ara-C, and VCR each induced apoptosis, as shown by cellular shrinkage, DNA fragmentation, and increases in annexin V-binding, caspase-3/7 activity, ROS production, and mitochondrial membrane depolarization. Whereas Ara-C and VCR increased the percentages of cells in the G0/G1 and G2/M phases, respectively, Andro showed little or no detectable effect on cell cycle progression. The apoptotic activities of Andro were largely suppressed by NAC, an inhibitor of ROS production, whereas NAC hardly affected the apoptotic activities of Ara-C and VCR. Conclusions: Andro induces ROS-dependent apoptosis in monocytic leukemia THP-1 and multiple myeloma H929 cells, underlining its potential as a therapeutic agent for treating hematopoietic tumors. The high toxicity for (thus forming: The high toxicity for H929 cells, by a mechanism that is different from that of Ara-C and VCR, is encouraging for further studies on the use of Andro against multiple myeloma.) H929 cells, by a mechanism that is different from that of Ara-C and VCR, is encouraging for further studies on the use of Andro against multiple myeloma.

Inhibition of Cytosolic Phospholipase A2α Induces Apoptosis in Multiple Myeloma Cells.

Cytosolic phospholipase A2α (cPLA2α) is the rate-limiting enzyme in releasing arachidonic acid and biosynthesis of its derivative eicosanoids. Thus, the catalytic activity of cPLA2α plays an important role in cellular metabolism in healthy as well as cancer cells. There is mounting evidence suggesting that cPLA2α is an interesting target for cancer treatment; however, it is unclear which cancers are most relevant for further investigation. Here we report the relative expression of cPLA2α in a variety of cancers and cancer cell lines using publicly available datasets. The profiling of a panel of cancer cell lines representing different tissue origins suggests that hematological malignancies are particularly sensitive to the growth inhibitory effect of cPLA2α inhibition. Several hematological cancers and cancer cell lines overexpressed cPLA2α, including multiple myeloma. Multiple myeloma is an incurable hematological cancer of plasma cells in the bone marrow with an emerging requirement of therapeutic approaches. We show here that two cPLA2α inhibitors AVX420 and AVX002, significantly and dose-dependently reduced the viability of multiple myeloma cells and induced apoptosis in vitro. Our findings implicate cPLA2α activity in the survival of multiple myeloma cells and support further studies into cPLA2α as a potential target for treating hematological cancers, including multiple myeloma.

Effects of Artesunate Combined with Arsenious Acid on Proliferation and Apoptosis of Multiple Myeloma Cells via PI3K/AKT Signaling Pathway

To investigate the effect of artesunate and arsenous acid and their combination on the proliferation and apoptosis of human multiple myeloma cells and their mechanism. Human multiple myeloma cell line RPMI 8226 cells were cultured and treated with 0, 1, 2, 4, 8 nmol/L arsenous acid and 0, 40, 80, 160, 320 μmol/L artesunate, respectively. The inhibition of cell growth was detected by CCK-8 assay. The apoptosis rate was detected by flow cytometry. QPCR was used to detect the mRNA expression of cell proliferation and apoptosis-related factors. The expression of cell proliferation, apoptosis-related factors and PI3K/AKT pathway protein were detected by Western blot. CCK-8 assay showed that the growth of multiple myeloma cells was inhibited by arsenous acid and artesunate. The IC50 of arsenous acid was 1.96 nmol/L, and artesunate was 153.96 μmol/L. Flow cytometry showed that arsenous acid, artesunate, and their combination could significantly increase the apoptosis of multiple myeloma cells. QPCR and Western blot showed that the arsenous acid, artesunate, and their combination treatment could inhibit the expression of mRNA and protein of cell proliferation-related factors interleukin-6 and vascular endothelial growth factor (P<0.05), inhibit apoptosis-related factor BCL-xl and promote the expression of mRNA and protein of Bax (P<0.05), and reduce the protein expression of p-PI3K and p-AKT in PI3K/AKT signaling pathway (P<0.05). Moreover, the combination effect of the two drugs on cell apoptosis and the above mentioned factors was obviously stronger than that of the two drugs alone. Artesunate combined with arsenous acid inhibits proliferation and promotes apoptosis of tumor cells through PI3K/AKT signaling pathway, and is superior to the effect of two drugs alone.

