Treated Myeloma Cells Research Articles

Chinese Herbal Medicine 'Diwu' Suppresses Myeloma Bone Disease Via ERK/MAPK Signaling Pathway By Targeting CCL3

Background： Multiple myeloma (MM) associated bone disease (MBD) is largely dependent on the bone marrow microenvironment (BME). The cross-talk between MM cells, bone marrow mesenchymal cells and myeloid progenitors affects the osteoclastogenesis. Traditional Chinese medicine 'Diwu' is effective for patients with arthritis and inflammation, also occasionally uses as one ingredient treating MM. But the effects of 'Diwu' in MBD are largely unknown. The aim of this study is to investigate the role and underlying mechanism of 'Diwu' in regulating BME and osteoclast differentiation. Methods： Myeloma cell lines RPMI-8226, NCI-H929, U266 and MM1 were treated with 'Diwu' extract. Killing effects was evaluated by the half inhibitory dose (IC50) value and proportion of apoptotic MM cells. RNA-seq was applied in 'Diwu' treated myeloma cells to identify MBD associated signaling pathways. RPMI-8226 was co-cultured with mesenchymal stromal cell (MSC) line HS5. Human monocyte cell line THP-1 was treated with CCL3 and M-CSF to form osteoclasts determined by tartrate-resistant acid phosphatase (TRACP) staining. Primary human MSCs were cultured into osteogenic or adipogenic differentiation, determined by alizarin red staining and oil red staining, respectively. RNAi technology was applied to knock down CCL3. The expression of osteoclast-specific genes TRACP and MAPK pathway-related genes was tested using quantitative real-time PCR. Phosphorylation and expression of MEK1, ERK, FOS were measured by Western Blot. The myeloma-bearing NOD/SCID mice were treated with intragastric 'Diwu'. Serum C-terminal telopeptide of type 1 collagen (CTXI) and propeptide of type I procollagen (PINP) was tested by ELISA. The tibia of mice was fixed and stained to analyze the differentiation and maturation of osteoblasts and osteoclasts. Micro-CT was used for bone destruction measurement. Results： 'Diwu' inhibited MM cells proliferation (IC50, 29.22-364.7μg/ml) in a dose-dependent manner and promoted apoptosis by upregulating protein expression of CASP3, BCL2. 'Diwu' decreased the secretion of chemokine (C-C motif) ligand 3 (CCL3) and receptor activator of nuclear factor-kappa B ligand (RANKL) in myeloma-MSC co-cultured system by diminishing the direct attachment and interaction of these two cells. The mRNA and protein levels of osteoclast-specific genes such as TRACP, and the phosphorylation and expression of MEK1, ERK, FOS in osteoclast were all reduced after 'Diwu' treatment. We observed improved osteogenic activation rather than lipid formation in hMSC differentiation. The level of TRACP isoform 5b (TRACP-5b) was significantly downregulated in mouse serum after 'Diwu' treatment for 14 days. Conclusions: Altogether, our results demonstrated that effective extract of Chinese herbal medicine 'Diwu' could simultaneously inhibit myeloma cells and suppress osteoclast formation, indicating an important role of 'Diwu' in regulating BM microenvironment. It is possible to translate 'Diwu' into clinical application as an oral agent for myeloma bone disease. Key words： 'Diwu'; Myeloma Bone Disease; Bone Marrow Microenvironment; CCL3; ERK/MAPK

MAFA Expression Promotes Proliferation of Myeloma Cells and Its Protein Stability Is Regulated By Proteasome Inhibitors

IntroductionMultiple myeloma (MM) patients with a t(14;16) or t(14;20) which result in high expression of MAF and MAFB, respectively, have a poor prognosis, and have not benefitted from the recent advances in MM therapy. Our previous work showed that post-translational modifications (PTM) further up-regulated MAF (Blood, 2016) and MAFB proteins (ASH Abstract #2091, 2014) conferring innate resistance to proteasome inhibitors (PI). The large MAF family of proteins contains a third member, MAFA. Little is known about its biological role in MM pathogenesis, as a probeset for MAFA is not present in gene expression profiling (GEP) analysis of molecular subgroups, and MAFA probes are not routinely used in fluorescence in situ hybridization (FISH) analysis. In the present study we therefore investigated the biological role of MAFA in MM and its effect on PI resistance.MethodsThe MAFA translocation in primary MM cells was determined by FISH analysis in 19 newly diagnosed MM patients. MAFA protein expression and protein level in response to PIs were assessed by immunoblotting and immunofluorescence staining (IFS). The effect of MAFA on proliferation of MM cells was measured by MTT assay. Annexin V staining and BrdU incorporation, analyzed by FACS analysis, was performed to determine the molecular action of MAFA on the survival of MM cells. MAFA expression was down-regulated in human MM cell lines (HMCLs). HMCLs using a lentiviral shRNA expression system. qRT-PCR analysis was used to determine MAFA mRNA expression in 32 HMCL and primary CD138+ plasma cells from 14 MM patients, and MAFA levels after shRNA treatment.ResultsFour levels of MAFA mRNA expression (relative to GAPDH) by RT-PCR analysis were determined: high levels were detected in 5 HMCLs, intermediate levels (100 to 500 ) in 10, low levels (10<100) in 7 and negative (<10) in 10 HMCLs. Among primary CD138+ patient cells (n=14), high levels were found in one patient, intermediate levels in 3, low levels in 4 and negative in 6 patients. FISH analysis found MAFA /t(8;14)(q24;q32) translocation in one out of 19 patients, which was also in the MF group by GEP analysis suggesting common downstream targets of MAFA with MAF and MAFB . MAFA protein was detected in the 6 HMCLs which expressed the highest MAFA mRNA levels by RT-PCR but not in the others.To determine if MAFA is required for MM cell survival, we knocked-down MAFA expression in three HMCLs that expressed high levels of MAFA . sh MAFA lentiviral supernatant infected HMCLs had 75% lower levels of MAFA mRNA and protein level compared with the cells infected with scrambled shRNA. Additionally, bioinformatically defined MAFA target genes were significantly decreased, including TLR4 (2.4-fold, P<0.01), NUAK1 (2.3-fold, p<0.01) and SPP1 (osteopontin, 2.2-fold, P<0.01), compared with HMCLs infected with control viral vector. Silencing MAFA expression significantly decreased the proliferation of HMCLs (62.5% decrease, P=2.1x10-4). Among 6 high MAFA expressing HMCLs, 3 co-expressed MAF and 2 co-expressed MAFB . To determine if MAFA synergized with MAF in promoting MM proliferation, we silenced MAFA and MAFB genes in cells co-expressing these genes. Simultaneously silencing MAFA and MAFB did not further decrease proliferation compared to either silencing MAFA or MAFB alone. These results indicate that MAFA promotes MM cell proliferation and that there is no synergetic effect between MAFA and MAFB in regulating MM proliferation.To determine if MAFA protein is regulated by PTM, we treated MM cells with PIs including bortezomib (Bzb) and carfilzomib (CFZ). Bzb induced stabilization of MAFA protein in a dose-dependent manner. Similar effects were seen with CFZ indicating that MAFA protein may be degraded by the ubiquitin proteasome system.ConclusionTaken together, our results indicate that increased levels of MAFA are seen in MM including the MAFA /t(8;14) translocation. High expression of MAFA mRNA and protein occurs in 18.7 % of MM cell lines. MAFA promotes myeloma proliferation and regulates TLR4, NUAK1 and SPP1 gene expression. The stability of MAF protein is regulated by PIs. These results provide new insights in understanding the role of the MAF family and, in particular, MAFA in MM pathogenesis. Experiments are ongoing to determine the function of MAFA in PI-resistance and to confirm MAFA targets in order to identify potential alternate approaches for the treatment of this poor performing molecular subgroup. DisclosuresMorgan:Bristol Myers: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding. Davies:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria.

