Resistant Multiple Myeloma Cell Lines Research Articles

New Insights for Polyphenolic Compounds as Naturally Inspired Proteasome Inhibitors.

Polyphenols, an important class of natural products, are widely distributed in plant-based foods. These compounds are endowed with several biological activities and exert protective effects in various physiopathological contexts, including cancer. We herein investigated novel potential mechanisms of action of polyphenols, focusing on the proteasome, which has emerged as an attractive therapeutic target in cancers such as multiple myeloma. We carried out a structure-based virtual screening study using the DrugBank database as a repository of FDA-approved polyphenolic molecules. Starting from 86 polyphenolic compounds, based on the theoretical binding affinity and the interactions established with key residues of the chymotrypsin binding site, we selected 2 promising candidates, namely Hesperidin and Diosmin. The further assessment of the biologic activity highlighted, for the first time, the capability of these two molecules to inhibit the β5-proteasome activity and to exert anti-tumor activity against proteasome inhibitor-sensitive or resistant multiple myeloma cell lines.

STMN1 promotes cell malignancy and bortezomib resistance of multiple myeloma cell lines via PI3K/AKT signaling

ABSTRACT Background This study investigates the biological functions of Stathmin1 (STMN1) involving drug resistance and cell proliferation in multiple myeloma (MM) and its related mechanisms. Methods Bone marrow aspirates were collected from 20 MM patients, and the bone marrow mononuclear cells (BMMCs) were separated by Ficoll-Hypaque density gradient centrifugation. Blood samples of 20 patients with monoclonal gammopathy of undetermined significance (MGUS) and 20 healthy donors were collected. Normal plasma cells sorted from the peripheral blood of MGUS patients and healthy subject as controls. Two bortezomib (BTZ)-resistant MM cell lines were established, namely NCI-H929/BTZ and KM3/BTZ cells, and then transfected with lentiviruses packaging sh-STMN1 to knock down STMN1 level in BTZ-resistant cells. Expression of STMN1 was assessed by RT-qPCR and western blotting. CCK-8 assays were performed to assess 50% growth inhibition (IC50) values. Green fluorescent protein in BTZ-resistant cells infected with lentiviruses was observed by fluorescence microscopy. Cell viability, proliferation, cell cycle, and apoptosis were evaluated through MTT assays, colony formation assays, flow cytometry analyses, and TUNEL staining. Results STMN1 was upregulated in MM cells and bone marrow aspirates of MM patients. Additionally, STMN1 depletion attenuated BTZ resistance in MM cells. Moreover, downregulation of STMN1 limited the malignant phenotypes of BTZ-resistant cells. Mechanistically, the PI3K/Akt signaling was inactivated by STMN1 downregulation in BTZ-resistant cells. Conclusion STMN1 silencing inhibits cell proliferation and BTZ resistance in MM by inactivating the PI3K/Akt signaling.

Identification of potential molecular players and therapeutic drug molecules in Melphalan resistant Multiple myeloma by integrated bioinformatics analysis

Multiple myeloma (MM), second most common haematological malignancy, still remains incurable with the acquiring of drug resistance. Melphalan therapy which has been used as one of the key therapies against MM, is impeded by the occurrence of Melphalan resistance. The precise underlying mechanism of this acquired Melphalan resistance in MM is not yet well deciphered. Therefore, this present study was aimed to identify the differentially expressed genes (DEGs) associated micro RNAs (miRNAs) and transcription factors (TFs) from the microarray dataset of Melphalan resistant and Melphalan sensitive MM cell lines, obtained from Gene Expression Omnibus (GEO) database. DEGs were analysed using GEO2R tool from the dataset. Then the gene ontology (GO) and pathway enrichment analysis were executed by using DAVID database. Protein-protein interaction (PPI) network of DEGs was constructed and analysed by using STRING database and Cytoscape tool. Genetic alterations in DEGs were studied through COSMIC database. Network of interaction of DEGs and miRNAs as well as TFs was constructed and analysed by using mirDIP, TRRUST and miRNet tools. Drug gene interaction was examined to identify potential drug molecules by DGIdb tool. Various DEGs that might play pivotal role in Melphalan resistant MM, were detected and selected for further analysis. miRNA analysis detected hsa-mir- 21-3p, hsa-mir-27a-5p, hsa-mir-129-2-3p that could interact with maximum target DEGs. One TF, STAT1 was found to regulate the expression of selected DEGs. The whole study may give a better understanding about the Melphalan resistance in MM.

Multiomics Analysis of IMiD/CELMoD Resistant Multiple Myeloma Models Uncovers Novel and Targetable Vulnerabilities in the SREBP Lipid Synthesis Pathway

Background Acquired resistance to immunomodulatory drugs (IMiDs)/cereblon (CRBN) E3 ligase modulators (CELMoDs) is a major challenge in multiple myeloma (MM) treatment. These drugs bind to the CRBN component of the CRL4CRBN E3 ubiquitin ligase and designate a new set of substrates for degradation via the proteasome. Generation of resistance is frequently associated with decreased CRBN expression and this is due to genetic alteration in ~1/3rd of patients. Alternative drivers of the low CRBN state, and other mechanisms of resistance, need to be elucidated. To tackle this complex problem, MM models with acquired IMiD/CELMoD resistance were generated and multiomics analysis performed. Methods IMiD/CELMoD resistant human MM cell lines were generated by treating MM1s and H929 cells with lenalidomide (Len), pomalidomide (Pom) or iberdomide (Iber) at ~10x GI50 concentration for ~12 weeks until resistance was achieved. Resistant cell lines and controls were characterised by whole exome sequencing (WES), RNA-Seq and proteomics. A genome-wide loss-of-function CRISPR screen (Brunello library) was carried out in Iber-resistant MM1s; gene effect scores were calculated with the Chronos model and compared to parental MM1s using data from the Broad Institute DepMap portal. Pathway analysis was performed using g:Profiler. The Membrane Bound Transcription Factor Peptidase, Site 1 (MBTPS1) inhibitor PF-429242 was used to inhibit activation of the Sterol Regulatory-Element Binding Protein (SREBP) pathway and the effect on cell viability measured using CellTiter-Blue®. The Multiple Myeloma Research Foundation CoMMpass database was used to explore the correlation between mRNA expression of SREBP pathway genes in newly diagnosed patients and progression-free survival (PFS). Results All models were resistant to the IMiD/CELMoD with which they were generated (to 20-100x the GI50 concentration) and exhibited cross-resistance to other IMiDs/CELMoDs. Functional assays showed that well-characterised effects of IMiD/CELMoD treatment, e.g. degradation of Ikaros/Aiolos, were abrogated. WES showed Pom-resistant MM1S and Pom- and Iber-resistant H929 cells had acquired mutations in CRBN predicted to have a high impact on function. Len-resistant H929 cells had new copy number loss at the CRBN locus but Len- and Iber-resistant MM1s cells had no genetic changes in CRBN. CRBN protein expression was reduced in all resistant lines compared to control (log2 fold changes (FCs) by proteomics ranging from -0.25 to -1.95, adj p <0.05). Together these models display diverse resistance mechanisms, reflecting the clinical picture. Proteomic analysis of the resistant lines identified key changes in the SREBP pathway. The proteomes of the resistant lines were heterogeneous and the only pathway with common significant enrichment was SREBP/fatty acid metabolism (on analysis performed per cell line of proteins with significantly decreased expression). Stearoyl-CoA Desaturase (SCD), a key effector of the SREBP pathway, was one of only 5 proteins with significantly altered expression in all 6 cell lines compared to control. SCD had log2FCs ranging from -0.37 to -1.50 (adj p <0.05). Other key pathway members are shown in Figure 1. A genome-wide CRISPR screen using Iber-resistant MM1s cells identified potential new dependencies (gene effect score <-1 in resistant cells and >-0.5 in parental cells) in 47 genes including SCD and MBTPS1. MBTPS1 is critical for activation of the SREBP pathway and demonstrated one of the largest changes in gene effect (-1.4 vs -0.3). The activity of PF-429242, an inhibitor of MBTPS1, was explored in the resistant lines. A significant difference in GI50 between Iber-resistant H929 cells and control was found (1uM and >10uM respectively) with greater activity in the resistant cells. The same pattern was observed with the other resistant H929 cell lines, but not the resistant MM1s. Other pathway inhibitors are being explored. The expression of genes encoding SREBP pathway components was explored in the CoMMpass dataset. High SCD or MBTPS1 mRNA expression was associated with significantly worse PFS (logrank p<0.01), providing a further rationale for targeting the pathway. Conclusions Models representing multiple different IMiD/CELMoD resistance mechanisms have unified alterations in the SREBP pathway, highlighting a role in resistance biology and potential novel and targetable vulnerabilities. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Investigating the potential molecular players and therapeutic drug molecules in carfilzomib resistant multiple myeloma by comprehensive bioinformatics analysis