A novel PDK1 inhibitor, JX06, inhibits glycolysis and induces apoptosis in multiple myeloma cells

Pyruvate dehydrogenase kinase 1 (PDK1) is a Ser/Thr kinase that inactivates mitochondrial pyruvate dehydrogenase (PDH), leading to switch of glucose metabolism from mitochondrial oxidation to aerobic glycolysis. We previously reported that PDK1 inhibition is a potent therapeutic strategy in multiple myeloma (MM). However, availability of PDK1 inhibitors, which are effective at low concentrations, are limited at present, making PDK1 inhibition difficult to apply in the clinic. In the present study, we examined the efficacy and mechanism of action of JX06, a novel PDK1 inhibitor, against MM cells. We confirmed that PDK1 is highly expressed in normal plasma cells and MM cells using publicly available gene expression datasets. JX06 suppressed cell growth and induced apoptosis against MM cells from approximately 0.5 μM JX06 treatment reduced PDH phosphorylation, suggesting that JX06 is indeed inhibiting PDK1. Intracellular metabolite analysis revealed that JX06 treatment reduced metabolites associated with glucose metabolism of MM cells. Additionally, JX06 in combination with a well-known proteasome inhibitor, bortezomib, significantly increased MM cell death, which raises the possibility of combination use of JX06 with proteasome inhibitors in the clinic. These findings demonstrate that PDK1 can be potentially targeted by JX06 in MM through glycolysis inhibition, leading to a novel therapeutic strategy in MM.

Root Bark of Morus alba L. and Its Bioactive Ingredient, Ursolic Acid, Suppress the Proliferation of Multiple Myeloma Cells by Inhibiting Wnt/β-Catenin Pathway.

The root bark of Morus alba L. has cytotoxic activity against several types of cancer cells. However, little is known about its chemopreventive mechanisms and bioactive metabolites. In this study, we showed that M. alba L. root bark extracts (MRBE) suppressed β-catenin response transcription (CRT), which is aberrantly activated in various cancers, by promoting the degradation of β-catenin. In addition, MRBE repressed the expression of the β-catenin/T-cell factor (TCF)-dependent genes, cmyc and cyclin D1, thus inhibiting the proliferation of RPMI-8226 multiple myeloma (MM) cells. MRBE induced apoptosis in MM cells, as evidenced by the increase in the population of annexin VFITC- positive cells and caspase-3/7 activity. We identified ursolic acid in MRBE through LC/mass spectrum (MS) and observed that it also decreased intracellular β-catenin, c-myc, and cyclin D1 levels. Furthermore, it suppressed the proliferation of RPMI-8226 cells by stimulating cell cycle arrest and apoptosis. These findings suggest that MRBE and its active ingredient, ursolic acid, exert antiproliferative activity by promoting the degradation of β-catenin and may have significant chemopreventive potential against MM.

MiR-335-laden B Cell-Derived Extracellular Vesicles Promote SOX4-Dependent Apoptosis in Human Multiple Myeloma Cells.

Multiple myeloma (MM) is characterized by the accumulation of malignant plasma cells in the bone marrow. Despite novel therapies, MM still remains an incurable cancer and new strategies are needed. Increased expression of the transcription factor Sex-determining region Y-related high-mobility-group box transcription factor 4 (SOX4) has been correlated with tumor development and progression through a variety of distinct processes, including inhibition of apoptosis, increased cell invasion and metastasis, and induction and maintenance of cancer-initiating cells. The role of SOX4 in MM is largely unknown. Since SOX4 is a known target of miR-335, we used miR-335 to assess whether SOX4 modulation could promote apoptosis in MM cells. Using an MM cell model we show that miR-335 acts both on SOX4-related genes (AKT, PI3K) and hypoxia-inducible factor 1-alpha (Hif1-α). In addition, we show miR-335-laden extracellular vesicles induced in B cells (iEVs) are also effective in targeting SOX4, causing apoptosis. Collectively, we propose that miR-335-laden iEVs could be developed as a novel form of gene therapy in MM.

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 DisclosuresNo relevant conflicts of interest to declare.