Upregulation of CD38 Expression on Multiple Myeloma Cells By the HDAC Inhibitor Ricolinostat Augments the Efficacy of Daratumumab

Background:The anti-CD38 antibody daratumumab is effective and increasingly being utilized to treat multiple myeloma. A clinical observation with daratumumab is that expression of CD38 on pretreatment myeloma cells correlates with efficacy, and patients with higher CD38 expression have a higher chance to respond. Moreover, during daratumumab therapy, there are reduced CD38 expression levels on myeloma cells, favoring immune escape and disease progression. We have recently shown that the histone deacetylase (HDAC) inhibitor panobinostat induces CD38 upregulation and augments the antibody-dependent cellular cytotoxicity (ADCC) of daratumumab (García-Guerrero E, Blood 2017). Here, we investigated the alternative HDAC inhibitor ricolinostat and all-trans retinoic acid (ATRA) to determine whether upregulation of CD38 is a class effect, and to identify the substance that works most synergistically with daratumumab.Methods:The myeloma cell lines MM1.S and OPM2 were treated with titrated doses of ricolinostat (0, 5, 10 µM), ATRA (0, 10, 25 nM) and panobinostat (0, 10, 25 nM). Expression of CD38, B-cell maturation antigen (BCMA) and SLAMF7 was analyzed by flow cytometry after 24, 48 and 72 h of exposure. ADCC against HDAC inhibitor-treated and untreated myeloma cells was analyzed after addition of PBMC at an effector to target ratio of 25:1 in the presence of daratumumab (1, 10, 50 ug/mL) or isotype control.Results:We first treated the myeloma cell line MM1.S with each of the HDAC inhibitors and analyzed their direct cytotoxic antimyeloma effect. We found the strongest effect with ricolinostat where live MM1.S myeloma cells had decreased to 50% and 20% after 48 h of exposure to 5 and 10 µM of the drug, respectively. Consistent with previous work the direct anti-myeloma effect of panobinostat (decrease of live MM1.S myeloma cells to 85% and 50% after 48 h of exposure to 10 and 25 nM, respectively) and ATRA (no cytotoxic effect) was lower compared to ricolinostat.We then analyzed expression of CD38 on residual live i.e. 7-AAD negative MM1.S cells by flow cytometry and observed a 2-fold (5 µM) and 2,5-fold (10 µM) increase in CD38 expression by mean fluorescence intensity (MFI) after treatment with ricolinostat-treated compared to untreated MM1.S cells. The increase in CD38 expression was already detectable after 24 h and further increased with longer exposure to the drug. Off note, withdrawal of ricolinostat resulted in rapid decline of CD38 expression to baseline levels but increased again to the same magnitude within 24 h of re-exposure to the drug. Overall, the increase in CD38 expression was strongest with ricolinostat followed by panobinostat and ATRA, which we confirmed with several myeloma cell lines and primary myeloma cells. We also confirmed that ricolinostat-induced upregulation of CD38 specifically occurred in myeloma cells and not in the lymphoma cell lines Raji and JeKo-1. In contrast to CD38, expression of BCMA and SLAMF7 after treatment of myeloma cells with ricolinostat had decreased.We then treated myeloma cells with ricolinostat for 48 hours to induce upregulation of CD38 and then performed ADCC assays with daratumumab in the presence of ricolinostat. We found, that the combination treatment ricolinostat plus daratumumab eliminated 95,6% of MM1.S cells after 20h; while 87,4% were eliminated with ricolinostat alone, and only 22,6% with daratumumab alone. Preliminary data show that ricolinostat does not affect expression of the complement inhibitor CD55 and induces a lower increase in CD59 expression compared to panobinostat. Experiments to evaluate complement-dependent cytotoxicity (CDC), and to confirm the synergistic antimyeloma effect of ricolinostat and daratumumab in a murine xenograft myeloma model, are ongoing.Conclusions:Our data demonstrate that the HDAC inhibitor ricolinostat induces upregulation of CD38 expression on myeloma cells. The increase in CD38 expression after treatment with ricolinostat is stronger compared to panobinostat and ATRA. Our data suggest that upregulation of CD38 on myeloma cells is a class effect of HDAC inhibitors, and identify ricolinostat as a highly synergistic combination partner for daratumumab, due to its considerable intrinsic antimyeloma activity. Our data suggest that combination treatment with ricolinostat and daratumumab could increase response rates and extend duration of responses of daratumumab therapy. DisclosuresNo relevant conflicts of interest to declare.