Multiple myeloma (MM), second most common hematological malignancy, still remains beyond cure because of acquirement of drug resistance. Proteasome inhibitor such as carfilzomib (Cfz) therapy which has been used as one of the key therapies against MM recently, is obstructed by the incidence of Cfz resistance. The underlying mechanism of this acquired Cfz resistance in MM is very little understood. Therefore, the current study was aimed to investigate the differentially expressed genes (DEGs), associated micro RNAs (miRNAs), and transcription factors (TFs) from the microarray datasets of Cfz resistant and Cfz sensitive MM cell lines, obtained from Gene Expression Omnibus (GEO) database. DEGs were detected using GEO2R tool from two datasets and common DEGs were identified through Venn diagram. Gene ontology (GO) and pathway enrichment analysis were performed on DAVID database. Through STRING database and Cytoscape tool, protein–protein interaction (PPI) network of DEGs was constructed. Genetic alterations in DEGs were investigated using COSMIC database. Interaction network between DEGs and miRNAs as well as TFs were obtained and constructed by using mirDIP, TRRUST, and miRNet tools. Drug gene interaction analysis was performed to identify potential drug molecules on DGIdb tool. Several common DEGs were identified in Cfz resistant MM. PDGF, VEGF, and Wnt signaling pathways were significantly enriched and might be involved in MM progression. miRNA analysis identified hsa-mir-124-3p, hsa-mir-26a-5p that can target DEGs. Various drug molecules such as dabrafenib, vemurafenib, and venetoclax that could potentially attenuate the MM pathophysiology, were detected. The entire study might provide a new understanding about the Cfz resistance in MM.

Deubiquitylase USP12 induces pro-survival autophagy and bortezomib resistance in multiple myeloma by stabilizing HMGB1.

Despite the establishment of novel therapeutic interventions, multiple myeloma (MM) remains invariably incurable due to development of drug resistance and subsequent relapse, which are attributed to activation of oncogenic pathways such as autophagy. Deubiquitinating enzymes (DUBs) are promising targets to overcome resistance to proteasome inhibitor-based treatment. Ubiquitin-specific protease-12 (USP12) is a DUB with a known prognostic value in several cancers. We found that USP12 protein levels were significantly higher in myeloma patient samples than in non-cancerous human samples. Depletion of USP12 suppressed cell growth and clonogenicity and inhibited autophagy. Mechanistic studies showed that USP12 interacted with, deubiquitylated and stabilized the critical autophagy mediator HMGB1 (high mobility group box-1) protein. Knockdown of USP12 decreased the level of HMGB1 and suppressed HMGB1-mediated autophagy in MM. Furthermore, basal autophagy activity associated with USP12/HMGB1 was elevated in bortezomib (BTZ)-resistant MM cell lines. USP12 depletion, concomitant with a reduced expression of HMGB1, suppressed autophagy and increased the sensitivity of resistant cells to BTZ. Collectively, our findings have identified an important role of the deubiquitylase USP12 in pro-survival autophagy and resultant BTZ resistance in MM by stabilizing HMGB1, suggesting that the USP12/HMGB1 axis might be pursued as a potential diagnostic and therapeutic target in human MM.

744 Multi-armed myxoma virus has therapeutic potential for treatment of multiple myeloma

BackgroundDespite improvements with new therapeutics, multiple myeloma (MM) patients still relapse and become refractory. Myxoma virus (MYXV) selectively replicates in human tumor cells and stimulates the immune system. MYXV selectively kills human patient MM cells and spares normal progenitors. MYXV also eradicates growth of a disseminated mouse MM in vivo. MYXV is a large, double stranded DNA poxvirus, and has a genome size amenable to insertion of multiple transgenes. We generated MYXV carrying IL-12 and decorin. IL-12 is an immune modulator that activates T- and NK-cells. Cellular responses to decorin include tumor cell intrinsic signaling effects, tumor matrix modulation, and inhibition of the TGF-beta pathway. This represents a promising therapeutic option for MM patients that do not respond well to immunotherapy. The current work suggests MYXV armed with IL-12 and decorin could be an effective anti-MM therapy.MethodsCytotoxicity assays were performed using a cell viability assay. Transgene expression levels were analyzed by microscopy, flow cytometry, and ELISA.ResultsHuman MM cell line U266 infected with MYXV (vMYX-hIL-12/Dec) carrying human IL-12, decorin, and green fluorescent protein (GFP) produced transgenes in a dose and time responsive manner. A panel of human MM cell lines was infected with vMYX-hIL-12/Dec and transgene expression in supernatant, cell killing EC50, and GFP levels were evaluated. Sensitive and resistant human MM cell lines were identified. The comparison of replication, cell killing capacity, and transgene expression highlighted the independent importance of these mechanisms in overall activity.ConclusionsThe current work describes the oncolytic activity and transgene expression following exposure to vMYX-hIL-12/Dec in human MM cell lines in vitro. Our initial studies suggest there is significant value in pursuing vMYX-hIL-12/Dec and other armed MYXV as a new approach towards MM therapy.

JaponiconeA induces apoptosis of bortezomib-sensitive and -resistant myeloma cells in vitro and in vivo by targeting IKK.

Objective:Multiple myeloma (MM) remains incurable with high rates of relapse. New therapeutic drugs are therefore urgently needed to improve the prognosis. JaponiconeA (JA), a natural product isolated from Inula japonica Thunb, has shown good anti-MM potential. A comprehensive study should therefore be conducted to identify both the in vitro and in vivo mechanisms of the anti-MM effects of JA.Methods:CCK8 assays and flow cytometry were used to detect the proliferation, apoptosis, and cell cycle of MM cell lines when treated with JA. In vivo experiments were conducted using subcutaneous xenograft mouse models. We also identified possible targets and the mechanism of JA using RNA-seq and c-Map databases, and identified the specific targets of JA in bortezomib-sensitive and -resistant MM cell lines using CETSA, DARTS, and rescue experiments. Furthermore, JA and bortezomib were used separately or together to characterize their possible synergistic effects.Results:In vitro, JA inhibited proliferation, and induced apoptosis and G2/M phase arrest in MM cell lines, and selectively killed primary CD138+ MM cells. In vivo, JA also demonstrated a strong anti-tumor effect with no observable toxicity. In addition, JA showed synergetic effects in combination with bortezomib, and enhanced the anti-tumor effect of bortezomib in bortezomib-resistant cells. CETSA and DARTS confirmed direct binding of JA to NF-κB inhibitor kinase beta (IKKβ), and overexpression of IKKβ or knockdown of IκBα partially rescued the apoptosis induced by JA.Conclusions:JA exhibited strong anti-tumor effects in MM. It sensitized myeloma cells to bortezomib and overcame NF-κB-induced drug resistance by inhibiting IKKβ, providing a new treatment strategy for MM patients.

