Adhesion Of Multiple Myeloma Cells Research Articles

MIF acting as a chaperone of SOD1 mediates MM cell intrinsic resistance to carfilzomib by modulating ROS-induced mitochondrial dysfunction

We previously discovered that high levels of macrophage migration inhibitory factor (MIF) were detected in multiple myeloma (MM) and associated with poor survival of patients. Knocking down MIF impaired MM cell adhesion to bone marrow stromal cells in vitro and led to the formation of extramedullary tumors more susceptible to chemotherapeutic treatment. Whether MIF directly regulates MM cell intrinsic resistance to chemotherapy has yet to be explored. We recently identified that MIF-knockout (KO) MM cell lines displayed greater apoptosis than control (CTR)-KO MM cells when treated with proteasome inhibitors, especially carfilzomib (CFZ).

TRAF6 Silencing Attenuates Multiple Myeloma Cell Adhesion to Bone Marrow Stromal Cells.

The bone marrow (BM) microenvironment plays an important role in supporting proliferation, survival and drug resistance of Multiple Myeloma (MM) cells. MM cells adhere to bone marrow stromal cells leading to the activation of tumour-promoting signaling pathways. Activation of the NFκB pathway, in particular, is central to the pathogenesis of MM. Tumour necrosis factor receptor-associated factor 6 (TRAF6) is a key mediator of NFκB activation and has previously been highlighted as a potential therapeutic target in MM. Here, we demonstrate that adherence of MM cell lines to stromal cells results in a reciprocal increase in TRAF6 expression. Knockdown of TRAF6 expression attenuates the ability of MM cells to bind to stromal cells and this is associated with a decrease in NFκB-induced expression of the adhesion molecules ICAM1 and VCAM1. Finally, we show that knockdown of TRAF6 sensitizes MM cells to treatment with bortezomib when co-cultured with stromal cells. Inhibiting TRAF6 represents a promising strategy to target MM cells in the BM microenvironment.

TBK1/Ikkε Inhibitor Amlx Blocks Multiple Myeloma Cell Growth in Vitro and In Vivo

Multiple myeloma (MM) is the most frequent cancer to involve the skeleton and remains incurable for most patients, thus novel therapies are needed. MM bone disease is characterized by osteolytic lesions that contribute significantly to patient morbidity and mortality. We showed that TBK1 signaling is a novel pathway that increases osteoclast (OCL) formation in Paget's disease, an inflammatory bone disease. Therefore, we hypothesized that TBK1 plays a similar role in MM induction of OCL. We found that MM conditioned media (MM-CM) dose-dependently increased bone marrow monocyte (BMM) expression of activated TBK1 protein and enhanced RANKL-driven OCL formation. TBK1 knockdown by shRNA transduction into BMM significantly attenuated the ability of MM-CM to increase OCL differentiation without altering OCL differentiation in control media. We found that the TBK1/IKKε inhibitor Amlexanox (Amlx) blocked normal and MM-enhanced OCL formation. Importantly, TBK1 mRNA expression in CD138+ plasma cells (PC) isolated from MM or PC leukemia patients is significantly higher as compared to PC from Monoclonal Gammopathy of Undetermined Significance (MGUS) patients.Therefore, we tested whether targeting the TBK1/ IKKε signaling pathways would also affect MM cells. We found that Amlx strongly decreased the viability of several MM cell lines and primary MM cells via induction of apoptosis. Amlx treatment of MM cell lines also induced a G1/S blockade, decreased activated ERK1/2, and increased translation of the dominant-negative C/EBPb-LIP isoform in several MM cell lines. The positive-acting C/EBPb-LAP isoform was previously shown to be a critical transcription factor for MM viability. Importantly, Amlx also enhanced the effectiveness of the proteasome inhibitors bortezomib and carfilzomib to kill MM cells in culture. Further, Amlx sensitized MM1.S cells to the induction of apoptosis by the autophagic inhibitor Bafilomycin A. Amlx dose-dependently inhibited tumor growth in a syngeneic MM mouse model in which 5TGM1 MM cells expressing secreted GLuc were injected subcutaneously into immunocompetent C57Bl/KaLwRij. Tumor growth was assessed by measuring tumor volumes and by the levels of secreted GLuc in the blood. Further, OCL formation ex vivo from bone marrow monocytes obtained from AMLX-treated mice versus controls was decreased. Amlx did not affect the viability of primary BMM, bone marrow stromal cells (BMSC), or splenocytes. Further, Amlx treatment of primary BMSC from MM patients or normal donors decreased expression of TNFα, IL-6 and RANKL, thereby decreasing BMSC support of MM survival and OCL differentiation. Amlx pretreatment of BMSC and murine pre-osteoblast MC4 cells also decreased VCAM1 expression and reduced MM cell adhesion, another mechanism for Amlx reduction of bone microenvironmental MM support.These data suggest that targeting TBK1/IKKε signaling may decrease MM bone disease by slowing MM growth, directly and indirectly, and preventing MM-induced osteolysis. DisclosuresGiuliani:Janssen Pharmaceutica: Other: Avisory Board, Research Funding; Celgene Italy: Other: Avisory Board, Research Funding; Takeda Pharmaceutical Co: Research Funding. Roodman:Amgen Denosumab: Membership on an entity's Board of Directors or advisory committees.