Abstract B73: A novel candidate for immunologic target in treatment of multiple myeloma

Abstract Background: Despite the development of novel drugs, including immune modulatory drugs (IMiDs) and proteasome inhibitors, multiple myeloma is still an uncurable disease. In particular, patients with high-risk chromosomal abnormalities, such as t (4;14) or del17, have poor prognosis. Recently, antibodies or CAR-T cells have been shown to be effective, suggesting that myeloma cells are sensitive to immune reactions. We have previously identified an HLA-A*24:02-restiricted epitope from a novel cancer-testis antigen CXorf48. In this study, we aim to characterize cytotoxic T lymphocyte (CTL) against this antigen and investigate the possibility of the CTLs for adoptive T-cell therapy in myeloma treatments. Methods: Expression of CXorf48 gene was examined by RT-PCR of myeloma cell lines. We then treated myeloma cells with low CXorf48 expressions by demethylating agents, 5-azacytidine (5-aza) or 5-aza-2’-deoxycytidine (DAC), and checked the expression level of this gene by quantitative RT-PCR. Furthermore, CXorf48-specific CTLs were induced from peripheral blood mononuclear cells of HLA-A*24:02-positive healthy donors and their antigen recognition was examined by ELISpot assay and 51Cr cytotoxicity assay. We also analyzed T-cell receptor (TCR) repertoire using dextramer-sorted CTLs and transduced the TCR genes into Jurkat cells. Results: Expression of CXorf48 gene was found in most myeloma cell lines including those with t (4;14) or del17. In addition, the gene expression was significantly increased by DAC in CXorf48-low myeloma cells, which caused enhanced recognition by anti-CXorf48 CTLs. We also identified CXorf48-specidfic TCR genes of CTLs from two donors, and generated TCR gene-transduced T cells. Conclusions: CXorf48 might be a useful target for immunotherapy in combination with demethylating agents for high-risk myeloma. Aiming at CXorf48-specific adoptive therapy, we are analyzing CXorf48-specific TCR-transduced human T cells. Citation Format: Maiko Matsushita, Saku Saito, Kanae Mori, Shinya Yokoe, Daiju Ichikawa, Yutaka Hattori. A novel candidate for immunologic target in treatment of multiple myeloma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B73.

Microbial antigens-loaded myeloma cells enhance Th2 cell proliferation and myeloma clonogenicity via Th2\u2013myeloma cell interaction

BackgroundMyeloma cells retain B cell functions, considered to be potential antigen presenting cells, yet there is little information regarding promoting Th2 cell proliferation or the direct effects to myeloma on the Th2 cells stimulated by microbial antigens-loaded myeloma cells.MethodsMixed lymphocyte reaction was used colorimetric assays via CCK8-kit. Surface molecular expression was performed by flow cytometry, cells sorting using microbeads. The concentrations of cytokines in serum were assessed using an ELISA kit. Clonogenic assay were performed in a methylcellulose culture system. Statistical analysis was assessed using the Student’s t-test or one-way analysis of variance for multiple comparisons test.ResultsThe expression of HLA-DR, CD80 and CD40 on RPMI8266 cell membrane surface was upregulated by interaction with interferon-γ and/or Bacillus Calmette-Guerin Vaccine (BCGV). RPMI8266 cells were able to induce the mixed lymphocyte reaction in a dose-dependent fashion. The Th2 ratio induced by RPMI8266 treated by BCGV and interferon-γ (treated-RPMI8266) cells was only slightly greater than by untreated-tumor cells, but the serum IL-4 level secreted by Th2 cells was markedly higher in treated-RPMI8266 cells group. Th2 cells stimulated by treated-myeloma cells could directly promote treated-myeloma cell clonogenicity in a dose-dependent manner. Anti-HLADR IgG2b completely blocked increased of IL-4 secretion by Th2 cells stimulated by treated-myeloma cells, while also blocked enhancing the clonogenicity of treated tumor cells stimulated by MM-Th2 cells.ConclusionsThese results indicate that a novel mechanism of myeloma pathogenesis in myeloma cells could act as an APC to present microbial Ags to Th2 cells, promoting Th2 cell proliferation, consequently facilitating tumor development by close interaction between Th2 myeloma cells. Taken together, the microbial Ag presenting course of MM-Th2-MM interactions—restricted by MHC class-II—may result in tumor development such that all factors involved in the system could have a potential for myeloma therapeutic intervention.