Targeting of Marcks By MiR-34a Overcomes Drug Resistance in Multiple Myeloma

Multiple myeloma (MM) is a plasma cell malignancy characterized by abnormal proliferation of clonal plasma cells in the bone marrow. Current therapies, such as the proteasome inhibitor bortezomib (BTZ), have improved the outcome of patients. Nevertheless MM remains an incurable disease with a high rate of relapse and development of drug resistance. So far, the pathogenic mechanisms underlying drug resistance of MM are not fully elucidated. Our previous study showed the overexpression of MARCKS (Myristoylated alanine-rich C-kinase substrate) protein in MM cells played an important role in drug resistance. However; the mechanism (s) underlying MARCKS overexpression and its association with drug resistance in MM have not been elucidated.In the current study, to investigate whether miRNAs play a role in aberrant upregulation of MARCKS in drug resistance, several miRNA-target prediction algorithms were exploited to generate a selective miRNA library for subsequent screening. Among 52 miRNA candidates that target MARCKS, the expression level of several miRNAs with higher score was assessed in drug resistant cell lines (8226-R5 and MM1.R) in comparison to the parental cells (8226 and MM1.S). We observed that miR-34a expression was most significantly downregulated in 8226-R5 and MM1.R cell lines relative to their parental lines, 8226 and MM1S. We also identified a negative correlation between MARCKS and miR-34a expression levels in 6 primary patient samples and two healthy donor samples. Next, to confirm direct targeting of MARCKS by miR-34a, two MM resistant cell lines (8226-R5 and MM1R) were transduced with lentiviral vectors harboring miR-34a precursor gene or scramble control and mRNA and protein level of MARCKS were assessed in miR-34a overexpressing cell lines in comparison to scramble control. Overexpression of miR-34a reduced MARCKS expression level in both mRNA and protein levels. In addition, the luciferase reporter assay was performed to validate MARCKS as a direct target of miR-34a. To this end, the 3' UTR sequence of human MARCKS was cloned into the luciferase-expressing vector pEZX-MT01 to the downstream of the firefly luciferase gene. Co-transfection of MARCKS-UTR or negative control luciferase reporter vectors together with miR-34a expressing or control vector showed that miR-34a overexpression significantly reduced the luciferase activity, indicating that miR-34a can directly target the MARCKS 3′ UTR.To examine the functional effect of miR-34a on cell proliferation and apoptosis in MM cells, the miR-34a overexpressing or scramble control cell lines were treated with BTZ. Overexpression of miR-34a in drug resistant cell lines re-sensitized them to BTZ. Furthermore, FACS analysis revealed that overexpression of miR-34a could induce apoptosis in drug resistant MM cell lines as indicated by an increase in the percentage of annexin-V positive cells compared to the control, suggesting that downregulation of miR-34a is associated with acquired BTZ-resistance in MM.To further elucidate the interaction of miR-34a and MARCKS, we knocked down MARCKS expression by transfecting shMARCKS lentiviral vector into 8226-R5 and MM1.R resistant cell lines and examined the miR-34a level in MARCKS-knockdown cells. We found that miR-34a is also regulated by MARCKS as silencing of MARCKS could restore the miR-34a expression level in MM resistant cell lines. In compatible with miR-34a overexpression experiments, following the knockdown of MARCKS, both cell lines had significantly lower viability after treatment with BTZ, in comparison to negative controls. We also showed that knockdown of MARCKS by shMARCKS silencing or suppression of MARCKS by miR-34a overexpression increased the mRNA and protein levels of some pro-apoptotic genes including PUMA which may be involved in induction of apoptosis in drug resistant MM cells.In conclusion, our data indicate that miR-34a is downregulated in drug resistant MM cell lines and primary MM cells, which contributes to the drug resistance of MM through upregulation of MARCKS. Moreover, regulatory interaction between miR-34a and MARCKS provides the rationale that restoration of miR-34a may serve as a promising therapeutic approach by targeting MARCKS in patients with refractory/relapsed MM. DisclosuresNo relevant conflicts of interest to declare.

Inhibition of the Epigenetic Modifier EZH2 By Mir-138 Upregulates Tumor Suppressor Gene Rbpms and Contributes to Drug Susceptibility in Preclinical Model of Multiple Myeloma

Multiple myeloma (MM) is a plasma cell malignancy driven by numerous genetic and epigenetic alterations including miRNA aberration. Several studies have shown that enhancer of zeste homologue 2 (EZH2) is overexpressed in MM. However, the detailed oncogenic mechanism of EZH2 in drug resistance of MM is not clear. Our previous study showed that miR-138 is an EZH2 target gene, which was epigenetically silenced by histone methylation in its promoter region in drug resistant MM cell lines and primary patient samples. Moreover, luciferase reporter assay confirmed direct targeting of EZH2 by miR-138 in MM cells, suggesting a double negative feedback loop between miR-138 and EZH2 in MM. We also identified that EZH2 and miR-138 expression were negatively correlated in primary patient samples. Here, we investigated how unbalanced miR-138/EZH2 regulatory axis is involved in pathogenesis and drug resistance of MM.To examine the functional effect of miR-138 on cell proliferation and apoptosis in MM cells, two resistant cell lines (8226-R5 and MM.1R) were transduced with miR-138 precursor or scramble lentiviruses and treated with different anti-myeloma drugs including bortezomib (BTZ) , lenalidomide (Len), dexamethasone (Dex) and doxorubicin (Dox). Overexpression of miR-138 in drug resistant cells could re-sensitize the resistant cells to anti-myeloma drugs. Also, reduction of cell proliferation upon overexpression of miR-138 was associated with apoptosis of the resistant cells. In addition, we explored whether EZH2 could rescue the effect of miR-138 on MM cells growth. To this end, we performed functional rescue assay by overexpressing EZH2 in 8226-R5 and MM.1R cells and co-transfecting with miR-138 mimics or scramble. EZH2 overexpression partially abolished the anti-tumor effect induced by miR-138 and BTZ confirming that combination of miR-138 and BTZ induces an inhibitory effect in MM cells by targeting EZH2.Furthermore, to investigate the downstream mechanism of EZH2 targeting in MM drug resistance, H3K27me3 ChIP-seq and genome-wide profiling datasets were analyzed and revealed that the EZH2 target gene, RNA-binding protein with multiple splicing (RBPMS), was significantly silenced in MM in comparison to other types of cancers. It was also downregulated in ISS stage III of MM compared to stage I and II. Notably, we identified that RBPMS was downregulated in 8226-R5 and MM1.R resistant cell lines in comparison to 8226 and MM1.S cells, respectively. The ChIP-qPCR also showed that the histone H3K27 was significantly methylated in the promoter region of RBPMS in drug resistant cells and primary patient samples in comparison to parental lines and normal donor samples, respectively. Additionally, functional study on RBPMS silenced cells revealed that knockdown of RBPMS could confer drug resistance to parental MM cells, suggesting a tumor suppressor activity for RBPMS in MM. Remarkably, targeting of EZH2 by miR-138 overexpression could restore the RBPMS expression level in drug resistant cells.In order to translate our findings to a therapeutic model, we next stablished a mouse xenograft model and investigated the effect of miR-138 on tumorigenesis of MM resistant cells in vivo. Intratumoral injection of synthetic miR-138 significantly suppressed tumorigenesis in combination with BTZ. The miR-138 or BTZ alone moderately prolonged survival compared to control treatment. However, the combination treatment of miR-138 mimics and BTZ significantly extended the overall survival (p= 0.0395). In addition, EZH2 protein and mRNA level were decreased in miR-138 mimics treated groups compared with controls whereas the expression of RBPMS was induced, indicating in vivo targeting of EZH2 by miR-138. IHC analysis of xenograft tumor sections showed that combination treatment of miR-138 mimics and BTZ resulted in a decrease in the proliferation index (Ki67) and an increase in the apoptotic index (Tunnel), compared to either BTZ or miR-138 mimics alone. Collectively, these results support that miR-138 functions as a tumor suppressor miRNA and contributes to the effect of EZH2 on MM cell growth and drug resistance.Our findings indicate that targeting of EZH2 by miR-138 sensitizes MM cells to anti-myeloma drugs and contributes to induction of apoptosis of MM cells, as well as suppression of MM tumor growth in vivo and could be a potential candidate for clinical application to the treatment of MM. DisclosuresNo relevant conflicts of interest to declare.