Silencing Long Non-Coding RNA ST3GAL6-AS1 Inhibits Adhesion and Migration of Myeloma Cells in Vitro

Introduction: Multiple myeloma (MM) is a fatal B-cell malignancy characterized by abnormal proliferation of plasma cells (PCs) in bone marrow (BM). Despite the remarkable improvements have been done in knowledge on the pathobiology of MM, treatment and patients care, MM is still an incurable disease. Long non-coding RNAs (lncRNAs) are a new sort of noncoding RNA longer than 200 nucleotides and without the function of translating into canonical functional proteins. Increasing evidence suggests that lncRNAs have huge impact on adjusting gene expression through the processes of transcription and post-transcription regulation, genomic imprinting, and chromatin modification. We have validated several dysregulated lncRNAs in MM by microarray and bioinformatic analysis. Among them, we focused on the function and mechanism of ST3 beta-galactoside alpha-2,3-sialyltransferase 6 antisense RNA 1(ST3GAL6-AS1), which was upregulated markedly in MM patients. In our previous study, we verified the upregulation level and the co-expression relationship of ST3GAL6-AS1 and ST3 beta-galactoside alpha-2,3-sialyltransferase 6 (ST3GAL6) in bone marrow samples from MM patients.Methods: MM cell lines MM1.S and RPMI-8226 were confirmed to have high expression levels of ST3GAL6-AS1 at RNA level compared with healthy controls. Knockdown of ST3GAL6-AS1 was conducted in MM1.S and RPMI-8226 cells using lncRNA Smart Silencer from Ribobio (Guangzhou, China). ST3GAL6-AS1 knockdown and ST3GAL6 expression were confirmed by quantitative real-time PCR (qRT-PCR) at RNA level. We next evaluated the effects of ST3GAL6-AS1 knockdown on MM cell adhesion to human umbilical vein endothelial cell (HUVEC), fibronectin and collagen I coated plates after MM1.S and RPMI-8226 was labeled by CFSE. Migration and invasion to complete medium were assessed using transwell plates comparing ST3GAL6-AS1 knockdown cells to NC controls. Matrigel matrix was coated at transwell plates in invasion analysis. Cell count in adhesion analysis was conducted by Image J software, while migration and invasion analysis were evaluated by flow cytometer.Results: ST3GAL6-AS1 (Genebank number: NR_046683.1) was a 769bp antisense RNA, which located in chromosome 3. Its expression level was decreased dramatically after transfection of ST3GAL6-AS1 smart silencer or control siRNA into MM1.S and RPMI-8226 cells (Figure 1A). There was a significant reduction in the ability of knockdown MM1.S and RPMI-8226 cells to adhere to HUVEC, fibronectin and collagen I in vitro compared to NC controls (Figure 1B). Migration and invasion ability of MM cells transfected with lncRNA smart silencer in response to complete medium were also decreased obviously (Figure 1C). However, there was no significant difference in the ability of proliferation and apoptosis between ST3GAL6-AS1 knockdown group and NC controls. Furthermore, the expression level of ST3GAL6 was decreased in ST3GAL6-AS1 knockdown MM cells (Figure 1D).Conclusions: Knockdown of ST3GAL6-AS1 in MM cells significantly inhibits adhesion, migration and invasion in vitro, indicating that ST3GAL6-AS1 may play an important role in the malignant behavior of MM cells. The co-expression between ST3GAL6-AS1 and gene ST3GAL6 has been demonstrated in our previous study, which was further confirmed in present study. Researches are ongoing to address the potential mechanism among them in MM. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Targeting c-met receptor tyrosine kinase by the DNA aptamer SL1 as a potential novel therapeutic option for myeloma.