Disulfiram May Contribute to Apoptosis of Myeloma Cells By Reactivating of Cell Cycle Regulating Gene CDKN2A

Objective: Multiple myeloma (MM) is an incurable plasma-cell dyscrasia characterized by uncontrolled growth of malignant plasma cells in the bone marrow. Proteasome inhibitor bortezomib is a frontline MM drug, however acquired resistance to bortezomib remains a clinical hurdle. Disulfiram (tetraethylthiuram disulfide, DSF) belongs to promising cancer-killing drugs, and although the mechanism of its anticancer activity remains unclear, it has been suggested that this drug inhibits proteasome activity. Furthermore, disulfiram contains strong thio-reactive functional groups and since the catalytic mechanism of DNA methyltransferases (DNMTs) involves the covalent attack at the C6 position of cytosine by the thiol group of the catalytic cysteine on the DNMT enzyme, the DSF can function as DNMT non-nucleoside inhibitor with possible gene demethylation´s effect. On the other hand, DNMT inhibitors, 5-azacytidine (AZA) and 5-aza-2´-deoxycytidine (DAC) are nucleoside analogs, whose mechanism of action involves incorporation of the aza-modified base into DNA during DNA synthesis with subsequent covalent trapping of the DNMT enzyme. Therefore, these nucleoside analogs (AZA and DAC) can have significant cytotoxicity and can lead to major adverse effects, including myelosuppression, when administered to patients.Methods: RPMI8226 and U266 myeloma cells were exposed to DSF (0, 1 µM and 1 µM), DAC (0, 5 µM, 3, 75 µM and 5 µM), JNJ-7706621 (0, 5 µM and 5 µM) and 5 µM SAHA (suberoylanilide hydroxamic acid). Subsequently, we assessed for: i/ cell viability assay using 3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide agent (MTT); ii/ DNA content by flow cytometry analysis; iii/ apoptosis determination by Annexin V and activity of caspase-3 and -7 quantification by cleavage of fluorogenic substrate CellEvent™ Caspase-3/7 Green Detection Reagent; iii/ CDKN2A and CDKN2B methylation status by methylation-specific PCR and pyrosequencing; iiii/ CDKN2A and CDKN2B gene expression analysis by quantitative RT-PCR.Results: To determine the influence of DSF, DAC and two cyclin dependent kinase (CDK) inhibitors JNJ-7706621 and SAHA on cell cycle regulation, RPMI8226 and U266 cell lines were treated with the same scheme as for methylation and expression analysis. U266 cell treatments with 1 µM DSF (18, 9 %) and 5 µM JNJ (21, 6 %) show similar parameters in sub-G1 cell population, but lower when they compared to SAHA treatment (29, 7 %). Analysis of apoptosis of cells treated with 1 µM DSF show 18,51 % of apoptotic cells, while 26,22 % and 28,4 % apoptotic cells after 5 µM SAHA and 5 µM JNJ treatments were detected. The proportions of cells in apoptosis in both 0, 1 µM and 1 µM DSF treatments are 21,8 % and 18,5 %. We have found unmethylated 162 bp CDKN2B analysed gene region, while 150 bp analysed CDKN2A gene region is methylated. Unmethylated state of the CDKN2B gene was confirmed by pyrosequencing of 223 bp promoter and 138 bp 1.exon gene regions. Expression of the unmethylated CDKN2B gene is not influenced, while the 0,1 µM DSF treatment causes increased expression of the CDKN2A gene (Graph1).Conclusion: In treated myeloma cells, we have examined apoptotic effect of DSF in comparison to CDK inhibitors JNJ-06621 and SAHA through detection of methylation and expression status of CDKN2A and CDKN2B genes. Our results indicate DSF pro-apoptotic effect comparable to used CDK inhibitors. The 5-aza-2´-deoxycytidine with demethylation activity and DSF do not affect an expression of the unmethylated CDKN2B gene, its expression effectively increases CDK inhibitors (Graph2). On the other hand, the expression of the methylated CDKN2A gene is only slightly increased in DSF treated cells in comparison to other treatments.Thisstudy was supported in part by NV18-03-0050 from the Ministry of Health of the Czech Republic and LF_2018_001 from Palacky University Olomouc. DisclosuresNo relevant conflicts of interest to declare.

Myeloma Drug Resistance Induced by Binding of Myeloma B7-H1 (PD-L1) to PD-1.

B7 homolog 1 (B7-H1)-expressing myeloma cells not only inhibit myeloma-specific cytotoxic T lymphocytes (CTL), but also confer a proliferative advantage: resistance to antimyeloma chemotherapy. However, it remains unknown whether B7-H1 expressed on myeloma cells induces cellular responses associated with aggressive myeloma behaviors. To address this question, we analyzed the proliferation and drug sensitivity of B7-H1-expressing myeloma cells transfected with B7-H1-specific short-hairpin RNA or treated with programmed cell death (PD)-1-Fc-coupled beads. Knockdown of B7-H1 expression in myeloma cells significantly inhibited cell proliferation and increased apoptosis induced by the chemotherapeutic alkylating agent melphalan, with downregulation of the expression of cell cycle-related genes (CCND3 and CDK6) and antiapoptotic genes (BCL2 and MCL1). B7-H1 molecules thus contributed to myeloma cell-cycle progression and suppression of drug-induced apoptosis. B7-H1-expressing myeloma cells had a higher affinity for PD-1 than for CD80. PD-1-Fc bead-treated myeloma cells also became resistant to apoptosis that was induced by melphalan and the proteasome inhibitor bortezomib. Apoptosis resistance was associated with the PI3K/AKT pathway. Both myeloma cell drug resistance and antiapoptotic responses occurred through the PI3K/AKT signaling pathway, initiated from "reverse" stimulation of B7-H1 by PD-1. Therefore, B7-H1 itself may function as an oncogenic protein in myeloma cells. The interaction between B7-H1 on myeloma cells and PD-1 molecules not only inhibits tumor-specific CTLs but also induces drug resistance in myeloma cells through the PI3K/AKT signaling pathway. These observations provide mechanistic insights into potential immunotherapeutic benefits of blocking the B7-H1-PD-1 pathway. Cancer Immunol Res; 4(9); 779-88. ©2016 AACR.

