Primary Patient Multiple Myeloma Cells Research Articles

Extracellular vesicles derived from immortalized human natural killer cell line NK3.3 as a novel therapeutic for multiple myeloma.

Over the last decade, there have been many advancements in the therapeutic treatment of multiple myeloma (MM), including the use of natural killer (NK) cells. However, despite promising results from clinical trials, there are concerns over the use of NK cell-based therapy. Cells often undergo growth arrest, limiting their experimental utility; donor cells are extremely heterogeneous, resulting in content variability; and patients receiving allogeneic cells are at risk for graft-versus-host disease and/or cytokine release syndrome. Extracellular vesicles (EVs) have emerged as a new natural therapeutic tool. EVs are known to carry cargo derived from the parent cell from which they originate. NK cells play an important role in the innate immune system, targeting and killing tumor cells. This has led many researchers to isolate EVs from NK cells for their cytotoxic potential. In this study, we isolated EVs from the NK cell line, NK3.3, which was derived from the peripheral blood of a healthy donor. Currently, it is the only normal human NK cell line reported with all the functional characteristics of healthy NK cells. To address the issue of growth arrest, we immortalized NK3.3 cells with lentivirus encoding the catalytic subunit of human telomerase htert (NK3.3-LTV). EVs from these cells were isolated using a modified polyethylene glycol (PEG)-acetate precipitation protocol to simplify processing and increase EV yield. We demonstrated that NK3.3-LTV EVs target both sensitive and drug-resistant MM cell lines as well as primary patient MM cells in vitro, decreasing proliferation and inducing apoptotic cell death as well as or better than EVs from non-immortalized cells with no toxicity towards normal cells. This study is the first step towards developing an immunotherapeutic product designed to treat patients with relapsed/refractory MM.

Apobec Family of Proteins Mediate Genomic Changes in Multiple Myeloma By Inducing DNA Damage and C>T Mutations

Multiple myeloma (MM) is a heterogeneous disease characterized by unstable genome. Spontaneous DNA damage coupled with dysregulated repair accentuates the genomic instability seen in MM. The resulting clonal heterogeneity plays a critical role in resistance to treatment and relapse. Understanding the molecular factors that contribute to genomic instability in myeloma is therefore of great importance in developing better treatment strategies. Recently, a role for the AID/APOBEC family of cytidine deaminases in generating somatic mutations has been described with in MM as well as number of other cancers. We and others have previously shown that significant number of mutations bear APOBEC signature and interestingly, the APOBEC mutation signature correlates with sub clonal diversity in MM. We have now further explored role of APOBEC in MM with special focus on its ability to induce clonal diversity.We observed that APOBECs 3A, 3B, 3C, and 3G are highly expressed in MM. Using a fluorescent oligonucleotide based APOBEC deaminase assay to measure deaminase activity, we also observed significantly increased APOBEC activity in MM cell lines as well as primary patient MM cells. Using shRNA-mediated knockdown, we report that depletion of APOBECs in MM cell lines results in decreased DNA double strand breaks. We next investigated the impact of overexpression of APOBEC genes on genome integrity in MM cells. Over expression of APOBECs 3B or 3G in U266 and LP1 MM cell lines led to 3-5 fold increase in deaminase activity in MM cell extracts measured by APOBEC deaminase assay. Furthermore, APOBEC overexpression resulted in elevated double strand breaks as evidenced by increased gH2AX nuclear foci and by western blot. In order to determine the functional impact of APOBEC-induced mutations, we co-transfected EGFP plasmid along with APOBEC expression vectors in myeloma cells. 72-hrs after transfection, we isolated the EGFP plasmid, transformed bacteria, and sequenced the plasmid DNA isolated from 5 colonies from each group. Overexpression of APOBECs resulted in increased number of C>T mutations (3-5 fold) in the EGFP plasmid co-transfected into these cells. Interestingly, APOBEC over expression also resulted in the loss of GFP fluorescence in 20-30% of the cells compared to control cells, suggesting that overexpression of APOBECs can cause deleterious mutations leading to loss of function.APOBEC-mediated deamination of cytosine to uracil leads to generation of abasic sites that are cleaved by abasic endonuclease (APE1), a regulator of homologous recombination pathway dysregulated in MM contributing to genomic instability. Therefore, we investigated how modulating APOBEC activity affects downstream DNA damage repair pathways in the gain-of and loss-of-function cells. Using in vitro activity assays, we measured APE1 and HR activity in extracts collected from APOBEC overexpression and knockdown cells. Extracts from APOBEC overexpressing cells showed elevated APE1 and HR activity (>50%), whereas depletion of APOBEC proteins resulted in up to 80% reduction in HR and APE1 activity in the extracts. Interestingly, using a small molecule HR inhibitor we found that inhibiting HR pathway can reduce the DNA breaks induced by APOBEC overexpression. Furthermore, we investigated the acquisition of genomic changes such as amplifications, deletions, and SNVs over time, in control and APOBEC knockdown cells, using SNP arrays and whole genome sequencing (WGS). Our results show that knockdown of APOBECs reduces the accumulation of copy number changes, structural variants and mutations over time. This suggests that elevated expression of APOBECs may be contributing to genomic instability in MM cells.In summary, our results suggest that APOBEC mediated DNA damage could contribute to increased dysregulation of these important repair pathways and genome stability in myeloma. Inhibiting APOBEC activity could reduce the rate of accumulation of the ongoing genomic changes. DisclosuresNo relevant conflicts of interest to declare.

Targeting Proteasome- Associated Ubiquitin Receptor PSMD4/Rpn10 in Multiple Myeloma

The LeBow Institute for Myeloma Therapeutics and Jerome Lipper Myeloma Center, Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MABackground and Rationale Normal cellular homeostasis is maintained by a balanced regulation of protein synthesis and degradation via the ubiquitin proteasome system (UPS). Deregulation of the UPS is linked to pathogenesis of various human diseases, including cancer. Targeting the 20S proteasome in the UPS via proteasome inhibitors bortezomib, carfilzomib, or ixazomib has proven effective therapy for relapsed, and newly diagnosed multiple myeloma (MM); however, dose-limiting toxicities and the development of resistance limit their long-term utility. Therapeutic strategies directed against ubiquitin receptors upstream of the 20S proteasome may allow for more specific targeting of the UPS, with fewer off-target activities and toxicities. Ubiquitin-tagged proteins are recognized by 19S proteasome-associated ubiquitin receptors; the ubiquitin moiety is then removed from the substrate protein via deubiquitylating (DUB) enzyme enzymatic activity; target protein is then unfolded by the AAA-ATPases; followed by degradation of proteins through 20S proteolytic activities. Here we examined the role of ubiquitin receptor Rpn10/PSMD4 in MM using RNA interference strategies.Materials and Methods We utilized MM cell lines, patient tumor cells, and peripheral blood mononuclear cells (PBMCs) from normal healthy donors. MM.1S cells were transiently transfected with control short interfering RNA (siRNA) or Rpn10-siRNA ON TARGET plus SMART pool using the cell line Nucleofector Kit V. Signal transduction pathways were evaluated using immunoblotting. Cell viability were assessed using WST-1 assay. Proteasome activity was measured as previously described (Chauhan et al., Cancer Cell 2005, 8:407-419). Statistical significance of data was determined using a Student's t test.Results1) A statistically significant inverse correlation was noted between Rpn10 levels and overall patient survival (n=175) (p= 0.00064). 2) Analysis of PSMD4/Rpn10 gene expression showed a significantly higher level in primary patient MM cells versus normal plasma cells or PBMCs (p = 0.023 or p = 0.001, respectively). 3) Rpn10 protein is more highly expressed in MM cells than in normal cells. 4) Functional studies showed that blockade of Rpn10 using RNA interference (siRNA and shRNA) significantly decreased MM cell viability (p < 0.001; n=3). 5) Rpn10-shRNA knockdown decreased MM cell proliferation. These findings were further confirmed in doxycycline-inducible Rpn10-shRNA knockout cells. 6) Transfection of Rpn10-siRNA in MM cells increased accumulation of poly-ubiquitylated proteins. 7) To test whether Rpn10 knockdown blocks cellular proteasome function, we used a reporter cell line expressing ubiquitin-tagged GFP that is constitutively targeted for proteasomal degradation. Rpn10-siRNA increased accumulation of the Ub-GFP reporter indicating impaired proteasome degradation. 8) Mechanistic studies show that Rpn10-siRNA-triggered MM cell death is associated with: (i) cell cycle arrest and apoptosis; (ii) increase in poly-ubiquitylated proteins; and (iii) activation of caspases and induction of endoplasmic stress response signaling.Conclusion Our preclinical data validates targeting 19S proteasome ubiquitin receptor Rpn10 upstream of proteasome in the ubiquitin proteasomal cascade, and provides the framework for development of proof-of-concept inhibitors targeting Rpn10 for future evaluation in clinical trials. DisclosuresChauhan:Triphase accelerator: Consultancy. Anderson:Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; MedImmune: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Other: scientific founder; Oncopep: Other: scientific founder.