CircPVT1 Inhibit Apoptosis and Enhance Drug Resistance in Multiple Myeloma

Objective: Circular RNAs (circRNAs) comprise a novel class of widespread non-coding RNAs that could regulate gene expression in eukaryotes. Glucocorticoid resistance is the primary factor of refractory relapse in patients with multiple myeloma (MM). This study was designed to reveal the role of circPVT1 in mediating glucocorticoid resistance in MM through regulating its target genes to inhibit apoptosis of MM cell and promote tumor stem cell survival.Methods and results: We determined with RT-PCR assay that circRNA PVT1 is differentially expressed between glucocorticoid resistant and sensitive MM cells (Figure A), and is closely correlated with prognosis (Figure G and H). We further investigated the role of circPVT1 in MM cell apoptosis and proliferation as well as its downstream signaling pathway mechanism. Silencing circPVT1 enhances sensitivity to glucocorticoid treatment (Figure D), promotes apoptosis and inhibits cell proliferation in resistant MM cell lines and myeloma cells in NOD-SCID mice, resulting up-regulation of the apoptosis pathway as measured by increased Caspase3, PARP and decreased Bcl-2 (Figure E). Measured by CCK-8 cell viability assay, ectopic expression of circPVT1 contributed to inducing proliferation and suppressing apoptosis in sensitive MM cell lines. Using Hoechst Staining, we found that MM stem cell population increases upon ectopic expression and decreases while silencing circPVT1, which revealed circPVT1 could facilitate the proliferation of stem cells to enhance glucocorticoid resistance (Figures F and I).Conclusion: As a downstream of circPVT1, it promotes c-myc nuclear translocation, which intermediated by miRNAs to enhance its targets thus promotes the proliferation of stem cells and inhibits apoptosis. We concluded that circPVT1 may mediate MM glucocorticoid resistance and its downstream signaling pathway may represent a novel target for MM treatment.Figure (A)RT-PCR showing the differential expression of circPVT1 and linear RNA PVT1 between MM.1R and MM.1S cell lines.** P <0.01. Error bars, S.D. (n=3). (B)RT-qPCR results showing the abundance of circPVT1 and PVT1 in MM.1R cells treated with RNase R. The levels of CircPVT1 and PVT1 were normalized to the values measured after mock treatments.** P <0.01. Error bars, S.D. (n=3). (C)The expression of circPVT1 was detected by RT-PCR and was validated by Sanger sequencing. (D)RT-PCR analysis of circPVT1 expression after treatment with siRNA targeting at the circPVT1.** P <0.01. Error bars, S.D. (n=3). (E)Western blot analysis of the levels of PARP, caspase 3, Bcl-2, control of β-actin in MM.1R and MM.1S transfected with circPVT1 siRNA or control siRNA, in absence of dexamethasone 1μM/L for 24 hours. (F)Using Hoechst Staining, analysis of side population of MM.1R transfected with circPVT1 siRNA or control siRNA. (G)RT-PCR showing the differential expression of circPVT1 of GC sensitive and resistant patients. ** P <0.01. Error bars, S.D. (n=28). (H)Kaplan-Meier analysis of the correlation between GC sensitivity and overall survival. (I)Diagram showing c-myc nuclear translocation facilitated by circPVT1, intermediated by miRNAs to enhance the targets of c-myc thus promotes the proliferation of stem cells and inhibits apoptosis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Leflunomide Inhibits c-Myc Signaling and Impairs Myeloma Cell Growth

Introduction:Despite recent advances in treatment that have improved the prognosis for patients with multiple myeloma (MM), the disease remains incurable. There is a need for MM treatments with new mechanisms of action. Leflunomide (Lef), a commercially available oral immunosuppressive agent that has been FDA-approved for treatment of rheumatoid arthritis was evaluated as a potential MM therapeutic. In vitro anti-MM activity of Lef has previously been reported; however, the molecular mechanisms of action of Lef in MM are incompletely understood. Inhibition of Src kinase-family members and blocking of dihydroorotate dehydrogenase, an enzyme involved in pyrimidine synthesis are among the few known targets of Lef. To identify novel direct targets of Lef and to further our understanding of Lefs' mode of action on MM cells, we performed in vitro kinase screening assays and RNA/miRNA sequencing analysis of cells treated with or without Lef.Methods and Results:Our pre-clinical studies with teriflunomide (Ter), the active metabolite of Lef, showed that it inhibited growth of MM cell lines (MM.1S, MM.1R, U266, H929, RPMI-8226, OPM-2, L-363) and primary MM patients' (CD138+) plasma cells at IC50s ranging from 30 uM to 140 uM. Pharmacokinetics analysis accompanying a phase 1 dose-escalation clinical trial in MM at our institute has demonstrated that Ter serum concentrations of 500 uM can be obtained. We also found that Ter induces cell-cycle arrest in both, glucocorticoid-sensitive (MM.1S) and resistant (MM.1R) MM cell lines at <150 uM. At higher in vitro concentrations (100 uM to 200 uM), Ter induces apoptosis in MM cells. Moreover, Ter synergized with dexamethasone as well as lenalidomide in in vitro growth inhibition of MM cell line MM.1S. In vivo mouse studies with Lef as single agent or in combination with the immune modulatory drug lenalidomide or with the proteasome inhibitor ixazomib are currently ongoing and results will be presented at the meeting. To identify novel direct targets of Ter, a screening assay described by Anastassiadis et al. (PMCID: PMC3230241) was applied to test the inhibitory activity of Ter on 600 known kinases. We identified 55 kinases that were inhibited >50% at 200 uM Ter. Subsequent dose-response kinase assays identified, among others, Aurora A and PIM family members, all of which are involved in MM pathogenesis, as novel direct targets of Lef, with IC50s ranging from 35 uM to 53 uM. To identify MM-associated mRNAs and miRNAs whose expression levels are frequently altered upon Lef exposure, 4 MM cell lines (RPMI-8226, U266, MM.1S, NCI-H929) and CD138-enriched primary MM cells were exposed to 200 µM Ter or DMSO control for 24h before extraction, purification and sequencing of mRNA and microRNA. RNA-sequencing analysis and DAVID functional annotation revealed that similar changes were present between patient samples and cell lines. Pathways that were predominantly affected by Ter included purine/pyrimidine metabolism, DNA-replication and cell-cycle regulation. Gene set enrichment analysis revealed that c-Myc, an oncogene that contributes to the malignant phenotype of MM and a known promising target in MM, signaling was impaired upon Ter treatment, in MM.1S cells. Differential expression analysis, real-time PCR and western blot analysis showed that c-Myc mRNA, c-Myc protein and c-Myc-regulated miRNAs belonging to the oncomir-1 cluster miR-17-92 were all down-regulated upon Ter exposure in the majority of the MM cells. Since PIM family members are involved in c-Myc signaling in cancer cells, we performed in silico molecular docking studies using Ter/PIM. Our results suggest that the active metabolite of Lef, Ter, can directly bind to the PIM family of proteins by binding to the ATP-binding site, thereby inhibiting its kinase activity. Since pan-PIM inhibitors such as PIM447 have been reported to be cytotoxic for myeloma cells due to decrease in c-Myc levels, these results suggest that decrease in c-Myc induced by Ter may be due to PIM inhibition.Conclusion:Leflunomide impairs in vitro MM cell growth - at least in part - through inhibition of c-Myc signaling. A clinical trial with leflunomide in MM has been initiated and a separate abstract with encouraging initial results has been submitted for presentation at this meeting. DisclosuresKrishnan:Onyx: Speakers Bureau; Sutro: Consultancy; Janssen: Consultancy, Speakers Bureau; Takeda: Speakers Bureau; Celgene: Consultancy, Equity Ownership, Speakers Bureau. Rosenzweig:Celgene: Speakers Bureau.