Elevated APEX1 Disrupts G2/M Checkpoint, Contributing to Evolution and Survival of Myeloma Cells

Cell cycle checkpoints provide the cell with time to repair chromosomal DNA damage before its replication (G1) and also prior to its segregation (G2), thus ensuring integrity, maintenance and protection of genome. Although proper functioning of both checkpoints is essential, G2/M has a special significance as a potentially lethal double-strand break in DNA escape repair and persist from G2 into mitosis, it may recombine in G1 to produce gene rearrangements. Moreover, G2 is the phase where homologous recombination (HR) can utilize a sister chromatid as a template to provide error-free repair. There is ample evidence that supports the role of defective G2/M checkpoint and dysregulated HR in genomic rearrangements and evolution in cancer. Previously, we have shown that elevated APEX1 contributes to dysregulated HR and genome stability in multiple myeloma (MM), and its upregulation leads to genomic instability and tumorigenesis in animal model. To further understand the role of APEX1 in myeloma, we investigated the impact of elevated APEX1 on cell cycle checkpoint/s and in the cellular response to genotoxic exposure. Our investigation using antibody array and subsequent confirmation with immunoprecipitation experiments demonstrated that APEX1 interacts with cyclin B and PLK in myeloma cells. A key step in progression from G2 to mitosis is the activation of cyclin B-CDK1 complex, which subsequently activates PLK to ensure G2/M progression. Based on observed interaction of APEX1 with cyclin B/PLK, we hypothesized that elevated APEX1 disrupts G2 checkpoint by mediating progression into mitosis. To test this, we inhibited APEX1 in myeloma cells by a small molecule as well as by shRNA targeting this gene, and investigated the impact on cell cycle checkpoints using a unique phospho-antibody array which allows investigation of 238 relevant proteins and their phosphorylation status. APEX1 inhibition by small molecule led to downregulation (> 2-fold) of many proteins/phosphorylations involved in the activation of cyclin B-CDK1 complex and other mediators of G2/M progression (including CDC25A, CDC25A, CDK1, ABL1), and upregulation of proteins/phosphorylations involved in G2/M arrest, including CDK1-phospho-Tyr15, 14-3-3 zeta-phospho-Ser58, p53-phospho-Ser15, and MYT and WEE which are involved in negative regulation of cyclin B-CDK1 complex. To further investigate the role of APEX1 in G2/M progression, myeloma cell lines (ARP, RPMI 8226, MM1S, LR5, H929) were treated with APEX1 inhibitor and subjected to cell cycle analysis using flow cytometery. Compared to control cells, all five APEX1-inhibitor treated cell lines showed a strong G2/M block, ranging from 5- to 10-fold increase in the fraction of cells in G2 phase in a dose dependent manner. The G2/M cell cycle arrest of APEX1-treated myeloma cells was further supported by reduced cell viability of treated myeloma cells (RPMI, H929, MM1S, ARP and U266); IC50 of inhibitor in myeloma cell lines ranged from 1.2 to 4 µM. Co-treatment with APEX1 inhbitor also sensitized myeloma cells to Melphalan. Consistent with these data, shRNA-mediated knockdown (KD) of APEX1 in RPMI cells was associated with 4-fold increase in the fraction of cells in G2, relative to control cells. APEX1-KD was also associated with reduction in cell viability (by 40%) and sensitization to melphalan. Our results therefore suggest that elevated APEX1 disrupts G2 checkpoint and sets a stage for genomic rearrangements by allowing persistance of DNA damage from G2 into mitosis. Dysfunctional G2 checkpoint, combined with APEX1-mediated dysregulation of HR, could be attributed to APEX1 associated genomic instability and oncogenic transformation. Therefore, inhibitors of APEX1, alone or in combination with other agents, have potential to make myeloma cells static. DisclosuresNo relevant conflicts of interest to declare.

Abstract 4102: The involvement of EMT in heparanase promoted bone-metastasis in multiple myeloma

Abstract Multiple myeloma (MM), a hematologic malignancy, preferentially grows in bone marrow and frequently metastasizes between bones. Our previous studies have demonstrated that heparanase (HPSE), an enzyme overexpressed in MM cells, promotes MM bone-metastasis and progression. However, the mechanism of HPSE action is unclear. Epithelial-mesenchymal-transition (EMT) is a process giving epithelial cells the features of mesenchymal cells which has been shown to be critical in the metastatic process of epithelial-origin tumors. Although MM is not an epithelial-derived malignancy, MM cells indeed express epithelial marker E-cadherin and mesenchymal marker Vimentin, and have the ability to adhere to and interact with the extracellular matrix (ECM). In the present study, we determined the role of EMT in HPSE-induced MM bone metastasis via: (1) Using Western blot to evaluate the expression of EMT markers E-cadherin, Vimentin and Fibronectin in MM cells that were transfected with empty vector (HPSE-low) or HPSE cDNA (HPSE-high), as well as MM cells treated with recombinant human HPSE (rHPSE) for 48hrs; (2) staining HPSE and EMT markers on tumors established by HPSE-low or HPSE-high cells in SCID mice (n = 10), and on bone marrow biopsies of 35 MM patients; (3) knocking down Vimentin in HPSE-high-luc MM cells by Vimentin shRNA (VIM k/d-luc cells) and testing bone homing of VIM k/d-luc cells in SCID mice by i.v. and s.c. injection of these cells. Tumor growth and bone-metastasis was determined by measuring human kappa levels (a soluble marker of MM cells) in mouse sera and weekly bioluminescent imaging. Western blots demonstrated HPSE-high and rHPSE treated MM cells expressed significantly higher levels of the mesenchymal markers Vimentin and Fibronectin, and lower levels of the epithelial marker E-cadherin, compared to HPSE-low or PBS treated MM cells. Significantly increased expression of Vimentin and decreased expression of E-cadherin was revealed in tumors formed by HPSE-high cells compared with the tumors formed by HPSE-low cells. Immunostaining of bone marrow biopsies of MM patients indicated HPSE and Vimentin expression in MM cells have a significant positive correlation (rs = 0.414, p = 0.014). Finally, both SCID-i.v. injection and SCID-s.c. injection models showed that knocking down mesenchymal marker Vimentin in HPSE-high MM cells successfully blocked HPSE-induced bone metastasis and inhibited tumor growth, compared with mice bearing shRNA control HPSE-high MM cells. In conclusion, our studies demonstrated (1) EMT-like features are a novel biomarker of poor prognosis in MM; (2) HPSE promotes MM metastasis to bone and disease progression, at least in part, through inducing EMT-like features in MM cells; (3) Inhibition of Vimentin expression in MM cells impairs HPSE-induced tumor growth and bone metastasis. Note: This abstract was not presented at the meeting. Citation Format: Qianying Pan, Juan Li, Patrick D. Rowan, Timothy N. Trotter, Yang Yang. The involvement of EMT in heparanase promoted bone-metastasis in multiple myeloma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4102. doi:10.1158/1538-7445.AM2015-4102