Exploiting Protein Translation Dependence in Multiple Myeloma with Omacetaxine-Based Therapy.

The prognosis of patients with multiple myeloma who are resistant to proteasome inhibitors, immunomodulatory drugs (IMiD), and daratumumab is extremely poor. Even B-cell maturation antigen-specific chimeric antigen receptor T-cell therapies provide only a temporary benefit before patients succumb to their disease. In this article, we interrogate the unique sensitivity of multiple myeloma cells to the alternative strategy of blocking protein translation with omacetaxine. We determined protein translation levels (n = 17) and sensitivity to omacetaxine (n = 51) of primary multiple myeloma patient samples. Synergy was evaluated between omacetaxine and IMiDs in vitro, ex vivo, and in vivo. Underlying mechanism was investigated via proteomic analysis. Almost universally, primary patient multiple myeloma cells exhibit >2.5-fold increased rates of protein translation compared with normal marrow cells. Ex vivo treatment with omacetaxine resulted in >50% reduction in viable multiple myeloma cells. In this cohort, high levels of translation serve as a biomarker for patient multiple myeloma cell sensitivity to omacetaxine. Unexpectedly, omacetaxine demonstrated synergy with IMiDs in multiple myeloma cell lines in vitro. In addition, in an IMiD-resistant relapsed patient sample, omacetaxine/IMiD combination treatment resensitized the multiple myeloma cells to the IMiD. Proteomic analysis found that the omacetaxine/IMiD combination treatment produced a double-hit on the IRF4/c-MYC pathway, which is critical to multiple myeloma survival. Overall, protein translation inhibitors represent a potential new drug class for myeloma treatment and provide a rationale for conducting clinical trials with omacetaxine alone and in combination with IMiDs for patients with relapsed/refractory multiple myeloma.

The JAK-STAT pathway regulates CD38 on myeloma cells in the bone marrow microenvironment: therapeutic implications

AbstractAnti-CD38 monoclonal antibody (MoAb) treatments including daratumumab (DARA) are effective therapies for both newly diagnosed and relapsed multiple myeloma (MM). In this study, we examined the soluble factors that modulate CD38 expression and are associated with sensitivity to DARA-mediated antibody-dependent cellular cytotoxicity (ADCC) in the bone marrow (BM) microenvironment. Importantly, primary BM stromal cell (BMSC) culture supernatant (BMSC-sup) and interleukin-6 (IL-6) downregulated CD38 expression and reduced DARA-mediated ADCC. Both cytokine profiling of the BMSC-sup and genome-scale clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) knockout screening in MM cell lines identified and validated the JAK-STAT3 signaling pathway mediating CD38 downregulation, whereas the JAK-STAT1 pathway mediated CD38 upregulation. STAT3 knockdown abrogated BMSC-sup– and IL-6–induced CD38 downregulation on MM cell lines. We also confirmed that STAT3 and CD38 is negatively correlated in primary MM cells. To assess potential clinical relevance, pharmacological inhibition of the JAK-STAT pathway on BMSC-sup–induced CD38 downregulation was further examined. JAK inhibitor ruxolitinib inhibited STAT3 phosphorylation in MM cell lines, upregulated CD38 expression in MM cell lines and primary patient MM cells, and augmented DARA-mediated ADCC against MM cell lines. Taken together, our results suggest that CD38 expression on MM cells in the BM microenvironment is regulated by both STAT1 (positively) and STAT3 (negatively), and that inhibition of the JAK-STAT3 pathway represents a novel therapeutic option to enhance CD38 expression and anti-CD38 MoAb-mediated MM cytotoxicity.

Nanobody-based CD38-specific heavy chain antibodies induce killing of multiple myeloma and other hematological malignancies.

Rationale: CD38 is a target for the therapy of multiple myeloma (MM) with monoclonal antibodies such as daratumumab and isatuximab. Since MM patients exhibit a high rate of relapse, the development of new biologics targeting alternative CD38 epitopes is desirable. The discovery of single-domain antibodies (nanobodies) has opened the way for a new generation of antitumor therapeutics. We report the generation of nanobody-based humanized IgG1 heavy chain antibodies (hcAbs) with a high specificity and affinity that recognize three different and non-overlapping epitopes of CD38 and compare their cytotoxicity against CD38-expressing hematological cancer cells in vitro, ex vivo and in vivo.Methods: We generated three humanized hcAbs (WF211-hcAb, MU1067-hcAb, JK36-hcAb) that recognize three different non-overlapping epitopes (E1, E2, E3) of CD38 by fusion of llama-derived nanobodies to the hinge- and Fc-domains of human IgG1. WF211-hcAb shares the binding epitope E1 with daratumumab. We compared the capacity of these CD38-specific hcAbs and daratumumab to induce CDC and ADCC in CD38-expressing tumor cell lines in vitro and in patient MM cells ex vivo as well as effects on xenograft tumor growth and survival in vivo.Results: CD38-specific heavy chain antibodies (WF211-hcAb, MU1067-hcAb, JK36-hcAb) potently induced antibody-dependent cellular cytotoxicity (ADCC) in CD38-expressing tumor cell lines and in primary patient MM cells, but only little if any complement-dependent cytotoxicity (CDC). In vivo, CD38-specific heavy chain antibodies significantly reduced the growth of systemic lymphomas and prolonged survival of tumor bearing SCID mice.Conclusions: CD38-specific nanobody-based humanized IgG1 heavy chain antibodies mediate cytotoxicity against CD38-expressing hematological cancer cells in vitro, ex vivo and in vivo. These promising results of our study indicate that CD38-specific hcAbs warrant further clinical development as therapeutics for multiple myeloma and other hematological malignancies.