Hepatocyte growth factor (HGF)/c‐met pathway activation has been implicated in the pathogenesis of multiple myeloma (MM), and blocking this pathway has been considered a rational therapeutic strategy for treating MM. Aptamers are single‐stranded nucleic acid molecules that fold into complex 3D structures and bind to a variety of targets. Recently, it was reported that DNA aptamer SL1 exhibited high specificity and affinity for c‐met and inhibited HGF/c‐met signaling in SNU‐5 cells. However, as the first c‐met‐targeted DNA aptamer to be identified, application of SL1 to myeloma treatment requires further investigation. Here, we explore the potential application of SL1 in MM. Our results indicated that c‐met expression is gradually increased in MM patients and contributes to poor outcomes. SL1 selectively bound to c‐met‐positive MM cells but not to normal B cells and suppressed the growth, migration and adhesion of MM cells in vitro in a co‐culture model performed with HS5 cells, wherein SL1 inhibited HGF‐induced activation of c‐met signaling. In vivo and ex vivo fluorescence imaging showed that SL1 accumulated in the c‐met positive tumour areas. In addition, SL1 was active against CD138+ primary MM cells and displayed a synergistic inhibition effect with bortezomib. Collectively, our data suggested that SL1 could be beneficial as a c‐met targeted antagonist in MM.

Role of RACK1 on cell proliferation, adhesion, and bortezomib-induced apoptosis in multiple myeloma

Receptor for activated C kinase 1 (RACK1), a scaffold protein, plays a crucial role in the progression of various cancers. However, the biological function and underlying mechanism of RACK1 in multiple myeloma (MM) cells remain unclear. The present study aimed to explore the function of RACK1 on the cell proliferation, adhesion, and bortezomib-induced apoptosis in MM. We found that RACK1 was significantly overexpressed in myeloma cell lines and primary myeloma cells compared with normal bone marrow plasma cells. Moreover, immunofluorescence revealed that RACK1 was primarily expressed in the cytoplasm of MM cells. Knockdown of RACK1 impaired growth of MM cells, blocked entry into the S-phase of the cell cycle, and resulted in reduced cell adhesion rates. More importantly, knockdown of RACK1 decreased the proliferation of MM cells by activating P-P38 and P-ERK in the MAPK/ERK signaling pathway. We also found that altered expression of RACK1 is associated with bortezomib-mediated MM cell apoptosis. In summary, these results may provide a possible target for therapy in MM.

Daratumumab induces CD38 internalization and impairs myeloma cell adhesion

ABSTRACTDaratumumab (Dara), a human immunoglobulin G1 kappa (IgG1κ) monoclonal anti-CD38 antibody, has been approved by the U.S. Food and Drug Administration for the treatment of relapsed multiple myeloma (MM) as a single agent as well as in combination with immunomodulatory drugs (IMiDs) and proteasome inhibitors (PI). Although the scientific rationale behind the use of Dara in combination with IMiDs has been extensively explored, the molecular mechanisms underlying Dara-PI regimens have not yet been investigated. Here, we demonstrate that CD38 on the surface of MM cells is rapidly internalized after Dara treatment; we also show that Dara treatment impairs MM cell adhesion, an effect that can be rescued by using the endocytosis inhibitor Dynasore. Finally, we show that Dara potentiates bortezomib (BTZ) killing of MM cells in vitro and in vivo, independent of its function as an immune activator. In conclusion, our data show that Dara impairs MM cell adhesion, which results in an increased sensitivity of MM to proteasome inhibition.

Screening and characterization of an Annexin A2 binding aptamer that inhibits the proliferation of myeloma cells

Multiple myeloma (MM) is a malignant plasma cell disease and is considered incurable. Annexin A2 (ANXA2) is closely related to the proliferation and adhesion of MM. Using protein-SELEX, we performed a screen for aptamers that bind GST-ANXA2 from a library, and GST protein was used for negative selection. The enrichment of the ssDNA pool was monitored by filter-binding assay during selection. After nine rounds of screening and high-throughput sequencing, we obtained six candidate aptamers that bind to the ANXA2 protein. The affinities of the candidate aptamers for ANXA2 were determined by ELONA. Binding of aptamer wh6 to the ANXA2 protein and to the MM cell was verified by aptamer pulldown experiment and flow cytometry, respectively. Aptamer wh6 binds the ANXA2 protein with good stability and has a dissociation constant in the nanomolar range. The binding specificity of aptamer wh6 was confirmed in vivo in nude mouse xenografts with MM cells and with MM bone marrow aspirates. Furthermore, aptamer wh6 can block MM cell adhesion to ANXA2 and block the proliferation of MM cells induced by ANXA2. In summary, wh6 can be considered a promising candidate tool for MM diagnosis and treatment.