Studies of the Proteasome Inhibitor Sensitivity Modulator Tight Junction Protein 1 Highlight a Role for Signaling through the Epidermal Growth Factor Receptor in Determining Proteasome Capacity

Background:Proteasome inhibitors (PIs) are important agents against myeloma, but innate and acquired resistance limit their effectiveness. We previously found Tight junction protein (TJP)-1 to be a modulator and potential biomarker of PI sensitivity, via studies of bortezomib resistant models and clinically annotated gene expression profiling (GEP) databases of patient samples. Also, we showed that its expression reduced EGFR/JAK1/STAT3 pathway activation and proteasome capacity. However, the molecular mechanisms by which TJP1 expression suppressed EGFR signaling and enhanced PI sensitivity were not understood.Methods:We performed co-immunoprecipitation (co-IP) studies to understand interactions of TJP1 with EGFR, and evaluated the impact of various cytokines on proteasome capacity. Also, genetic and pharmacologic approaches were used to modulate EGFR/JAK/STAT activity to determine the effects on PI sensitivity. Finally, gene set enrichment analyses (GSEA) were performed to determine if EGFR activation signatures could be identified in primary samples, and if they correlated with outcomes.Results:Previous studies of TJP1 focused on myeloma models, but since bortezomib is also approved for mantle cell lymphoma, we sought to determine if TJP1 influenced sensitivity here as well. Suppression of TJP1 using shRNAs reduced the sensitivity of JeKo-1 and MINO cells to bortezomib. In addition, murine embryonic stem cells (mESCs) with TJP1 knockout were more resistant to PIs than was the case for wild-type mESCs. Moreover, the TJP1 knockout mESCs expressed higher levels of both activated EGFR and immunoproteasome subunits PSMB8 and 9, and had greater levels of proteasome chymotrypsin-like (ChT-L) activity. Also, re-introduction of TJP1 into the knockout mESCs ehanced their PI sensitivity, and reduced the ChT-L activity. Co-IP studies showed that IP of TJP1 also led to detection of EGFR, and vice versa. Phospho-specific antibodies showed that TJP1 precipitated predominantly the non-phosphorylated, inactive EGFR. Indeed, when cells were treated with EGF and extracts were subjected to IP for TJP1, reduced levels of EGFR were precipitated. Evaluating other receptor tyrosine kinases, we found that TJP1 precipitated IGF-1R but not IL-6R. Therefore, we treated myeloma cells with EGF, IL-6, or IGF-1; all reduced PI sensitivity, but only EGF enhanced proteasome ChT-L activity and capacity. Moreover, shRNA-mediated EGFR knockdown, or pharmacologic inhibition with erlotininb in myeloma cells reduced JAK1/STAT3 activity, levels of PSMB8 and 9, and ChT-L activity, and enhanced the activity of bortezomib. Since the PSMB8 and PSMB9 promoters have consensus STAT3 binding sites, we knocked down STAT3 and found that this also reduced PSMB8 and 9 and ChT-L activity, and enhanced the efficacy of bortezomib. In a mouse model of lytic bone destruction by xenografted RPMI 8226 cells, the ability of bortezomib to increase bone trabecular volume was blunted by TJP1 suppression, consistent with a lower anti-myeloma effect. Lastly, we used publically available EGF-stimulated and EGFR inhibitor gene signatures to examine the clinically annotated GEP data from myeloma patients. This showed that: 1) TJP1 expression in myeloma samples from patients on the University of Arkansas Total Therapy studies was positively correlated with known signatures of inhibitors of EGFR signaling; 2) in GSEA, signatures of EGF and EGFR signaling were significantly positively enriched when genes were ranked by correlation with TJP1; and 3) an EGFR-related gene set had significant negative correlation with survival duration in the Total Therapy 3 dataset.Conclusions:Taken together, our data support the role of TJP1 as a negative regulator of EGFR signaling through its ability to specifically bind, and possibly stabilize the non-phosphorylated form of EGFR. Furthermore, they demonstrate that TJP1 influences proteasome capacity through EGFR/JAK/STAT signaling, and that approaches to suppress this pathway hold promise to overcome PI resistance and achieve PI sensitization. Finally, they validate a heretofore underappreciated role for EGF/EGFR signaling in myeloma pathobiology and patient outcomes. DisclosuresMulligan:Millennium Pharmaceuticals: Employment. Usmani:Celgene Corporation: Consultancy, Honoraria, Research Funding; Millennium: Consultancy, Honoraria; Onyx: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy; Array BioPharma: Research Funding; Janssen: Research Funding; Pharmacyclics: Research Funding. Orlowski:Bristol-Myers Squibb: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Onyx Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Millennium: The Takeda Oncology Company: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Spectrum Pharmaceuticals: Research Funding; JW Pharmaceutical: Research Funding; Array BioPharma: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity’s Board of Directors or advisory committees; Janssen Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees.