Targeting Myeloma Cell Metabolism Via Disruption of the Lnc-17-92 Transcriptional Program: Druggable New Vulnerability in Multiple Myeloma

Long noncoding RNAs (lncRNA) are major regulators of chromatin dynamics and gene expression. We have recently performed deep RNA sequencing of CD138+ cells from 360 uniformly-treated, newly-diagnosed multiple myeloma (MM) patients (IFM/DFCI 2009) and described the lncRNA landscape and their role as independent risk predictors for clinical outcome in MM. Moreover, we have identified one of these lncRNAs - lnc-17-92 - as an independent risk predictor highly correlating with EFS and OS in newly-diagnosed MM providing rationale to define its molecular role in MM. Lnc-17-92 is generated at MIR17HG gene locus and is known for being involved in the biogenesis of miR-17-92 cluster of microRNAs. We here establish, for the first time, role of this transcript as a lncRNA with microRNA-independent function and molecular and biological implications in MM. Having confirmed its expression in MM cell lines and primary MM cells, we have utilized antisense oligonucleotides (n=3) to suppress lnc-17-92 expression in large panel of human MM cell lines (HMCLs) (n=12) and primary patient MM cells (n=13). Lnc-17-92 inhibition impaired MM cell proliferation leading to apoptotic cell death. This inhibitory effect was not rescued by ectopic expression of miR-17-92 microRNAs, confirming independent activity of lnc-17-92 on MM cell growth and viability. The microRNA-independent role of lnc-17-92 in transcriptional control was further confirmed using DROSHAKOcells. Analysis of transcriptomic changes after lnc-17-92 modulation in HMCLs and primary MM cells identified bona fide transcriptional targets of lnc-17-92. Using two independent MM RNA-seq datasets, we observed high correlation (R&amp;gt; 0.4) between lnc-17-92 expression and the expression of 12 of the transcriptional targets identified above. Interestingly, these genes were significantly enriched within metabolic pathways, suggesting an unexplored role for lnc-17-92 in MM cell metabolism. Further analysis using an RNAi-based loss-of-function screening in 3 HMCLs revealed Acetyl-CoA Carboxylase Alpha (ACC1) as a novel myeloma vulnerability. ACC1 encodes the limiting enzyme in the de novo lipogenesis pathway. Analysis of incorporation of C14-radiolabeled glucose into lipids in MM cells revealed that inhibition of ACC1 or lnc-17-92 strongly inhibited de novo lipogenesis in HMCLs and in primary MM cells. We have used ACC1 conditional KD MM cells expressing IPTG-inducible ACC1 shRNAs and confirmed significant role of ACC1 in MM cell growth and survival, both in vitro and in vivo in SCID mice model. Importantly, supplementation of palmitate, the main downstream product of ACC1 activity, significantly reverses the growth inhibitory effect of either ACC1 or lnc-17-92 suppression in MM cells. These data suggest an important role for lipogenesis pathway on lnc-17-92-promoted MM cell growth. We have further investigated mechanism by which lnc-17-92 may exert its transcriptional control. Protein-RNA pulldown assay established MYC as interacting partner of lnc-17-92. This interaction was confirmed by immunoprecipitation of MYC-bound RNA followed by qRT-PCR with specific primers for detection of lnc-17-92. ChIP-seq analysis revealed a direct binding of MYC at regulatory regions of ACC1 in MM.1S cells; these data were corroborated by the decreased ACC1 expression observed in MYC KD MM cells. Taken together, these data suggest that lnc-17-92 may function as a scaffold between MYC and the E-box motifs present on ACC1 intronic sequences, facilitating MYC binding and its transcriptional activity on ACC1. Finally, for translational application, we have pre-clinically investigated ND-646, a clinically applicable small molecule inhibitor of ACC1. Analysis of incorporation of C14-radiolabeled glucose into lipids confirmed its effect on lipogenesis in MM, which was associated with a significant in vitro growth inhibitory activity in large panel of HMCLs and primary patient MM cells. In vivo studies in murine model of human MM, using this oral agent, are ongoing and will be presented. In conclusion, we here report for the first time the microRNA-independent role of lnc-17-92 in MM pathobiology with direct impact on transcriptional control of lipogenesis. The availability of oral inhibitor of this pathway may allow the clinical application of this unique targeted therapy in MM. Disclosures Anderson: Janssen: Other: Advisory Board; Gilead Sciences: Other: Advisory Board; OncoPep: Other: Scientific founder ; Sanofi-Aventis: Other: Advisory Board; C4 Therapeutics: Other: Scientific founder . Munshi:Abbvie: Consultancy; Adaptive: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Adaptive: Consultancy; Oncopep: Consultancy; Celgene: Consultancy; Takeda: Consultancy; Janssen: Consultancy; Oncopep: Consultancy; Takeda: Consultancy; Amgen: Consultancy; Abbvie: Consultancy.

Functional Role of Linc-RNAs in Multiple Myeloma: Linc-MIR17HG Affects Fatty Acid Biosynthesis Via transcriptional Regulation of ACC1 with Potential Therapeutic Implications

Besides the well described function of RNA to produce proteins, a large volume of transcribed product has non-coding function. A recent analysis of RNA repertoire has identified a family of non-coding transcripts with sequence longer than 200 nucleotides, the long intergenic non-coding RNAs (lincRNAs). Although lincRNAs have been considered to provide regulatory functions, their precise role in cellular biology remains unclear. Using our RNA-seq data from 360 newly-diagnosed patients and 18 normal plasma cells, we have recently described the landscape of lincRNAs in multiple myeloma (MM) and reported their role as independent risk predictors for survival outcome.We have now studied the functional role of a lincRNA, the miR-17-92 primary precursor linc-MIR17HG, present in our lincRNA profile and highly correlated with overall survival in MM. We observe that inhibition of linc-MIR17HG by antisense LNA GapmeRs (n=2) leads to apoptosis in 12 genotypically distinct MM cell lines as well as in 13 primary patient MM cells. These effects are not fully rescued by expression of miR-17-92 microRNAs, suggesting a distinct biological function for linc-MIR17HG in MM. We therefore performed gene expression profile in 2 MM cell lines (AMO1 and NCI-H929) and in 2 primary patient MM cells after short-term suppression of linc-MIR17HG; and, at these early time points (18-36h), we found significant downregulation of a subset of genes (FC>2; p<0.05) including ACC1, EXT1, EPT1, ANO6, CCDC91 and KIA1109, but not miR-17-92 microRNAs. These transcriptional changes were validated by qRT-PCR in MM cell lines and primary MM cells exposed to different LNA GapmeRs targeting linc-MIR17HG with a non-overlapping spectrum of off-target effects. Importantly, our RNA-seq analysis of 360 newly-diagnosed MM patients from IFM/DFCI 2009 clinical study showed that expression of linc-MIR17HG strongly correlated with the expression of each of these genes (R2>0.4; p<0.01) in MM patients, further suggesting a regulatory function by linc-MIR17HG at transcriptional level. Using CRISPR interference (CRISPRi), we have also identified that the linc-MIR17HG with transcriptional regulatory functions is not produced from the canonical transcript isoforms MIR17HG-201/-203; these isoforms, rather, appear to be involved in production of microRNAs, leaving an alternative transcription start site usage as possible source for the transcriptional regulator isoform(s).We next investigated whether regulation of these early targets may contribute to the activity of linc-MIR17HG. We performed a RNAi-based screening in 2 MM cells lines (AMO1 and NCI-H929) by silencing each of the linc-MIR17HG downstream target genes with at least 2 different highly-specific siRNAs. This approach revealed that silencing of acetyl-CoA carboxylase 1 (ACC1, also known as ACACA), a gene encoding the limiting enzyme in the biosynthesis of fatty acids, significantly affects MM cell growth and viability. These results were validated using stable knock-down via shRNAs, confirming ACC1 as a novel vulnerability in MM. These results provide a molecular basis for reported role of fatty acid metabolism in MM cell growth and survival. We have now evaluated two orally available inhibitors of ACC1 activity, ND-630 and ND-646, in a panel of 10 MM cell lines, an report a potent time- and dose-dependent anti-proliferative effect. The activity of these inhibitors and linc-MIR17HG on fatty acid biosynthesis in MM cells is under investigation and will be presented.We have also begun to investigate molecular pathway used by linc-MIR17HG to modulate ACC1 function. Our preliminary data suggest that linc-MIR17HG may function as a scaffold between MYC and the E-box motifs present on ACC1 intronic sequences, facilitating MYC binding and its transcriptional activity.In conclusion, we highlight a transcriptional regulatory activity of a lincRNA in MM with significant functional impact that can be therapeutically translated. DisclosuresAnderson:Bristol Myers Squibb: Consultancy; Millennium Takeda: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; OncoPep: Equity Ownership, Other: Scientific founder; C4 Therapeutics: Equity Ownership, Other: Scientific founder; Celgene: Consultancy. Munshi:OncoPep: Other: Board of director.