Adenylate Kinase 2 Is a Selective Multiple Myeloma Cell Dependency That Is Preferentially Essential in NSD2-Overexpressing Cells

Background: Multiple myeloma (MM) is the second most common hematologic malignancy and remains incurable. Advances in MM therapy have come about due to therapies that target vulnerabilities of the plasma cell such as high protein load (proteasome inhibitors; PIs), dependence on specific transcription factors such as IKZF1 and IKZF3 which are degraded by immunomodulatory drugs (IMiDs), the susceptibility of B cells to glucocorticoids and the presence of specific B cell markers that can serve as targets for monoclonal antibodies and CAR-T cells. Gene editing screens offer a way to identify novel MM therapeutic targets.Objectives: The molecular heterogeneity of MM imposes challenges to discovering generalized therapeutic targets. Therefore, identification of selective dependencies associated with a particular recurrent genetic lesion is a promising strategy to personalize therapy. Here, we aim to identify vulnerabilities linked to the chromosomal translocation t(4;14), a recurrent rearrangement in MM characterized by overexpression of the histone methyltransferase NSD2.Methods: Genome-wide CRISPR-based loss-of-function screens were performed in NSD2-high and low isogenic cells derived from the t(4;14) MM cell line KMS11 to define selective dependencies associated with NSD2 overexpression. High-confidence hits were corroborated by in vitro competitive growth assays where individual candidates are genetically knocked out or suppressed or chemically inhibited. Detailed investigation was performed for selected candidates using various molecular and biochemical assays to elucidate mechanisms by which these genes contribute to MM cell fitness.Results: A fitness screen in NSD2-high and low isogenic MM cells identified 1118 essential genes which are common between the cell pair. We further revealed 282 genes whose loss is more detrimental to cells overexpressing NSD2 and 139 genes that are preferentially essential when NSD2 levels are low. Pathway analysis of NSD2-high selectively essential genes indicated that these cells are more dependent on mitochondrial processes including oxidative phosphorylation. Although proteasomal degradation is essential for all MM cells, our screens indicated that NSD2-high cells are more dependent on the proteasome, which was validated by increased sensitivity to the PI bortezomib. One of the high-confidence selective NSD2-high hits was the mitochondrial adenine nucleotide regulator adenylate kinase 2 (AK2). Analyzing the dependence of hundreds of human cell lines on AK2 using the cancer dependency map portal (depmap.org/portal/), we found that AK2 is not a common essential gene. The top enriched linages with AK2 dependency included MM with notable representation of t(4;14)-positive cell lines. Analysis of the multiple myeloma research foundation (MMRF)-CoMMPass data demonstrated that MM patients with high NSD2 expression, despite poor prognosis, display enhanced overall survival when AK2 levels are low. In vitro competitive growth assays in NSD2-high and low MM cells confirmed the increased dependence of NSD2-overexpressing cells on AK2. In addition, NSD2-high MM cells displayed elevated sensitivity to AK2 inhibitors. Moreover, AK2 knockdown in t(4;14) MM cell lines increased sensitivity to the PI bortezomib. Mechanistically, we showed that AK2 disruption activates apoptotic unfolded protein response (UPR) signaling in MM cells. Metabolomic profiling in NSD2-high and low MM cells revealed accumulation of purine metabolites and reduction of pyrimidine metabolites upon NSD2 overexpression. Intriguingly, purine supplementation rescued MM cell depletion due to AK2 loss. These observations suggested that MM cells, especially those with NSD2 overexpression, are addicted to elevated purine levels and that lethality of MM cells upon AK2 loss is due to perturbed purine metabolism. How impaired purine metabolism activates UPR signaling is currently under investigation.Conclusions: Our work indicated that NSD2 overexpression resulting from chromosomal translocation t(4;14), despite its oncogenic role, generates metabolic dependencies in MM cells. Our findings further suggest that inhibition of AK2, a mitochondrial enzyme involved in purine metabolism, can induce UPR-mediated apoptosis in MM cells and could be used in combination with PI therapy to treat MM patients with t(4;14) translocations. DisclosuresLicht: Epizyme: Research Funding.