A Novel Aryl-Guanidinium Based Compound Targets the STAT3 Signalling Pathway, Downregulates MCL-1 Expression and Induces Anti-Myeloma Activity

Multiple myeloma (MM) is a hematological malignancy characterized by the proliferation of malignant plasma cells in the bone marrow microenvironment. Increased understanding of the pathophysiology of MM and advances in therapeutics have led to an increased survival of patients in the past decade. However, despite the effectiveness of the first-line treatments, patients invariably relapse and become drug refractory due to genetic heterogeneity and induction of prosurvival signalling cascades in the bone marrow microenvironment. There is, therefore, a need for novel therapeutics which target key prosurvival signalling pathways in MM. The Janus kinase (JAK)/Signal transducer and activator of transcription 3 (STAT3) pathway in MM can be both constitutively active due to autonomous mutations or activated by growth and survival factors, such as interleukin-6 (IL-6), secreted by bone marrow stromal cells and other cells within the bone marrow microenvironment. STAT3 is upregulated in MM and increases the expression of genes involved proliferation, angiogenesis and evasion of apoptosis. STAT3 target genes include prosurvival BCL2 family members, such as MCL-1 which is overexpressed in MM, leading to apoptosis resistance and is associated with shorter patient survival. Several studies have shown promise in targeting prosurvival BCL2 family members with BCL2 homology domain 3 (BH3) mimetics in MM. We have previously described a member of a series of guanidinium based compounds which inhibits BRAF, a serine/threonine kinase component of the RAS/ERK signalling pathway, through a putative type-III allosteric mechanism [Diez-Cecilia et al, (2015) Bioorg. Med. Chem. Lett. 25, 4287-4292]. Here, we describe a new derivative of this series, VP79s, which exhibits cytotoxicity in MM cell lines with IC50 values in the low micromolar range; however, VP79s does not inhibit RAS/ERK signalling suggesting a novel mechanism of action. VP79s was found to induce apoptosis in NCI-H929 and U266B1 MM cell lines in a dose- and time-dependent manner. VP79s was more potent in inducing cell death in a panel of drug sensitive and drug resistant MM cell lines compared to standard and emerging therapeutic agents for MM. VP79s rapidly inhibited both constitutively active and IL-6-induced STAT3 signalling in MM cells. Examination of upstream mediators of STAT3 signalling suggested a mechanism for VP79s inhibition of STAT3 activation via inhibition of JAK2 phosphorylation and downregulation of the IL-6 receptors, CD126 and CD130. Analysis of downstream STAT3 targets demonstrated that treatment with VP79s resulted in a rapid time-dependent decrease in expression of MCL-1. VP79s was shown to overcome adhesion mediated drug resistance in MM cells in a co-culture model system with bone marrow stromal cells which induces MM cell resistance to treatment with the proteasome inhibitor bortezomib. Combining VP79s with bortezomib synergistically enhanced apoptosis in MM cells compared with single agent treatment. VP79s showed selective cytotoxicity in patient derived MM cells compared to normal donor lymphocytes and reduced the viability of MM patient samples in a dose-responsive manner suggesting its potential as an anti-MM therapeutic. In conclusion, the novel agent VP79s inhibits the JAK/STAT3 signalling pathway resulting in rapid downregulation of MCL-1 expression and inducing potent anti-MM activity. This mechanism may suggest a novel strategy for targeting MCL-1, an emerging target in MM. Disclosures No relevant conflicts of interest to declare.

Overcoming Innate Resistance to a Beta-Catenin Inhibitor-Tegavivint- by Manipulating Autophagy in Multiple Myeloma

Multiple myeloma (MM) is an incurable haematological malignancy with patients eventually exhausting all available therapeutic modalities and succumbing to the disease. Various lines of evidence have demonstrated that the Wnt/β-catenin pathway is frequently up-regulated in advanced MM. Tegavivint (BC2059), an inhibitor of the Wnt/β-catenin pathway, that we have previously shown to have anti-MM activity in vitro, ex vivo and in vivo, is currently in clinical trials for the treatment of solid tumours. In this study, we further demonstrate the dose dependent pro-apoptotic activity of BC2059, including activity against the innately resistant human MM cell line (HMCL) LP1. We show that BC2059 causes down-regulation of β-catenin and it’s down-stream target survivin, in parallel with reactive oxygen species (ROS) generation. Coincident with this, BC2059 induces autophagy with a rise in LC3II and fall in p62. Moreover, chemical inhibition of autophagy was highly synergistic with BC2059 in all HMCL tested, with evidence of pro-apoptotic potentiation of BC2059 activity, with enhanced generation of active caspases 8, 9 and 3. Consistent with this, BC2059 treatment of LP1 in combination with knock down of the autophagy related protein Atg5 markedly enhanced cell killing. In conclusion, our results demonstrate that resistance to BC2059 can be partially attributed to the induction of cytoprotective autophagy and that inhibition of autophagy enhances BC2059-induced MM cell killing. The combination of Wnt/β-catenin pathway and autophagic inhibition represents a novel therapeutic strategy for advanced MM that warrants further evaluation.

Downregulation of MDM2 Leads to Anti-Proliferative Effects through Activation of p53-Associated Pathway Mediated By Both Dual Inhibitor MX69 and Mir-548c-3p in Multiple Myeloma