Dysregulation of Splicing in Multiple Myeloma: The Splicing Factor SRSF1 Supports MM Cell Proliferation Via Splicing Control

Gene expression profile has provided interesting insights into the disease biology, helped develop new risk stratification, and identify novel druggable targets in multiple myeloma (MM). However, there is significant impact of alternative pre-mRNA splicing (AS) as one of the key transcriptome modifier. These spliced variants increases the transcriptomic complexity and its misregulation affect disease behavior impacting therapeutic consideration in various disease processes including cancer.Our large well annotated deep RNA sequencing data from purified MM cells data from 420 newly-diagnosed patients treated homogeneously have identified 1534 genes with one or more splicing events observed in at least 10% or more patients. Median alternative splicing event per patient was 595 (range 223 - 2735). These observed global alternative splicing events in MM involves aberrant splicing of critical growth and survival genes affects the disease biology as well as overall survival. Moreover, the decrease of cell viability observed in a large panel of MM cell lines after inhibition of splicing at the pre-mRNA complex and stalling at the A complex confirmed that MM cells are exquisitely sensitive to pharmacological inhibition of splicing.Based on these data, we further focused on understanding the molecular mechanisms driving aberrant alternative splicing in MM. An increasing body of evidence indicates that altered expression of regulatory splicing factors (SF) can have oncogenic properties by impacting AS of cancer-associated genes. We used our large RNA-seq dataset to create a genome wide global alterations map of SF and identified several splicing factors significantly dysregulated in MM compared to normal plasma cells with impact on clinical outcome. The splicing factor Serine and Arginine Rich Splicing Factor 1 (SRSF1), regulating initiation of spliceosome assembly, was selected for further evaluation, as its impact on clinical outcome was confirmed in two additional independent myeloma datasets.In gain-of (GOF) studies enforced expression of SRSF1 in MM cells significantly increased proliferation, especially in the presence of bone marrow stromal cells; and conversely, in loss-of function (LOF) studies, downregulation of SRSF1, using stable or doxy-inducible shRNA systems significantly inhibited MM cell proliferation and survival over time. We utilized SRSF1 mutants to dissect the mechanisms involved in the SRSF1-mediated MM growth induction, and observed that the growth promoting effect of SRSF1 in MM cells was mainly due to its splicing activity.We next investigated the impact of SRSF1 on allelic isoforms of specific gene targets by RNA-seq in LOF and confirmed in GOF studies. Splicing profiles showed widespread changes in AS induced by SRSF1 knock down. The most recurrent splicing events were skipped exon (SE) and alternative first (AF) exon splicing as compared to control cells. SE splice events were primarily upregulated and AF splice events were evenly upregulated and downregulated. Genes in which splicing events in these categories occurred mostly did not show significant difference in overall gene expression level when compared to control, following SRSF1 depletion. When analyzing cellular functions of SRSF1-regulated splicing events, we found that SRSF1 knock down affects genes in the RNA processing pathway as well as genes involved in cancer-related functions such as mTOR and MYC-related pathways. Splicing analysis was corroborated with immunoprecipitation (IP) followed by mass spectrometry (MS) analysis of T7-tagged SRSF1 MM cells.We have observed increased levels of SRSF phosphorylation, which regulates it's subcellular localization and activity, in MM cell lines and primary patient MM cells compared to normal donor PBMCs. Moreover, we evaluated the chemical compound TG003, an inhibitor of Cdc2-like kinase (CLK) 1 and 4 that regulate splicing by fine-tuning the phosphorylation of SR proteins. Treatment with TG003 decreased SRSF1 phosphorylation preventing the spliceosome assembly and inducing a dose dependent inhibition of MM cell viability.In conclusions, here we provide mechanistic insights into myeloma-related splicing dysregulation and establish SRSF1 as a tumor promoting gene with therapeutic potential. DisclosuresAvet-Loiseau:Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding. Munshi:OncoPep: Other: Board of director.

Inhibitor of DNA Binding 2 (ID2) Plays a Key Tumor Suppressor Role in Promoting Oncogenic Transformation in Multiple Myeloma

Dysregulation of transcriptional control is a common phenomenon associated with oncogenesis. Inhibitors of DNA binding (ID) proteins are critical actors in lymphopoiesis, acting as regulators of transcription through a helix-loop-helix (HLH) domain which enables heterodimerization with basic HLH (bHLH) proteins inhibiting their binding to DNA. ID proteins have been implicated in malignant transformation, but their role in multiple myeloma (MM) is unknown. Here, we evaluated the role of ID proteins in biology and transcriptional dysregulation in MM.We first evaluated the expression of the four ID proteins in normal and malignant plasma cells using RNA sequencing data from a cohort of 360 newly diagnosed MM patients and 16 normal plasma cells. We observed significant downregulation of ID2 in primary patient MM cells in comparison to normal plasma cells (p 0.0013).To study ID2 function in MM cells, we next overexpressed ID2 in 2 MM cell lines (MM1S and NCIH929) and observed a significant decrease in proliferation rate, together with G0/G1 phase cell cycle arrest. We performed RNA-sequencing to evaluate the transcriptomic changes following ID2 overexpression. Gene set enrichment analysis (GSEA) revealed significant downregulation of genes involved in E2F pathway and significant changes in pathways related to immune response, regulation of cell death and cell proliferation. In addition, analysis of upstream cis-regulatory motifs of genes significantly dysregulated in both cell lines (>1.5 fold change) showed a highly significant enrichment for bHLH class I transcription factors (E proteins) binding motifs. Conversely, stable ID2 knockdown in 4 MM cell lines (MM1S, NCIH929, RPMI8226 and KMS11) expressing intermediate levels of ID2, showed an increased proliferation rate, assessed by cell counting, H3-thymidine incorporation and ATP production. RNA-sequencing after ID2 knockdown in MM1S and NCIH929 cells showed 600 common genes upregulated in both cell lines (>1.5 fold change). GSEA revealed upregulation of pathways involved in inflammatory response and epithelial-to-mesenchymal transition, while upstream cis regulatory motifs analysis showed a highly significant enrichment for binding motifs of bHLH class I transcription factors E proteins, in particular Tcf3 (p <0.0001).Next, we sought to investigate the mechanisms involved in ID2 downregulation in MM. Since the role of the microenvironment is critical in myelomagenesis, we evaluated the impact of BM microenvironment on ID2 expression in a co-culture system. Using bone marrow stromal cells (BMSC) derived from MM patients and stromal cell line (HS5) in co-culture with various MM cell lines, we observed that both cell-cell interactions and soluble factors secreted by BMSC or HS5 were able to significantly downregulate ID2 expression at the RNA and protein level. Furthermore, ID2 overexpression in MM cell lines (MM1S and NCIH929) abrogated the impact of BMSC on MM cell proliferation. Next, we evaluated ID2 promoter methylation profile and binding motifs using Sequenom mass array and the assay for transposase-accessible chromatin sequencing (ATAC-seq), respectively. While we didn't observe any increase in methylation of CpG islands located in ID2 promoter in co-culture, explaining ID2 downregulation, we identified several binding motifs corresponding to known driver transcription factors in MM. Especially, we identified SP1 binding motif and we confirmed SP1 binding to ID2 promoter by ChIP-sequencing in MM1S, NCIH929 and U266.These data demonstrate that in MM, ID2 acts as a tumor suppressor by promoting major transcriptomic changes and cell cycle arrest. Bone marrow stromal cells further induce significant downregulation of ID2 in myeloma cells suggesting that ID2/bHLH axis and other ID2 related pathways represent a potential new therapeutic target in myeloma. DisclosuresAnderson:Gilead: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Consultancy; Celgene: Consultancy; C4 Therapeutics: Equity Ownership, Other: Scientific founder; OncoPep: Equity Ownership, Other: Scientific founder; Millennium Takeda: Consultancy. Munshi:OncoPep: Other: Board of director.