Fatty Acid Binding Proteins As a Novel Therapeutic Target in Multiple Myeloma

The fatty acid binding protein (FABP) family is instrumental in fatty acid oxidation, lipid shuttling and signal transduction, but the roles of FABPs in multiple myeloma (MM) have not been explored previously, despite these being safely targetable and holding great translational promise. Using the Broad's Cancer Dependency Map (DepMap) Crispr screening tool, we found that MM, and in fact almost every cancer, shows dependency on FABP5. In patient microarray datasets, we found that increased FABP5 expression in MM cells correlates with poor overall survival (OS) and shorter relapse free survival (Zhan et al., 2006., Mulligan et al., 2006., Carrasco et al., 2006). Interestingly, MM cells from “proliferative” (most aggressive) patient subgroups have the highest expression of FABP5 when comparing the molecular MM subtypes (Zhan et al. 2006). We also found that MM cells from relapsed patients have increased FABP5 expression versus newly-diagnosed patients (Chng et al., 2007). Finally, we analyzed 779 patients in the MMRF CoMMpass dataset and confirmed worse progression-free survival (PFS) and OS in patients with higher than mean FABP5 expression levels in their tumor cells (log-rank-value for high vs. low expression <0.0001 for both PFS and OS) (Fig. 1). Overall, our data strongly suggest that elevated FABP5 is a novel risk factor for poor MM prognosis and that targeting the FABP family holds great promise as a new therapeutic avenue.We next demonstrated that FABP5 is the most highly expressed FABP family member in myeloma cell lines and patient samples, and that inhibition of FABP proteins with two inhibitors, BMS309403 and SBFI-26, negatively impacts MM cell cycle and cell number, and induces apoptosis in human (MM.1S, RPMI-8226, H929, and OPM2) and mouse (5TGM1 and Vk*Myc) MM cells in vitro in a dose- and time-dependent manner. Next, using the MM.1S/SCID-beige xenograft and the syngeneic 5TGM1-TK/C57BL/KaLwRij (KaLwRij) models, we found that pharmacological FABP inhibition improves survival and decreases tumor burden. In both models, mice were tail vein inoculated with MM cells (5x10^6 MM.1S or 1x10^6 5TGM1-TK) and started on intraperitoneal treatments the following day. SCID-beige mice received 5 mg/kg BMS309403, 1 mg/kg SBFI-26, both, or vehicle 3x/week; KaLwRij mice received 5 mg/kg BMS309403 or vehicle 3x/week. Mice were analyzed biweekly with live bioluminescent imaging of whole mice starting 10-12 days post inoculation. In the SCID-beige model, all treatments reduced tumor burden starting at day 21 and led to significantly increased survival, compared to the vehicle. Similar trends were also seen in the KaLwRij group for BMS309403 treatments; SBFI-26 and combinations are now being explored in this model.After demonstrating strong evidence that FABP inhibition reduced tumor growth in vitro and in vivo, we performed RNA-sequencing of human MM.1S cells treated with BMS309403, SBFI-26, both inhibitors, or neither, at 50 μM for 24 hours and analyzed gene and pathway alternations using dbString and Ingenuity pathway analyses with cutoffs of FC≤|1.2|. RNA-sequencing pathway analysis revealed that single or combination treatment decreased IRE1-mediated unfolded protein response, endoplasmic reticulum (ER) unfolded protein response, and chromatin silencing. At the individual gene level, major oncogenic genes demonstrated decreased expression including c-myc, KRAS and XBP1. Western blot confirmed that Myc protein was decreased with FABP inhibition in MM cells, illustrating a novel role for FABP proteins in tumor cell survival and proliferation. To confirm that FABP inhibition has negative effects on MM cells, Myc signaling and ER stress, and to rule out inhibitor off-target effects, we also generated FABP5 knockout MM.1S cell pools using Crispr/Cas9, which we are analyzing with RealTime-Glo and RNA-sequencing.In conclusion, FABP inhibition slowed tumor growth and induced tumor cell death in vitro and in two mouse models. RNA-sequencing revealed two potential, novel mediators of FABP action in MM cells: Myc pathway inhibition and enhanced ER stress. Dysfunction in these two pathways via FABP inhibitors potentially triggered cell cycle arrest, decreased proliferation, and increased apoptosis in MM cells. Combined with clinical data from CoMMpass and other datasets, this suggests that FABP inhibition in MM is an untapped therapeutic avenue that should be further explored and developed. [Display omitted] DisclosuresMarinac: GRAIL Inc: Research Funding; JBF Legal: Consultancy.