Bortezomib Could Reduce Cell Viability by Activation of Heme Oxygenase 1 in Multiple Myeloma Cells

AbstractAbstract 5005Bortezomib (PS-341, Velcade®) specifically targeting the ubiquitin-proteosome pathway, is the first defined therapeutic proteosoma inhibitor approved by FDA for treatment of Multiple Myeloma (MM). We explored the hypothesis that the activity of Bortezomib in MM cells may involve the heme oxygenase 1 (HMOX1), a enzyme with pleiotropic effects, catalysing the degradation of heme to carbon monoxide, biliverdin and ferrous iron.After 24h of treatment with Bortezomib at 10nM, the U266 and SKM-M1 MM cell lines showed a decrease of cell viability around 50% and an increase of HMOX1 protein and mRNA expression of 40 fold (p<0. 001). Using a colorimetric assay we observed that Bortezomib increased also the HMOX1enzymatic activity (p<0. 03). To elucidate the significance of the HMOX1 increase after bortezomib treatment, we treated myeloma cells with Bortezomib plus an inducer (Hemin 10uM) or an inhibitor (Tin, Sn-Mesoprotoporphyrin, 10uM) of the enzyme activity. Treatment with Hemin and Tin alone not changed the cell viability. Cell viability was reduced (p<0. 003) by addition of Hemin and was increased (p<0. 002) by Tin addiction with respect to Bortezomib alone. Conclusion:Bortezomib increases HMOX1expression and its enzymatic activity and this increase could be an important mechanism of Bortezomib induced cytotoxicity in MM cells. Modulation of HMOX1 activity has effect on Bortezomib induced cell death. Disclosures:Off Label Use: Eltrombopag is a thrombopoietin receptor agonist indicated for the treatment of thrombocytopenia in patients with chronic immune (idiopathic) thrombocytopenic purpura (ITP).

MiR-34a Replacement As a Novel Therapeutic Approach for Multiple Myeloma: Preclinical In Vitro and In Vivo Evidence

Abstract 2910Multiple myeloma (MM) remains an incurable disease despite important therapeutic advances in the last few years. Small non-coding RNAs (miRNAs) synthetic mimics are a new class of biological agents which have recently demonstrated preclinical activity against a variety of human neoplasms. miRNA antitumor activity has been related to their capacity to interfere with mRNA stability and protein transducing activity. miR-34a has tumor suppressor activity and is transcriptionally regulated by p53. We investigated the in vitro and in vivo therapeutic potential of pre-miR-34a mimics against human MM cells. Transient expression of pre-miR-34a mimics, after electroporation of SKMM1 and RPMI-8226 MM cell lines which display low constitutive miR-34a levels, triggered antiproliferative effects, as demonstrated by MTT and long-term soft-agar colony assays. 48 hours after cell transfection, apoptotic events were detected in both cell lines exposed to miR-34a mimics. In parallel experiments, MM cells stably transduced with miR-34a gene cloned in a lentiviral vector showed significant growth reduction as compared to empty vector-transduced cell colonies, providing additional evidence of miR-34a tumor suppressor activity in MM cells. qRT-PCR analysis of treated MM cells showed that pre-miR-34a mimics induced down-regulation of mRNAs coding for Notch1 and the cell-cycle dependent kinase 6 (CDK6), validated miR-34a targets. Furthermore, decreased anti-apoptotic Bcl-2 and CDK6 proteins was detected after pre-miR-34a mimic expression, evidence by western blotting analysis. The anti-MM activity of pre-miR-34a mimics was also evaluated in vivo using xenografted SCID models of human MM. Intra-tumor delivery of pre-miR-34a was performed by a novel formulation with Neutral Lipid Emulsion (NLE). Following 4 injections (3 days apart) of pre-miR34a formulated in NLE particles, a highly significant inhibition of tumor growth was detected in SKMM1 xenografted SCID mice. At day 13 after the first treatment, tumors in mice treated with formulated pre-miR-34a were significantly smaller than tumors in mice treated with the formulated scrambled sequence (P=0.0002) or vehicle (P=0.0002) or PBS (P=0.0001). Interestingly, at day 21a three mice enrolled in the miR-34a treated group showed complete regression of tumors. Formulated synthetic pre-miR-34a also produced a significant increase of mice survival (P=0.01 versus formulated scrambled sequence). A similar in vivo tumor growth inhibition was observed in mice xenografted with SKMM1 MM cells stably transduced with a miR-34a lentiviral construct, as compared to cells transduced with the empty vector. We here provide the first proof-of-principle demonstrating that replacement of miR-34a produces therapeutic activity against MM cells with low constitutive miR-34a expression. Our findings provide a framework for development of miR-34a-based therapeutic strategies in MM.Supported by AIRC 5 per mille, Molecular Clinical Oncology Program No. 9980 Disclosures:Anderson:Millennium Pharmaceuticals, Inc.: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Onyx: Consultancy; Merck: Consultancy; Bristol-Myers Squibb: Consultancy; Actelion: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Investigation of the Molecular Action of Bortezomib Treatment In Multiple Myeloma