Abstract B08: Biomimetic Culture Platform for Ex Vivo Preservation of Patient-Derived Multiple Myeloma Cells

Abstract Multiple myeloma (MM) is an incurable B-cell malignancy with a typical survival of 5 to 7 years, and is characterized by: (1) the accumulation of IgG (κ or λ) producing monoclonal plasma cells in bone marrow (BM) and (2) the progression of metastatic osteolytic lesions in bones. We have developed a new approach for the extended culture (&amp;gt; 3 weeks) of difficult-to-preserve primary patient multiple myeloma cells (PMMCs) present in bone marrow aspirates, using an osteoblast (OSB)-derived 3D tissue scaffold constructed in a perfused microfluidic environment [1]. In contrast, the ex vivo viability of PMMCs cannot be preserved using conventional culture techniques beyond a few days. This biomimetic model was used to show, for the first time, that: (1) the adhesion of PMMCs to OSB, mediated by osteoblastic N-cadherin, and the long-term viability of OSBs were critical factors in maintaining the ex vivo viability and proliferative capacity of PMMCs; (2) perfusion flow enhances the survival of OSBs, and thus increases the long-term viability of PMMCs; and noteworthy, (3) PMMCs can acquire drug-resistance due to the adhesive interactions with OSBs, a phenomenon called cell adhesion-mediated drug-resistance (CAM-DR) [2]. These findings support that the in vitro model is capable of recapitulating critical MM tumor microenvironment associated with the survival and drug-resistance of PMMCs. Upon further development, our ex vivo model may provide a new means of selecting the best therapy for an individual patient based on the ex vivo response of his or her PMMCs to drug treatment options as well as novel immunotherapeutic treatments such as chimeric antigen receptor (CAR) T-cell therapy. This precision medicine approach is expected to provide a transformative means of: (1) reducing ineffective or unnecessary therapies; (2) minimizing excess toxicity and costs; and (3) inhibiting the development of tumor cells' cross-resistance to additional drugs, as a critical part of the long-term care of MM patients. Also, we anticipate that our approach can be used for: (1) preclinical evaluation of new therapeutics, (2) studying the development of drug-resistant PMMC subpopulations, (3) identifying novel therapeutic targets, and (4) developing ex vivo models of breast and prostate cancers that metastasize to BM via similar mechanisms. Currently, we are in the early stage of prospectively correlating the ex vivo chemosensitivity and resistance of PMMCs with short-term clinical response, as a means of validating the predictive capability of our ex vivo MM culture platform. We also plan to incorporate the 3D-reconstructed network of primary human osteocytes [3] to study interactions of osteocytes with PMMCs and other BMMCs including immune cells.

A New Class of T-Cell Bispecific Antibodies for the Treatment of Multiple Myeloma, Binding to B Cell Maturation Antigen and CD3 and Showing Potent, Specific Antitumor Activity in Myeloma Cells and Long Duration of Action in Cynomolgus Monkeys

A New Class of T-Cell Bispecific Antibodies for the Treatment of Multiple Myeloma, Binding to B Cell Maturation Antigen and CD3 and Showing Potent, Specific Antitumor Activity in Myeloma Cells and Long Duration of Action in Cynomolgus Monkeys

Activation of Lysosomal Function and Reactive Oxygen Species Play Crucial Roles in SAR650984-Induced Direct Killing of Human Multiple Myeloma Cells with Mutated p53, Which Is Further Augmented By Pomalidomide

The anti-CD38 monoclonal antibody (mAb) SAR650984 (SAR) is showing promising clinical activities in ongoing trials in relapsed/refractory multiple myeloma (MM), a setting in which p53 is often mutated. Besides effector-mediated antibody-dependent cellular cytotoxicity and complement-mediated cytotoxicity, we here define additional molecular mechanisms of SAR-directed MM cell death, and further delineate enhanced anti-MM activity triggered by combining SAR with pomalidomide (Pom) or lenalidomide (Len). Since CD38 expression on MM cell lines is not as high as on primary patient MM cells, we first used lentivirus to overexpress CD38 in 3 MM cell lines with lower CD38 levels and p53 mutations. Without Fc-cross-linking agents or effector cells, SAR, but not other anti-CD38 mAb, induced homotypic aggregation (HA)-associated MM cell death in these cell lines, which was dependent on CD38 level, membrane lipid raft, and actin cytoskeleton. SAR and its F(ab)'2 fragments triggered comparable caspase 3/7 activation in CD38-highly expressing MM cells, indicating that SAR-induced caspase-dependent apoptosis is both CD38-specific and Fc-independent. Most importantly, SAR and its F(ab)'2 fragments induce lysosome-dependent cell death (LCD) in CD38 overexpressing MM cells even with p53 mutations, evidenced by enlarged lysosomes and increased lysosomal membrane permeabilization (LMP) along with leakage of cathepsin B and lysosome associated membrane protein 1 (LAMP1), in the presence or absence of IL-6 or bone marrow stromal cells. SAR significantly induced mobilization of cathepsin B and LAMP1 from lysosomes to the cytosol, particularly at the intercellular junctions, further confirming that lysosomes relocalize to sites of cell adhesion following SAR treatment. Conversely, the lysosomal vacuolar H+-ATPase inhibitor blocks SAR-induced LCD. SAR stimulated reactive oxygen species (ROS), whereas the antioxidant N-acetylcysteine (NAC) blocks SAR-induced ROS and MM cell death. Since NAC does not inhibit HA or lysosome enlargement or LMP, SAR-induced ROS occurs downstream of HA and activation of lysosomal function. SAR does not induce autophagy, indicating that autophagy is not required for this unique mode of cell death. Finally Pom, more potently than Len, enhances SAR-induced direct and indirect killing of MM cells, even resistant to Pom/Len. Moreover, combined treatment of MM cells with SAR and Pom augments caspase 3/7 activity induced by either agent alone. Taken together, these data show that SAR is the first naked therapeutic mAb with convincing direct killing of MM cell lines and patient MM cells, including those with p53 mutations and drug resistance, mainly via lysosome activation and ROS. Coupled with augmented both caspase 3/7-dependent and effector-mediated MM cytotoxicity when combined with Pom, our data further provide the framework for combination clinical trials. DisclosuresSong:Sanofi: Employment. Yang:Sanofi: Employment. Adrian:Sanofi: Employment. Richardson:Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees.