Final Overall Survival Results from BELLINI, a Phase 3 Study of Venetoclax or Placebo in Combination with Bortezomib and Dexamethasone in Relapsed/Refractory Multiple Myeloma

Final Overall Survival Results from BELLINI, a Phase 3 Study of Venetoclax or Placebo in Combination with Bortezomib and Dexamethasone in Relapsed/Refractory Multiple Myeloma

P-079: IL10R inhibition reprograms tumor-associated macrophages and reverses drug resistance in Multiple Myeloma

<h3>Background</h3> Multiple myeloma (MM) is a cancer of plasma cells within the bone marrow (BM) and remains to be incurable. Tumor-associated macrophages (TAMs) are a major immunosuppressive component in the tumor microenvironment and display the pro-tumor M2 phenotype, supporting tumor proliferation, survival, and drug resistance. Targeting TAMs to block their pro-tumor functions represent a promising class of cancer immunotherapy. IL10 is a key immunosuppressive cytokine that leads to recruitment and development of TAMs. In this study, we investigate the role of IL10 in MM TAM development, and hypothesize that inhibition of IL10/IL10R signaling in TAMs will reprogram them for MM killing and overcome drug resistance. <h3>Methods</h3> Macrophage (MPP) polarization to M1 (CD80+) or M2 (CD163+) is determined by antibody staining and flow cytometry. In vitro and ex vivo studies were carried out in our lab's proprietary 3D tissue-engineered BM model (3DTEBM). First, we compared TAM M2/M1 ratio as well as IL10 cytokine levels in the BM of healthy and MM subjects. To study the role of IL10 in TAM development, we co-cultured MΦ with increasing MM cells or rhIL10 in the 3DTEBM for 3 days and analyzed the resulting M2/M1 ratio. To determine the effect of IL10R inhibition, MM and MΦ co-cultures, or unsorted patient BM were treated with or without 5ug/ml α-IL10R monoclonal antibody (mAb), and TAM phenotype was analyzed after 3 days. Additionally, humanized huCD34-NCG mice were inoculated with MM cells and treated with α-IL10R mAb 3 times/week for 2 weeks; mice femurs were flushed and BM cells were analyzed for TAM phenotype. Finally, we investigated α-IL10R mAb as combination treatment with chemotherapy in MM-GFP and MΦ co-cultures; MM survival was determined by count of GFP+ cells and apoptosis was determined by Annexin/PI staining with flow cytometry. <h3>Results</h3> Compared to healthy subjects, M2/M1 ratio in MM patient TAMs were 3-fold higher, and BM IL10 level was 3.8-fold higher. Additionally, increasing MM cell ratio in co-cultures induced TAM polarization to M2, and was contributed by IL10. Importantly, α-IL10R mAb was able to reverse MM induced M2 TAM phenotype in vitro, ex vivo, and in vivo. Moreover, the presence of TAMs resulted in significant MM proliferation and chemotherapy resistance toward lenalidomide and dexamethasone. However, combination treatment with α-IL10R mAb completely reversed the drug resistance and abrogated cancer cells in vitro, partially due to induction of apoptosis in MM cells. <h3>Conclusions</h3> In summary, we have shown that MM induced M2 polarization of TAMs in an IL-10 dependent fashion, and disruption of IL10/IL10R signaling reversed M2 phenotype in TAMs, and overcame TAM-supported drug resistance. Future studies are warranted to examine mechanism behind overcoming drug resistance as well as the effect of α-IL10R immunotherapy in vivo and in patients.

P-088: Indirubin-3’-monoxime acts as an alternative proteasome inhibitor and confers new regimens for the treatment of Multiple Myeloma

<h3>Background</h3> Multiple myeloma (MM) is still an incurable malignancy of plasma cells. Despite the therapeutic benefit of the proteasome inhibitor (PI) bortezomib, drug resistance still accounts for majority of tumor relapses and cancer-related death in MM patients. Thus, exploring the mechanism underlying BTZ resistance and investigating a novel treatment strategy are urgent requisites for clinical practise. Indirubin-3'-monoxime (Id-3) is an active ingredient of a traditional Chinese medicine. <h3>Method</h3> In the present study, we firstly investigated Id-3 effects on myeloma treatment in patient primary samples and myeloma cell lines. We also investigated the molecular mechanism of Id-3 treatment on MM cells via in vivo and in vitro study. <h3>Results</h3> We first reported that Id-3 is an effective cytotoxicity agentia against MM cells growth by inducing the MM cells apoptosis. Id-3 treatment also effectively induces the cell death in bortezomib-resistant MM cell lines and primary myeloma patient samples. Stringently, we found there is a synergistic anti-MM effect between Id-3 and bortezomib. The treatment with lower dosage combination of Id-3 and bortezomib exhibited a notably inhibition of the growth of myeloma cells both in xenograft mouse model and MM-PDX model. Mechanism study revealed that Id-3 treatment efficiently inhibited the chymotrypsin-like and caspase-like activity of proteasome in MM cells. The level of the proteasome 11s activator, PA200 (PSME4) and PA28y (PSME3) was significantly down-regulated both in mRNA and protein level. GEO datasets analysis showed that the high levels of PSME4 and PSME3 were over-expressed in relapsed patients and MM cell lines which correlated with the drug-resistance of MM cells. Kaplan-Meier analysis showed that patients with high-levels of PSME3 or PSME4 had the shortened survival. Knocking-down the level of PSME4 or PSME3 by shRNA noteworthy inhibited the cell growth and enhanced the sensitivity to bortezomib of MM cells. In vivo study also found that down-regulated the level of PSME3 or PSME4 can suppress the proliferation of MM cells and prolong the survival of MM-bearing mouse. <h3>Conclusions</h3> Taken together, our study supported that Id-3 is an effective agentia against the survival of MM cells both in bortezomib-sensitive and -resistance ones. We further identified that the 11s proteasome activators, PSME3 and PSME4, were down-regulated effectively by Id-3 treatment. High-levels of PSME3 or PSME4 were correlated with the inferior outcome of MM patients. Down-regulating PSME3 and PSME4 by Id-3 or shRNA could efficiently suppressed the growth of MM cells and improve the survival of MM-bearing mice. Our study provides the rational evidence for the clinical application of Id-3 in the treatment of MM.