Multiple myeloma (MM) is a plasma cell malignancy characterized by abnormal proliferation of clonal plasma cells in the bone marrow. MM remains an incurable disease with a high rate of relapse and development of drug resistance. Mouse double minute 2 homolog (MDM2) has been characterized as an oncogene that is associated with cancer development and radio/chemotherapy resistance in cancer. However, the mechanism(s) underlying MDM2 overexpression and its association with drug resistance in MM have not been fully explored. In addition, the effect of the newly discovered dual inhibitor MX69, which targets MDM2 and XIAP, is unknown in MM. To assess the effects of MDM2 overexpression in MM patients, we performed GEO data (GSE6477, GSE31161) differential expression analysis on bone marrow samples from three patient groups- normal donors, newly diagnosed MM and relapsed MM. We found that relapsed MM patients expressed high MDM2 compared to newly diagnosed MM patients, and both groups expressed higher MDM2 compared to normal donors (p&lt;0.001). Furthermore, immunoblotting and qRT-PCR showed upregulated MDM2 expression in drug resistant MM cell lines (MM1R, 8226R5) as compared to their parental drug sensitive cells (MM1S, 8226S). XIAP is an important inhibitor of apoptotic proteins caspases 3, 7 and 9. Elevated expression of XIAP is involved in chemotherapy resistance in cancer. To determine whether MDM2 and XIAP downregulation by dual inhibitor MX69 could induce apoptosis in MM cells, we treated two drug resistant MM cell lines with MX69. Cell viability assay showed that MX69 reduced viability of MM cells in a time- and dose-dependent manner. Since MDM2 is an antagonist of tumor suppressor p53, we measured the expression of p53 following MX69 treatment and found that MX69-mediated downregulation of MDM2 significantly enhanced p53, PUMA and p21 expression in MM cell lines harboring wild-type p53. In MM p53 null cells, we found that p53 family pro-apoptotic factors p63 and p73 were increased upon MX69 treatment. To investigate the downstream mechanism of MDM2 targeting in MM drug resistance, we knocked down MDM2 in MM cells via siRNA-mediated silencing. Cell viability assay indicated that siMDM2 reduced cell proliferation compared to negative control (P=0.009). Following MDM2 silencing, XIAP expression was decreased and p53 expression was increased in p53 positive MM cell lines, whereas p63, p73 and p21 were increased in both p53 positive and p53 null MM cells. Furthermore, combined treatment of MX69 with dexamethasone (Dex), doxorubicin (Dox) or lenalidomide (Len) displayed synergistic toxic effects (combination index ranging from 0.437 to 0.8) in drug resistant MM cell lines. MX69 alone or in combination with Dex, Dox and Len significantly increased apoptosis and decreased proliferation of MM cells. To explore whether miRNAs play a role in regulating MDM2 overexpression and MM drug resistance, several miRNA target prediction algorithms were exploited to generate a selective miRNA library for subsequent screening. Four miRNAs (miR-890, miR-211-5p, miR-429 and miR-548c-3p) were common among GSE49261, GSE39571, GSE16558 and GSE17498 GEO datasets. These four miRNAs were downregulated in MM patients and negatively correlated with MDM2. Furthermore, analyzing prediction score revealed that miR-548c-3p had the highest binding score with MDM2 3'UTR. Moreover, MiR-548c-3p showed a significant downregulation in drug resistant cells in comparison with their parental sensitive cells (p&lt;0.0001). To determine whether MDM2 expression is selectively regulated by miR-548c-3p, we transfected two MM drug resistant cells with miR-548c-3p mimics and found that MDM2 mRNA and protein levels were suppressed and the cell viability was significantly reduced compared to the scrambled controls. In conclusion, our results indicate that MDM2 overexpression is associated with disease progression and drug resistance in MM. Treatment of drug resistant MM cells with MDM2/XIAP dual inhibitor MX69 sensitizes the cells to anti-myeloma drugs and induces apoptosis. Our finding of the regulatory interaction between miR-548c-3p and MDM2 reveals key insights into the dysregulation of MDM2 in MM and provides a new therapeutic approach to target MDM2 by restoration of miR-548c-3p. Disclosures No relevant conflicts of interest to declare.

In silico Prediction Followed By in Vitro validation Identifies the Survivin Inhibitor YM155 As a Potent Secondary Drug Against Pi-Resistant Myeloma

Drug resistance is a major cause of concern in cancer chemotherapy that not only reduces efficacy but also causes toxicity through overdosing. Multiple myeloma (MM) is the second-most common haematological malignancy in the USA that remains a challenging disease to cure due to the presence of significant complexity and heterogeneity at molecular level with high treatment cost. Proteasome inhibitor, the standard of care drug, has proven to remarkably improve myeloma patient outcomes but is associated with dose limiting toxicities and drug resistance. Therefore, new combination treatment regimens are urgently needed for the treatment of PI-resistant MM that can lower the required dose of standard-of-care drug without compromising on treatment efficacy. We have created an optimization-regularization based computational prediction method called secDrug that uses large-scale pharmacogenomics databases to identify novel secondary drugs against PI-resistance when used as single agent or in combination. We applied secDrug to PIs-resistant cell lines (&gt;100) in the Genomics of Drug Sensitivity in Cancer (GDSC) database and predicted the top drugs that can be best combined with PIs to overcome PI-resistance in MM. These include: Survivin inhibitor (YM155), Nicotinamide phospho ribosyl transferase or Nampt inhibitor (FK866), PIKfyve inhibitor (YM201636), Raf inhibitor (PLX-4720), Bcl2 inhibitor (Navitoclax),HSP90 inhibitor (17-AAG), AKT inhibitor (KIN00102), HDAC inhibitors (Panobinostat, SAHA), S6K1-specific inhibitor (PF-4708671), and the neddylation inhibitor (MLN4924). To validate our prediction results, we treated human multiple myeloma cell lines (HMCLs) highly resistant to the proteasome inhibitors Bortezomib, Carfilzomob, Oprozomib and Ixazomib and &gt;10 clonally derived acquired resistant cells with the top predicted best secondary drugs. Next, we treated three pairs of parental PI-sensitive (P) and clonally-derived PI-resistant (R) cell lines from PI-sensitive (P) HMCLs representing acquired PI-response with the top predicted drug combinations. Our preliminary in vitro data confirmed our in silico predictions of secondary drugs based on secDrug analysis. Among these, YM155 and YM155 + Ixazomib showed strikingly very high in vitro cytotoxicity against PI- resistant MM cell lines (Figure 1). The results, based on Chou-Talalay's combination index (CI) theorem, show that this combination works highly synergistically where the IC50 of ixazomib in MM cells was significantly reduced in presence of YM155. Similar results were obtained for the other top predicted combination regimens. Our findings provide a strong case for combining YM155 with ixazomib to enhance sensitivity or overcome resistance to ixazomib and thereby improve patient outcome. Further, this study provides additional support towards using secDrug for predicting secondary drugs in cancers resistant to the standard-of-care therapy. Currently, we are exploring the molecular mechanisms behind the synergism between proteasome inhibitors and the top drugs identified by our algorithm as candidates for secondary combination therapies using next-gen transcriptomics and single-cell analysis approaches. Earlier studies have shown that YM155 potentially induces selective cell death in human pluripotent stem cells. Further, the presence of cancer stem cells sub population/ MM-CSCs in drug-resistant tumors have been shown to significantly contribute to the development of PI-resistance in MM. Hence, we propose that YM155 reverses PI-resistance by reducing the stem cell load in multiple myeloma cell line model system. Therefore, our future plan is to assess the effects of YM155 on MM-cancer stem cells like subclones/ sub population/ MM-CSCs including CD19- CD138- quiescent stem cells, ALDH+ and side populations/SP) in drug-resistant tumors. Overall, our in vitro validation results corroborated with our in silico prediction of secondary drugs. Disclosures No relevant conflicts of interest to declare.