Bidirectional Notch Signaling Between Multiple Myeloma (MM) Cells and Osteocytes As a Potential Target to Inhibit Tumor Growth and Osteoclast Recruitment in MM

Multiple myeloma (MM) is characterized by monoclonal plasma cells that induce devastating bone disease, by markedly increasing osteoclastic bone destruction, and severely suppressing bone formation. Although the contributions of bone marrow stromal cells, immune cells, and osteoclasts to tumor growth and bone disease in MM have been extensively studied, little information is available on the role of osteocytes (Ots) in MM, which are the primary regulators of bone homeostasis and comprise 95% of bone cells. We recently showed that MM cells and Ots physically interact in vivo through the osteocytic lacunar-canalicular network. This network allows direct cell-to-cell communication and distributes Ot-secreted molecules among cells in bone and the bone marrow to regulate osteoblast and osteoclast activity. Further, apoptotic Ots recruit osteoclast precursors to specific areas of bone for localized bone resorption.In the current study we report that Ot-MM cell interactions activate bidirectional Notch signaling between Ots and MM cells which induce: 1) Ot apoptosis, that is maintained by MM-derived TNFα; 2) stimulate Ot and MM cells Rankl production, and 3) markedly increase MM cell growth. For these studies we used co-cultures of murine Ot-like MLO-A5 cells with murine and human MM cell lines or primary patient MM cells in vitro, 6wk-old female SCID mice injected intratibially with human JJN3 MM cells or saline in vivo, and a novel ex vivo calvarial organ culture system with murine 5TGM1 MM cells to provide a more realistic bone microenvironment with an intact osteocytic network.We found that the prevalence of Rankl-expressing Ots was significantly higher in JJN3-injected mice compared to controls. Similarly, increased Rankl expression in osteocytic cells was observed in co-cultures of Ots with JJN3, 5TGM1 or MM patient cells. Further, inhibition of MM-induced Ot apoptosis with DEVD significantly reduced Rankl levels as did anti-TNFα treatment. We next determined the effects of Ot apoptosis on osteoclast precursor recruitment. Conditioned media (CM) from JJN3 and MLO-A5 cells co-cultured in direct contact enhanced osteoclast precursor chemotaxis by 50% compared to CM from MLO-A5 or JJN3 cells cultured alone. This effect was inhibited by blocking Ot apoptosis with DEVD or by a combination of the Notch inhibitor GSIXX and anti-TNFα. Taken together, these results show that MM-induced Ot apoptosis increases Ot Rankl expression and potentiates Ot-mediated recruitment of osteoclast precursors.Importantly, we found that the Ot-MM Notch signaling was bidirectional. Direct contact of MM cells with Ot-like cells increased the expression of the Notch target genes Hes1/Hey1 in MM patient and 5TGM1 cells, which was abolished by the Notch inhibitor GSIXX. Moreover, expression of Rankl was increased in 5TGM1 and MM patient cells cultured in direct contact with MLO-A5 cells. Further, MM-Ot-like cell-to-cell contact specifically increased Notch receptor (R) 3 expression in patient MM cells, and Notch R1-3 as well as induced R4 in 5TGM1 cells. Finally, cell-to-cell contact between MM patient cells or 5TGM1 cells with MLO-A5 cells increased MM cell proliferation that was blocked by GSIXX. Culture of 5TGM1 cells in an ex vivo bone organ system containing authentic murine Ots also activated MM cell Notch signaling, with increased expression of Hey1, and increased the expression of Notch R3 and Rankl. All these effects were inhibited by the addition of Notch inhibitor GSIXX to the ex vivo cultures.These results indicate that bidirectional Notch signaling between MM cells and Ots increases MM tumor growth, Ots apoptosis and the expression of molecules that regulate osteoclast activity and chemotaxis. They further show that Ots can regulate tumor growth and osteoclast activity in MM and suggest that development of pharmacologic agents that selectively target NotchR3-mediated signaling in primary MM cells should decrease tumor growth and avoid the serious side-effects associated with generalized Notch inhibition. DisclosuresNo relevant conflicts of interest to declare.

Targeting 19S-Proteasome Deubiquitinase Rpn11/POH1/PSMD14 in Multiple Myeloma

Introduction Deregulation of the ubiquitin-proteasome system (UPS) is linked to pathogenesis of various human diseases, including cancer. Targeting the proteasome is an effective therapy in multiple myeloma (MM) patients. Recent research efforts led to the discovery of newer agents that target enzymes modulating protein ubiquitin-conjugation/deconjugation rather than the proteasome itself, with the goal of generating more specific and less toxic antitumor therapies. Ubiquitylation is a dynamic reversible process coordinated by many enzymes: ubiquitin ligases attach ubiquitin to proteins allowing for their degradation, whereas deubiquitylating (DUB) enzymes deconjugate ubiquitin from target proteins, thereby preventing their proteasome-mediated degradation. Rpn11 is a DUB enzyme associated with the 19S regulatory particle lid of the proteasome that removes ubiquitin from target proteins to facilitate protein degradation by 20S proteasome core particle. Here we examined the role of Rpn11 in MM using both biochemical and RNA interference strategies.Materials and Methods Cell viability and apoptosis were assessed using WST and Annexin V staining, respectively. MM.1S MM cells were transiently transfected with control short interfering RNA (siRNA), RPN11 ON TARGET plus SMART pool siRNA using the cell line Nucleofector Kit V. Isobologram analysisand CalcuSyn software program were utilized to assesssynergistic/additive anti-MM activity. Ub-AMC assay Proteasome activity was measured, as in our prior study (Chauhan et al., Cancer Cell 2005, 8:407-419). Signal transduction pathways were evaluated using immunoblotting. Statistical significance of data was determined using a Student's t test. O-phenanthroline (OPA) was purchased from EMD Millipore, USA; and bortezomib, lenalidomide, and pomalidomide were purchased from Selleck chemicals, USA.Results Gene expression (GEP) analysis of Rpn11 showed a significantly higher level in primary patient MM cells (n=73) versus normal plasma cells (n=15) (p < 0.05). We found a statistically significant inverse correlation between Rpn11 levels and overall patient survival (p =0.035). Western blot analyses show higher Rpn11 levels in MM cell lines and patient cells compared to normal cells. Rpn11-siRNA significantly decreased MM cell viability (p < 0.001; n=3). To further validate our siRNA data, we utilized Rpn11 inhibitor O-phenanthroline (OPA) (Verma et al., Science 2002, 298:611-5). Treatment of MM cell lines (MM.1S, MM.1R, RPMI-8226, ARP-1, Dox40, LR5, INA6, ANBL6.WT, and ANBL6.BR) and primary patient cells for 48h significantly decreased their viability (IC50 range 8µM to 60µM; p < 0.001 for all cell lines; n=3) without markedly affecting PBMCs from normal healthy donors, suggesting specific anti-MM activity and a favorable therapeutic index for OPA. Tumor cells obtained from patients whose disease was progressing while on bortezomib, dexamethasone, and lenalidomide therapies remained sensitive to OPA. Moreover, the cytotoxicity of OPA was observed in MM cell lines sensitive and resistant to conventional (dex) and novel (bortezomib) therapies. Furthermore, OPA inhibits proliferation of MM cells even in the presence of BM stromal cells or pDCs. OPA inhibits Rpn11 DUB activity without blocking 20S proteasome activities. Mechanistic studies show that OPA-triggered MM cell death is associated with 1) accumulation of cells in early and late apoptotic phase; 2) increase in polyubiquinated proteins; and 3) activation of caspases mediating both intrinsic and extrinsic apoptotic pathways. Importantly, OPA-induced apoptosis in MM cells occurs in a p53-independent manner, since OPA triggered significant apoptosis in both p53-null (ARP-1) and p53-mutant (RPMI-8226) MM cells (p < 0.004). Finally, combining OPA with lenalidomide, pomalidomide, or bortezomib induces synergistic/additive anti-MM activity, and overcomes drug resistance.Conclusion Our preclinical data showing efficacy of OPA in MM disease models validates targeting 19S proteasome-associated DUB Rpn11 upstream of the proteasome in the ubiquitin proteasomal cascade to overcome proteasome inhibitor resistance, and provides the framework for clinical evaluation of Rpn11 inhibitors to improve patient outcome in MM. DisclosuresChauhan:Stemline Therapeutics: Consultancy.