AbstractAbstract 5038Bortezomib (Velcade®) is a 26S proteasome inhibitor currently used for the treatment of relapsed multiple myeloma (MM) and more recently mantle cell lymphoma patients. Clinical trials are under way to evaluate its efficacy in other hematological malignancies diseases including diffuse large B cell lymphoma (DLBCL). Although it was widely accepted that bortezomib acts as a NF-κB inhibitor, this view has recently been challenged by work by Hideshima et al. (Blood 2009) who found that treated myeloma cells actually displayed down-regulation of NF-κB inhibitors (IκBα), increased activity of pro-NF-κB molecules (RIP2 and IKKβ) as well as increasing levels of NF-κB DNA binding. Additionally, although bortezomib has a potent anti-tumor activity, not all MM patients are responsive. The identification and understanding of the molecular mechanisms leading to bortezomib-resistance will help with the development of new approaches and will improve the treatment of these patients. In order to investigate the direct effect of bortezomib on lymphomas, we treated different cell lines with this drug and levels of cell proliferation and apoptosis were measured. Bortezomib was shown to significantly inhibit proliferation and promote apoptosis in MM cell lines (RPMI-8226, NCI-H929, JJN-3 and Thiel) and DLBCL cell lines (SU-DHL-4 and SU-DHL-10, HLY-1, HLB-1 and RIVA). Since MM cell lines were shown to be the most sensitive to the bortezomib, we selected them to carry out further studies. We next employed whole genome expression arrays (Affymetrix U113plus2.0) and microRNA arrays to identify molecules associated with treatment in these cell lines. Using unsupervised cluster analysis, we observed that the cells treated with bortezomib have a distinct microRNA and gene expression profile from their counterpart controls. Additionally, we used a shRNA whole-genome library to carry out a loss-of-function screen for cells resistant to bortezomib-induced apoptosis. The RPMI-8226 cell line was transduced with the shRNA library. One half of the cells was then treated with bortezomib. Relative gene expression levels were measured by hybridizing samples to custom-made arrays. Unsupervised cluster analysis identified a number of shRNAs which might be involved in mechanisms of resistance to bortezomib. Further functional studies will be necessary to investigate the role of these microRNAs in the mechanism of action of bortezomib. Disclosures:No relevant conflicts of interest to declare.

Anti-proliferative and cytotoxic effects of Strychnos nux-vomica root extract on human multiple myeloma cell line – RPMI 8226

Multiple myeloma (MM) is an incurable hematological malignancy with high incidence in the elderly. The currently used chemotherapeutic drugs show severe side effects, dose-limiting toxicity and development of resistance. In search of novel plant derived anti-cancer agents, Strychnos nux-vomica L. (SN) root extract was screened using the human MM-cell line, RPMI 8226. SN-extract exhibited anti-proliferative activity in a dose and time dependent manner. The morphological assessment of SN-extract treated cells showed significant features associated with apoptosis. Cell cycle analysis using flow cytometry of cells stained with propidium iodide revealed accumulation of cells at sub-G 0/G 1 phase. In addition, disruption of mitochondrial membrane potential and subsequent leakage of mitochondrial cytochrome c was observed in SN-extract treated myeloma cells. The anti-proliferative and cytotoxic activity could be due to the alkaloids strychnine and brucine, which have been identified by LC–mass spectral analysis of the SN-extract in comparison to the reference standards analyzed under identical conditions.

Gene Expression Profiling in Multiple Myeloma Cells Treated with the Novel Anti-Myeloma Agents Zoledronate and Fluvastatin.

We have shown in vitro that multiple myeloma (MM) cells can be destroyed by treating them with the mevalonate pathway inhibitors zoledronate and fluvastatin. While the efficacy of these compounds singly and combination have been demonstrated, their exact modes of action remain largely unknown. The present study aimed to use microarray and quantitative real-time PCR (QRT-PCR) techniques to analyse gene expression in treated myeloma cells to identify novel genes and pathways involved in the anti-myeloma action of these compounds. The human MM cell line NCI-H929 was treated with zoledronate and fluvastatin singly and in combination, and RNA was extracted and used to interrogate oligonucleotide microarrays consisting of 19,000 features representing known and unknown genes. Quantitative real-time PCR was subsequently used to confirm the expression of several genes of interest. Flow cytometry with Annexin V FITC staining was used to detect apoptosis. It was observed that genes related to apoptosis (caspases and p53-related genes), cell cycle control (cyclins), GTPase signalling (Rabs), and growth and proliferation (growth factors) were particularly affected by zoledronate and fluvastatin, and some of these genetic effects were synergistic when a combination of zoledronate and fluvastatin was used. QRT-PCR confirmed the effects on the caspase- and p53-related apoptotic pathways, and these effects were correlated with increased apoptosis in the myeloma cells. The mevalonate pathway inhibitors fluvastatin and zoledronate are highly efficient at killing MM cells, and their effects appear to be synergistic. Our microarray and QT-PCR analyses demonstrated that the expression of specific groups of genes important to the survival and proliferation of myeloma cells are affected by these compounds. p53 and caspase-dependent pathways appear to be the key apoptotic cascades stimulated. Insights into the mechanisms of these novel therapeutics are important as they might help to define their roles in the treatment of multiple myeloma.

Inhibition of Catalase Activity with 3-Amino-Triazole Enhances the Cytotoxicity of the Alzheimer’s Amyloid-β Peptide

Amyloid-β (Aβ) is a cytotoxic peptide implicated in the pathology of Alzheimer’s disease. The antioxidant enzyme catalase has been suggested to protect against Aβ cytotoxicity in both neuronal and non-neuronal cell types. Inhibition of endogenous catalase using 3-amino-1,2,4-triazole (3AT) in neuronal (NT-2) and myeloma (SP2/0-Ag-14) cell lines increases Aβ toxicity, suggesting that any protective role for endogenous catalase requires active enzyme. In Aβ treated myeloma cells there was a significant decrease in the total cell catalase activity and immunoreactivity. However, when the surviving live cell population was isolated following Aβ treatment the levels of catalase were significantly increased. The surviving live cell population from groups treated with both 3AT and Aβ contain elevated immunoreactive catalase levels suggesting that the protective role for endogenous catalase may have a component independent of the antioxidant activity, possibly by acting as an Aβ binding protein. Amyloid-β (Aβ) cytotoxicity can be prevented by Vitamin E treatment or an anti-Aβ monoclonal antibody (ALI01), both of which also prevent Aβ cytotoxicity in cells treated with 3AT. These observations suggest that Aβ mediated cell death in both neuronal and non-neuronal cells is mediated in part by actions to increase hydrogen peroxide. Catalase has a protective role, as a hydrogen peroxide-degrading enzyme and catalase inhibition by Aβ is not the direct cause of cytotoxicity.