Proteomic Profiling and Mechanistic Investigating of a Novel Anti-Cancer Small Molecule Inhibitor of Sec61

Secreted and transmembrane (TM) proteins play key roles in malignant transformation and tumor growth, including autocrine growth factor expression, receptor oncogene signal transduction pathways, metastasis, and immune system evasion. During translation, the majority of such proteins require translocation through the Sec61 translocon into the Endoplasmic Reticulum (ER) for further processing. This process is negotiated by unique signal sequences of the translating protein. Therefore, Sec61 represents a novel therapeutic target for cancer treatment through selective blockade of protein secretion. We generated Sec61 inhibitors and assessed their potential against target proteins using HEK293 cell lines stably expressing secreted or TM proteins of interest fused to a luciferase reporter. Additionally, anti-tumor activity was determined across both solid and liquid tumor cell lines in vitro and in mouse models. KZR-8834, a lead candidate identified through a medicinal chemistry campaign, induced cell death in multiple tumor cell lines in vitro, including multiple myeloma (MM), and was effective in xenograft models at doses that did not induce significant body weight loss or clinical signs of toxicity. We utilized quantitative proteomic methods to study KZR-8834 for inhibition of protein secretion and global modulation of protein homeostasis in sensitive and resistant tumor cell lines. Multiple tumor cell types were tested at various doses and time courses followed by subcellular fractionation of cytosolic and membrane/ER proteomes. Subsequent proteomic profiling was performed with Stable Isotope Labeling by/with Amino acids in Cell culture (SILAC) and/or Tandem Mass Tag 6-plex (TMT-sixplex). Sensitive targets from both proteomes were further verified using downstream biochemical methods. Sec61 client proteins showed both time- and dose-dependent inhibition upon compound treatment and proteomic results were verified via western blot analysis. Approximately 20% of the total Sec61 clientome and 25% of total proteins detected in a sensitive multiple myeloma (MM) cell line, H929, were significantly down-regulated in response to KZR-8834 treatment at concentrations leading to cell death. IPA pathway analysis suggested that activation of the ER stress response gene ATF4 was induced by KZR-8834 treatment in H929 cells. In a resistant MM cell line, U266, only 13% of the total Sec61 clientome and 5% of total protein detected were significantly down-regulated in response to the same compound treatment. A distinct profile of down-regulated Sec61 clientome was noted with overlap in only 11 of 394 commonly expressed proteins across those two cell lines. Interestingly, in compound treated cells, 39 down-regulated Sec61 client proteins in H929 were either unchanged or upregulated in U266 cells. Conversely, 38 upregulated H929 Sec61 clients were either unchanged or down-regulated in U266 cells. We further explored the ER stress response induced by KZR-8834 via comparative proteomic analysis in H929 cells treated with known ER stress inducers, Tunicamycin and Thapsigargin. These agents, which exert ER stress upon inhibition of N-linked glycosylation and blockade of ER Ca2+ flux, respectively, showed distinct cytosolic proteomic profiles in H929 cells relative to KZR-8834 treatment. These data suggest that KZR-8834-induced blockade of Sec61 results in a unique form of proteotoxic stress in sensitive MM cells. Collectively our results highlight quantitative proteomic profiling as a valuable tool toward elucidating the mechanism of pleiotropic acting molecules like KZR-8834. These studies constitute important first steps toward clarifying the anti-tumor mechanism inhibiting Sec61, a novel pathway agent, for the potential treatment of hematologic tumors. Disclosures Qian: Kezar Life Sciences: Employment, Equity Ownership. Johnson:Kezar Life Sciences: Employment, Equity Ownership. Kirk:Kezar Life Sciences: Employment, Equity Ownership. Lowe:Kezar Life Sciences: Employment, Equity Ownership. McMinn:Kezar Life Sciences: Employment, Equity Ownership. Millare:Kezar Life Sciences: Employment, Equity Ownership. Muchamuel:Kezar Life Sciences: Employment, Equity Ownership. Wang:Kezar Life Sciences: Employment, Equity Ownership.

Combined Inhibition of HDAC and AKT As a Strategy to Overcome Multi-Drug Resistance in Patients with Multiple Myeloma

Nowadays, patients with multiple myeloma (MM) have multiple choices of therapy including monoclonal antibodies, proteasome inhibitors, and immunomodulatory drugs (IMiDs), whereas some patients still develop resistance to these drugs and require novel therapeutic modalities. Here, we focused on inhibition of HDAC and AKT to overcome drug resistance. Lenalidomide (Len) selectively binds to cereblon (CRBN), which mediates recruitment of specific substrates like IKZF1 to E3 ubiquitin ligase and subsequent degradation, resulting in downregulation of IRF-4 and c-Myc. Then, we developed Len-resistant myeloma cells by RNAi-mediated downregulation of CRBN. Treatment of these cells with HDAC inhibitors reduced IKZF1 mRNA, suggesting potential efficacy of HDAC inhibitors against CRBN-low expressing or mutated MM. According to the integrated database for expression profile and disease prognosis (GenomicScape, http://www.genomicscape.com), higher expression of MICA was significantly associated with better overall survival in MM. MICA is an NK cell-activating ligand and plays an important role in ADCC. We observed that ADCC activity of both daratumumab and elotuzumab against MM cells was enhanced in the presence of HDAC inhibitors, which was compatible with our previous data that HDAC inhibitors upregulated MICA mRNA expression via inhibition of IKZF1 (ASH2018 abstract #4435). We also observed that HDAC inhibitors upregulated MICA mRNA in CRBN-deficient cells, suggesting promise of the combination of HDAC inhibitors and monoclonal antibodies against Len-resistant MM. Len-resistance is also affected by phosphorylation status of GSK-3. PI3K/AKT pathway is frequently activated in MM cells, and AKT inactivates GSK-3 by direct phosphorylation, resulting in c-Myc stabilization. Enhanced phosphorylation of GSK-3 was observed in CRBN-deficient H929 cells after long-term culture with Len, and such a phosphorylation status of GSK-3 was correlated with less CRBN amount and higher Len concentration (Figure 1). Afuresertib, an AKT inhibitor, suppressed GSK-3 phosphorylation (p-GSK-3) with or without ACY-1215, an HDAC inhibitor, leading to a substantial decrease of c-Myc (Figure 2). On the other hand, CHIR 99021, a GSK-3 inhibitor, partially counteracted to cytotoxic effect of afuresertib on H929 cells (Figure 3). These results suggest that increased p-GSK-3 is involved in acquired Len-resistance, and that combined inhibition of HDAC and AKT can overcome Len-resistance through decreased p-GSK-3. Furthermore, we examined the efficacy of CUDC-907, a dual HDAC and PI3K inhibitor. CUDC-907 had a cytotoxic effect on the MM cell lines including those had low CRBN expression. Bortezomib, doxorubicin, and dexamethasone resistant MM cell lines were also sensitive to CUDC-907. CUDC-907 upregulated MICA mRNA expression, but downregulated IKZF1 mRNA expression. Treatment of RPMI-8226 cells with CUDC-907 enhanced the ADCC activity of daratumumab (Figure 4). Furthermore, CUDC-907 was effective on primary MM cells which were resistant to bortezomib and Len (Figure 5). Thus, dual inhibition of HDAC and AKT with or without monoclonal antibodies is a promising therapeutic approach to multi-drug resistant MM. Disclosures Imai: Celgene: Honoraria, Research Funding; Janssen Parmaceutical K.K.: Honoraria, Research Funding; Bristol-Myers Squibb: Research Funding. Futami:Torii Pharmaceutical: Research Funding. Ri:Janssen Pharmaceutical: Honoraria, Research Funding; Takeda Pharmaceutical: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Daiichi Sankyo: Research Funding; Ono Pharmaceutical: Honoraria, Research Funding; Kyowa Kirin: Research Funding; Chugai Pharmaceutical: Research Funding; Sanofi: Honoraria, Research Funding; Abbvie: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; MSD: Research Funding; Novartis Pharma: Research Funding; Gilead Sciences: Research Funding; Astellas Pharma: Research Funding; Teijin Pharma: Research Funding. Yasui:TokioTHERA Holdings, Inc.: Equity Ownership. Iida:Teijin Pharma: Research Funding; Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Astellas: Research Funding; Gilead: Research Funding; Sanofi: Research Funding; MSD: Research Funding; Abbvie: Research Funding; Kyowa Kirin: Research Funding; Chugai: Research Funding; Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Daichi Sankyo: Honoraria, Research Funding. Tojo:Torii Pharmaceutical: Research Funding; AMED: Research Funding.