Apoptotic Extracellular Vesicles Ameliorate Multiple Myeloma by Restoring Fas-Mediated Apoptosis.

Apoptosis is critical for maintaining bodily homeostasis and produces a large number of apoptotic extracellular vesicles (apoEVs). Several types of cancer cells display reduced expression of Fas on the cell surface and are thus capable of escaping Fas ligand-induced apoptosis. However, it is unknown whether normal cell-derived apoEVs can regulate tumor growth. In this study, we show that apoEVs can induce multiple myeloma (MM) cell apoptosis and inhibit MM cell growth. Systemic infusion of mesenchymal stem cell (MSC)-derived apoEVs significantly prolongs the lifespan of MM mice. Mechanistically, apoEVs directly contact MM cells to facilitate Fas trafficking from the cytoplasm to the cell membrane by evoking Ca2+ influx and elevation of cytosolic Ca2+. Subsequently, apoEVs use their Fas ligand to activate the Fas pathway in MM cells, leading to the initiation of apoptosis. This study identifies the role of apoEVs in inducing MM apoptosis and suggests a potential for apoEVs to treat MM.

LBX2-AS1 as a Novel Diagnostic Biomarker and Therapeutic Target Facilitates Multiple Myeloma Progression by Enhancing mRNA Stability of LBX2.

Objective: Multiple myeloma (MM) represents a common age-associated malignancy globally. The function and underlying mechanism of antisense lncRNA LBX2-AS1 remain ambiguous in multiple myeloma (MM). Herein, we aimed to observe the biological implication of this lncRNA in MM.Methods: RT-qPCR was employed to examine circulating LBX2-AS1 and LBX2 in 60 paired MM and healthy subjects. Correlation between the two was analyzed by Pearson test. Under transfection with shLBX2-AS1, proliferation and apoptosis were evaluated in MM cells through CCK-8, colony formation and flow cytometry. LBX2 expression was examined in MM cells with shLBX2-AS1 or pcDNA3.1-LBX2 transfection. Following treatment with cycloheximide or actinomycin D, LBX2 expression was examined in pcDNA3.1-LBX2-transfected MM cells at different time points. Rescue assays were then presented. Finally, xenograft tumor models were established.Results: Circulating LBX2-AS1 was up-regulated in MM patients and positively correlated to LBX2 expression. Area under the curve (AUC) of LBX2-AS1 expression was 0.7525. Its up-regulation was also found in MM cells and primarily distributed in cytoplasm. LBX2-AS1 knockdown distinctly weakened proliferative ability and induced apoptosis in MM cells. Overexpressing LBX2-AS1 markedly strengthened LBX2 expression by increasing its mRNA stability. Rescue assays showed that silencing LBX2-AS1 distinctly weakened the pcDNA3.1-LBX2-induced increase in proliferation and decrease in apoptosis for MM cells. Silencing LBX2-AS1 markedly weakened tumor growth.Conclusion: Our data demonstrated that circulating LBX2-AS1 could be an underlying diagnostic marker in MM. Targeting LBX2-AS1 suppressed tumor progression by affecting mRNA stability of LBX2 in MM. Hence, LBX2-AS1 could be a novel therapeutic marker against MM.