Palmitic Acid, As Verified Using Lipid Profiling By Secondary Ion Mass Spectrometry, Demonstrates Anti-Tumor Activity Against Multiple Myeloma

Introduction Many recent studies have examined lipid metabolic changes in multiple myeloma (MM). Changes in lipid metabolism affect the survival of MM cells. Developments in imaging mass spectrometry (IMS) have facilitated research on the lipid profiles of tumors. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is an IMS technique that uses a focused ion beam as the primary source for ionization. TOF-SIMS imaging is used to analyze the surface of specimens at sub-micrometer resolution, enabling analyses of the subcellular distribution of molecules in individual cells. TOF-SIMS analysis has enabled the detection of multiple fatty acid groups from single cells. Therefore, we applied this method to human clinical specimens to analyze the membrane fatty acid composition and determine candidate molecules for MM therapies. Using the different lipid profiles for MM cells and normal plasma cells (PCs), we conducted a cytocidal assay with MM cell lines supplemented with the fatty acids screened out by the profiles to assess lipotoxicity against MM. The molecules demonstrating distinct differences among cell types (i.e., MM and PC) were considered candidates for which supplementation leads to imbalanced lipid metabolism and cell death in a tumor-specific manner. We further evaluated the induction of apoptosis.Methods Primary patient MM cells and normal PCs were isolated from the bone marrow aspirates of two patients and two healthy volunteers using fluorescence-activated cell sorting. These separated cells were analyzed with PHI TRIFT V (ULVAC-PHI, Inc.). Analyses were performed in negative ion mode, and signals in the mass range of m/z 0 to 1850 were monitored. We performed pairwise comparisons of mean signal intensities for five types of fatty acids between MM cells and PCs.MM cell lines (U266 and RPMI-8226) were treated with 0–1000 µM of palmitic acid, palmitoleic acid, linoleic acid, oleic acid, and stearic acid. The number of viable cells in suspension at 72 hours after treatment was determined by the trypan blue exclusion test. HS-5, a human bone marrow stromal cell line, was used in the co-culture experiment. Healthy volunteers’ normal peripheral blood mononuclear cells (PBMCs) were purified by Ficoll-Hypaque density-gradient centrifugation. The distribution of apoptotic and necrotic cells were analyzed by measuring AnnexinV binding and propidium iodide uptake.Results The amounts of MM cells and PCs relative to the total nucleated cells were 3.38%, 35.9% for MM cells, 0.0368% and 0.246% for PCs. Multiple ions, including phosphoric acid, and five species of fatty acids (palmitoleic acid, palmitic acid, linoleic acid, oleic acid, and stearic acid) were detected. The mean signal intensities of palmitoleic acid and palmitic acid of MM cells were significantly lower than those of normal PCs (P = .00081 and .0018, respectively). These results were replicated in a second pairwise comparison. We did not observe statistically significant differences in intensities for linoleic acid, oleic acid, or stearic acid.In the cytocidal assay, palmitic acid reduced U266 cell viability dose-dependently for doses of 50–1000 μM. High concentrations of the other fatty acids also reduced cell viability; however, the effect on cell death was not observed at the low dose of 50–100 µM, as it was for palmitic acid. Even in co-culture experiments, palmitic acid decreased the viability of MM cells. Moreover, the proportions of both apoptotic and necrotic cells increased and the proportion of viable cells decreased 24 hours after palmitic acid treatment in MM cells. Palmitic acid also reduced the viability of RPMI-8226 cell lines. Meanwhile, cell viabilities of normal PBMCs were not affected by palmitic acid, even at 100–500 µM.Conclusion We applied the single-cell TOF-SIMS lipid analysis effectively to a very small population of cells. Significantly smaller intensities of palmitoleic acid and palmitic acid were observed in MM cells compared to normal cells. We also demonstrated an inhibitory effect of palmitic acid on the survival of MM cells. Palmitic acid is a potential candidate for novel therapeutic agents that specifically attack MM and should be considered in future studies of MM in a lipid biology framework. DisclosuresNo relevant conflicts of interest to declare.

Targeting Proteasome Ubiquitin Receptor RPN13/ADRM1 in Multiple Myeloma

Background and Rationale Deregulation of the ubiquitin-proteasome system (UPS) is linked to pathogenesis of various human diseases, including cancer. Targeting the proteasome is an effective therapy in multiple myeloma (MM) patients. Recent research efforts led to the discovery of newer agents that target enzymes modulating protein ubiquitin-conjugation/deconjugation rather than the proteasome itself, with the goal of generating more specific and less toxic antitumor therapies. Ubiquitylation is a dynamic reversible process coordinated by many enzymes: ubiquitin ligases attach ubiquitin to proteins allowing for their degradation, whereas deubiquitylating enzymes deconjugate ubiquitin from target proteins, thereby preventing their proteasome-mediated degradation. RPN13 is ubiquitin receptor within the 19S regulatory particle lid of the proteasome that recognizes ubiquitylated proteins marked for degradation by 20S core particle. Here we examined the role of RPN13 in MM using both biochemical and RNA interference strategies.Materials and Methods We utilizedMM cell lines, patient tumor cells, and peripheral blood mononuclear cells (PBMCs) from normal healthy donors. Drug sensitivity/cell viability and apoptosis were assessed using XTT/MTT and Annexin V staining, respectively. MM.1S cells were transiently transfected with control short interfering RNA (siRNA), RPN13 siRNA ON TARGET plus SMART pool siRNA using the cell line Nucleofector Kit V. Synergistic/additive anti-MM activity was assessed by isobologram analysisusing “CalcuSyn” software program. Signal transduction pathways were evaluated using immunoblotting. Proteasome activity was measured as previously described (Chauhan et al., Cancer Cell 2005, 8:407-419). Statistical significance of data was determined using a Student’s t test. RA190 was purchased from Xcess Biosciences, USA; and bortezomib, lenalidomide, and pomalidomide were purchased from Selleck chemicals, USA.Results Analysis of RPN13/ADRM1 expression showed a significantly higher level in primary patient MM cells (n=73) versus normal plasma cells (n=15) (p < 0.004). Similarly, immunoblot analysis showed elevated RPN13 in MM cells versus normals. RPN13 siRNA knockdown significantly decreased MM cell viability (p < 0.001; n=3). To further validate our siRNA data, we utilized recently reported novel agent RA190 (bis-benzylidine piperidone) that targets RPN13. RA190 inhibits recognition of polyubiquitylated proteins and their deubiquitylation, which in turn prevents their degradation (Anchoori et al., Cancer Cell 2013, 24:791). Treatment of MM cell lines (MM.1S, MM.1R, RPMI-8226, ARP-1, ANBL6.WT, and ANBL6.BR) and primary patient cells for 48h significantly decreased their viability (IC50 range 200nM to 600nM; p < 0.001 for all cell lines; n=3) without markedly affecting PBMCs from normal healthy donors, suggesting specific anti-MM activity and a favorable therapeutic index for RA190. Tumor cells were obtained from patients whose disease was progressing while on bortezomib, dexamethasone, and lenalidomide therapies. Moreover, the cytotoxicity of RA190 was observed in MM cell lines sensitive and resistant to conventional (dex) and novel (bortezomib) therapies. Furthermore, RA190 inhibits proliferation of MM cells even in the presence of BM stromal cells. Mechanistic studies show that RA190-triggered MM cell death is associated with 1) accumulation of cells in early and late apoptotic phase; 2) increase in polyubiquinated proteins; and 3) activation of caspases mediating both intrinsic and extrinsic apoptotic pathways. Importantly, RA190-induced apoptosis in MM cells occurs in a p53-independent manner, since RA190 triggered significant apoptosis in both p53-null (ARP-1) and p53-mutant (RPMI-8226) MM cells (p < 0.004). Finally, combining RA190 with lenalidomide, pomalidomide, or bortezomib induces synergistic/additive anti-MM activity, and overcomes drug resistance.Conclusion Our preclinical data showing efficacy of RA190 in MM disease models validates targeting ubiquitin receptors upstream of the proteasome in the ubiquitin proteasomal cascade to overcome proteasome inhibitor resistance, and provides the framework for clinical evaluation of RPN13 inhibitors to improve patient outcome in MM. DisclosuresNo relevant conflicts of interest to declare.