Overexpression of Mir-21-5p Induces Apoptosis and Cell Cycle Arrest By Down-Regulating SKP2 and Overcomes Bortezomib Resistance in Multiple Myeloma

High expression of SKP2 (S-Phase Kinase Associated Protein 2) is associated with tumor cell growth and drug resistance in many solid tumors and hematological malignancies. However, the role of SKP2 in drug resistance/ relapse of multiple myeloma (MM) patients has not been fully investigated. To this end, we first analyzed two publicly accessible datasets (GSE6477, GSE9782) which demonstrated that relapsed MM patients showed higher expression of SKP2 compared to newly diagnosed patients (p=0.02). Furthermore, high expression of SKP2 was correlated with shorter overall survival compared to newly diagnosed MM patients with low SKP2 expression (p=0.0002). To investigate whether miRNAs could play a role in the dysregulation of SKP2 in MM, we exploited several miRNA-target prediction algorithms (Targetscan, miRDB, microT-CDS) to generate a selective miRNA library for subsequent screening. Over all, the screening results displayed 59 miRNA candidates predicated to target SKP2, 9 miRNAs were commonly identified by all three algorithms. Among these, miR-21-5p showed the highest potential binding score for SKP2 and was found to be in an inverse correlation with SKP2 in MM patient dataset (GSE16558). To address whether miR-21-5p/SKP2 axis may play a role in drug resistance in MM, we used two drug resistant MM cell lines (RPMI-8226R5 and MM.1R) to analyze the basal expression of SKP2 and miR-21-5p compared with that in their parental lines (RPMI-8226 and MM.1S, respectively). It turned out that the resistant MM cells displayed low expression of miR-21-5p and high expression of SKP2 whereas the parental lines showed the opposite pattern. Moreover, overexpression of miR-21-5p in both MM drug resistant cell lines significantly reduced both mRNA and protein levels of SKP2 further supporting that SKP2 could be a target of miR-21-5p. SKP2 mediates the ubiquitination of cyclin kinase inhibitor p27Kip1, a central negative regulator of cell cycle. In line with this, our immunoblot analysis confirmed that high SKP2 expressing drug resistant MM cells showed a low level of p27Kip1protein and low SKP2 expressing MM parental lines showed a high level of p27kip1 protein. To investigate the functional effects of miR-21-5p overexpression, we first performed FACS to determine if this overexpression would modulate cell cycle regulation of drug resistant MM cells. miR-21-5p overexpression increased in the proportion of cells at G1/G0 phase (RPMI-8226R5: 41.6 % to 59.1%, MM.1R: 19.5 % to 39.4%), decreased in the proportion of cells in S phase (RPMI-8226R5: 30.9 % to 25.5 %, MM.1R: 44.7 % to 32.2%) and G2/M phase (RPMI-8226R5: 15.5 % to 11.5 %, MM.1R: 27.6 % to22.4% ) compared with scrambled control. At the same time, immunoblot results showed that overexpression of miR-21-5p accumulated p27Kip1 and p21 (Cip1/Waf1), and suppressed CDK6 and Cyclin E cell cycle markers. Next, we examined the effect of miR-21-5p overexpression on cell proliferation and apoptosis in MM resistant cells using both MTT cytotoxicity assay and vital exclusion staining (trypan blue). In both assays, miR-21-5p mimics-transfected MM resistant cells showed significant reduction of cell viability and increase in apoptosis in a time-dependent manner compared with scrambled-transfected cells. Overexpression of miR-21-5p upregulated apoptotic markers: cleaved PARP-1, cleaved caspase-3, PUMA and BAX and down regulated anti-apoptotic markers: BCL-2 and survivin. Subsequently, efficacy of bortezomib (BTZ) was examined on miR-21-5p overexpressed MM resistant cells which were interestingly re-sensitized to BTZ in a dose- and time-dependent manner. In addition, our FACS experiment also confirmed that combination of BTZ and miR-21-5p mimics increased the percentage of annexin-V positive cells compared to scrambled and miR-21-5p mimics in the MM resistant cells. In conclusion, miR-21-5p is suppressed in MM resistant cells, which contributes to the drug resistance in MM through upregulation of SKP2. Overexpression of miR-21-5p induces apoptosis and cell cycle arrest in MM drug resistant cells by downregulating SKP2 gene expression and overcomes BTZ resistance in these cells. Our study provides a proof-of-concept that miR-21-5p could be utilized as a SKP2-targeting molecule which in combination with BTZ would be a promising therapeutic approach to overcome MM drug resistance. Disclosures No relevant conflicts of interest to declare.

Blockade of Ubiquitin Receptor PSMD4/Rpn10 Triggers Cytotoxicity and Overcomes Bortezomib-Resistance in Multiple Myeloma

Background and Rationale Proteasome inhibitors (PIs) are standard of care therapy for patients diagnosed with multiple myeloma (MM). However, prolonged treatment can be associated with toxicity and development of drug resistance. Our research efforts have therefore focused on designing therapeutic strategies that can overcome PI-resistance. In this context, our RNA-interference-based loss-of-function studies have identified 19S proteasome-associated Ubiquitin Receptor (UbRs) Rpn10 and Rpn13 as potential therapeutic targets. Both Rpn10 and Rpn13 UbRs are associated with the 19S regulatory lid of the proteasome that recognizes ubiquitylated proteins marked for degradation by 20S core particle. We have previously reported the role of Rpn13 in MM (Song et., al Luekemia 2016 Sep;30(9):1877-86). Here we functionally characterized the role of Rpn10 in MM.Materials and Methods Cytotoxicity assays were performed using a panel of MM cell lines, primary patient cells, and peripheral blood mononuclear cells (PBMCs) from normal healthy donors. Cell viability was assessed using WST-1, MTT, and Cell Titer-Glo assay.Signal transduction pathways were evaluated using immunoblotting. Proteasome activity was measured as previously described (Chauhan et al., Cancer Cell 2005, 8:407-419; Chauhan et al., Cancer Cell 2012, 22(3):345-358). Statistical significance of data was determined using a Student's t test.Results Functional characterization of Rpn10 in MM:Gene expression (GEP) analysis showed inverse correlation between Rpn10 and overall patient survival (n=175) (p= 0.00064). Immunoblot analysis showed high Rpn10 protein levels in primary patient cells and MM cell lines versus normal plasma cells or PBMCs. Rpn10 knockdown by siRNA significantly decreased MM cell viability in both bortezomib-sensitive and -resistant MM cell lines. To assess the Rpn10 function, we generated a doxycycline-inducible Rpn10-shRNA knockout stable MM cell line. Rpn10-shRNA knockdown decreased MM cell proliferation. Mechanistic studies show that Rpn10 knockdown-triggered MM cell death is associated with 1) accumulation of cells in early and late apoptotic phase; 2) increase in polyubiquinated proteins; 3) arrest of cell cycle; 4) induction of ER stress; and 5) activation of caspases mediating apoptotic pathways. In order to determine if blockade of Rpn10 affects cellular proteasome function, we next utilized a reporter cell line expressing ubiquitin-tagged GFP that is constitutively targeted for proteasomal degradation. Interestingly, Rpn10-siRNA increased accumulation of the Ub-GFP reporter, reflecting impaired proteasome-mediated protein degradation. Finally, treatment of MM cells with peptides targeting UIM2 domain of Rpn10 significantly decreased MM cell viability, suggesting a potential therapeutic approach in MM.ConclusionOur preclinical data validates targeting 19S proteasome ubiquitin receptor Rpn10 upstream of the proteasome in the ubiquitin proteasomal cascade, and provides the framework for clinical evaluation of Rpn10 inhibitors to overcome PI resistance and improve patient outcome in MM. DisclosuresAnderson:C4 Therapeutics: Equity Ownership, Other: Scientific founder; Celgene: Consultancy; Bristol Myers Squibb: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; Millennium Takeda: Consultancy; OncoPep: Equity Ownership, Other: Scientific founder.