Multiple Myeloma Cells Express IL-17A Creating An Autocrine Loop: An Attractive Therapeutic Target

We have previously demonstrated that Th17 cells, which produce IL-17A, are significantly elevated in peripheral blood and bone marrow (BM) of patients with Multiple Myeloma (MM) and IL-17A promotes MM cell growth and survival, both in vitro and in vivo via IL-17A receptor. We have recently evaluated and observed that anti-IL-17A monoclonal antibody (mAb) significantly inhibited MM cell growth in vitro, while IL-17A induced proliferation of MM cells compared to control. We have also observed significant down-regulation of IL-6 production by anti-IL-17A mAb in MM-BMSC co-culture. Importantly, the administration of anti-IL-17A mAb weekly for 4 weeks in the SCIDhu model of human myeloma, where MM cells grow within the human microenvironment in mice led to a significant inhibition of tumor growth compared to the control mice. This remarkable activity of anti-IL17 mAb raised the question of whether the myeloma cells themselves are a possible source of IL-17. In this study, we used transcriptome sequencing (RNA-Seq) data to evaluate the expression of IL-17A in primary CD138+ myeloma cells (N=17) compared to normal plasma cells (NPC) (N=5). Whereas none of the NPCs expressed IL-17A, it was significantly over-expressed in majority of MM cells. In addition, these data also showed that the expression of other IL-17 family members (IL-7B, C, D, E & F) and Th17-associated pro-inflammatory cytokines (IL-21, IL-22 & IL-23) were not significantly elevated in primary myeloma cells compared to normal donor plasma cells. We further validated these observations by IL-17 immunoblot showing IL17 expression in all MM cell lines and 10 out of 14 primary patient MM cells; confirmed IL-17 expression in MM cells by quantitative RT-PCR, and flow cytometry and by immuno-histochemistry and confocal microscopy. We observed that IL-17 knock down by IL-17-specific siRNA inhibited MM cell growth as well as their ability to induce IL-6 production in co-cultures with BMSC. Finally, expression profile data from 172 uniformly treated patients showed that patients with lower IL-17A expression had superior overall survival compared to those with higher expression. These data confirms that MM cells express IL-17 and targeting it with a mAb will abrogate the autocrine loop making it an attractive therapeutic target. Disclosures:No relevant conflicts of interest to declare.

CD147 regulates the expression of MCT1 and lactate export in multiple myeloma cells

Increased use of the glycolytic pathway, even in the presence of oxygen, has recently been recognized as a key characteristic of malignant cells. However, the glycolytic phenotype results in increased lactic acid production and, in order to prevent cellular acidosis, tumor cells must increase proton efflux via upregulation of pH regulators such as proton-pumps, sodium-proton exchangers, and/or monocarboxylate transporters (MCT) (e.g., MCT1, MCT4). Interestingly, expression of MCT1 and MCT4 has been previously shown to be dependent upon expression of the transmembrane glycoprotein CD147. Recently, we demonstrated that primary patient multiple myeloma (MM) cells and human MM cell lines (HMCLs) overexpress CD147. Therefore, the goal of the current study was to specifically determine if MCT1 and MCT4 were also overexpressed in MM cells. RT-PCR analysis demonstrated both primary patient MM cells and HMCLs overexpress MCT1 and MCT4 mRNA. Notably, primary MM cells or HMCLs were found to express variable levels of MCT1 and/or MCT4 at the protein level despite CD147 expression. In those HMCLs positive for MCT1 and/or MCT4 protein expression, MCT1 and/or MCT4 were found to be associated with CD147. Specific siRNA-mediated downregulation of MCT1 but not MCT4 resulted in decreased HMCL proliferation, decreased lactate export, and increased cellular media pH. However, western blot analysis revealed that downregulation of MCT1 also downregulated CD147 and vice versa despite no effect on mRNA levels. Taken together, these data demonstrate the association between MCT1 and CD147 proteins in MM cells and importance of their association for lactate export and proliferation in MM cells.

Targeting miR-21 InhibitsIn VitroandIn VivoMultiple Myeloma Cell Growth

Deregulated expression of miRNAs plays a role in the pathogenesis and progression of multiple myeloma. Among upregulated miRNAs, miR-21 has oncogenic potential and therefore represents an attractive target for the treatment of multiple myeloma. Here, we investigated the in vitro and in vivo anti-multiple myeloma activity of miR-21 inhibitors. Either transient-enforced expression or lentivirus-based constitutive expression of miR-21 inhibitors triggered significant growth inhibition of primary patient multiple myeloma cells or interleukin-6-dependent/independent multiple myeloma cell lines and overcame the protective activity of human bone marrow stromal cells. Conversely, transfection of miR-21 mimics significantly increased proliferation of multiple myeloma cells, showing its tumor-promoting potential in multiple myeloma. Importantly, upregulation of miR-21 canonical validated targets (PTEN, Rho-B, and BTG2), together with functional impairment of both AKT and extracellular signal-regulated kinase signaling, were achieved by transfection of miR-21 inhibitors into multiple myeloma cells. In vivo delivery of miR-21 inhibitors in severe combined immunodeficient mice bearing human multiple myeloma xenografts expressing miR-21 induced significant antitumor activity. Upregulation of PTEN and downregulation of p-AKT were observed in retrieved xenografts following treatment with miR-21 inhibitors. Our findings show the first evidence that in vivo antagonism of miR-21 exerts anti-multiple myeloma activity, providing the rationale for clinical development of miR-21 inhibitors in this still incurable disease.