UTX/KDM6A Loss Enhances the Malignant Phenotype of Multiple Myeloma and Sensitizes Cells to EZH2 inhibition

Loss or inactivation of the histone H3K27 demethylase UTX occurs in several malignancies, including multiple myeloma (MM). Using an isogenic cell system, we found that loss of UTX leads to deactivation of gene expression ultimately promoting the proliferation, clonogenicity, adhesion, and tumorigenicity of MM cells. Moreover, UTX mutant cells showed increased invitro and invivo sensitivity to inhibition of EZH2, a histone methyltransferase that generates H3K27me3. Such sensitivity was related to a decrease in the levels of IRF4 and c-MYC and an activation of repressors of IRF4 characteristic of germinal center B cells such as BCL6 and IRF1. Rebalance of H3K27me3 levels at specific genes through EZH2 inhibitors may be a therapeutic strategy in MM cases harboring UTX mutations.

Emerging combination therapies for the management of multiple myeloma: the role of elotuzumab.

Treatment options for patients with multiple myeloma (MM) have increased during the past decade. Despite the significant advances, challenges remain on which combination strategies will provide the optimal response for any given patient. Defining optimal combination strategies and corresponding companion diagnostics, that will guide clinical decisions are required to target relapsed or refractory multiple myeloma (RRMM) in order to improve disease progression, survival and quality of life for patients with MM. Elotuzumab is a humanized monoclonal antibody that targets signaling lymphocytic activation molecule F7 (SLAMF7), approved by the US Food and Drug Administration (FDA) in 2015 and the European Medicines Agency in 2016 for the treatment of MM. SLAMF7 is expressed in normal and malignant plasma cells and has lower expression on natural killer (NK) cells. Experimental evidence indicates that elotuzumab exhibits anti-myeloma activity through 1) antibody-dependent cell-mediated cytotoxicity, 2) enhancing NK cells cytotoxicity and 3) interfering with adhesion of MM cells to bone marrow stem cells (BMSCs). Although elotuzumab has no single agent activity in patients with RRMM who have received one to three prior therapies, the combination of elotuzumab with anti-myeloma agents, such as immunomodulatory drugs-lenalidomide, or proteasome inhibitors (PIs)-bortezomib, remarkably improved the overall response rates and progression-free survival in MM patients with only minimal incremental toxicity. In brief, the clinical data for elotuzumab indicate that targeting SLAMF7 in combination with the use of conventional therapies is feasible and effective with a tolerable safety profile for the treatment of RRMM.

Abstract 5005: Inhibition of E-selectin or E-selectin together with CXCR4 resensitizes multiple myeloma to treatment

Abstract INTRODUCTION: Multiple myeloma (MM) is a plasma cell malignancy localized in the bone marrow. Selectins and CXCR4 play a pivotal role in homing, adhesion, and proliferation of MM. Herein, we tested the role of E-selectin in the pathophysiology of MM by using a specific E-selectin antagonist (GMI-1271) and a dual E-selectin/CXCR4 antagonist (GMI-1359) on the proliferation and cell trafficking of MM in vitro and in vivo. PROCEDURES: Endothelial cells (HUVECs), stromal cells (HS5), MM patient-derived stromal cells (MSP1), and MM cell lines (MM1s, H929, U266 and RPMI8226) were tested for expression of E-selectin, CLA (E-selectin ligand) and CXCR4 by flow cytometry. The effect of GMI-1271 and GMI-1359 was studied on MM cell adhesion to stroma; chemotaxis to stromal media or SDF1; and MM cell survival by MTT assay, where MM cells were treated with lenalidomide or carfilzomib; in the presence, or absence of GMI-1271 or -1359. In a xenograft mouse model, the sensitivity of MM was tested in mice treated with vehicle, lenalidomide, GMI-1271, or a combination of GMI-1271 and lenalidomide (n=10 mice/group); and tumor growth was monitored weekly using bioluminescence imaging. SUMMARY: Expression of E-selectin was high in HUVECs, MSP1 and HS5; CLA was high only in RPMI8226; and CXCR4 was high in all MM cells. MM1s cell adhesion to stroma was decreased by GMI-1271 and even more profoundly decreased by GMI-1359 in a dose-dependent manner. Chemotaxis of MM1s cells towards conditioned media or SDF1 was modestly affected by GMI-1271, but greatly reduced by GMI-1359. MM cell proliferation was significantly inhibited by GMI-1359, only when MM cells were co-cultured with stroma, but not alone. Stroma-induced drug resistance of MM cells to carfilzomib and lenalidomide was reduced in the presence of the GMI compounds, while the effect of GMI-1359 was stronger than GMI-1271. In vivo, GMI-1271 and lenalidomide as single agents delayed tumor growth compared to vehicle by 40% and 35%, respectively; however, a combination treatment delayed tumor growth more profoundly than vehicle by 64% (p=0.04). CONCLUSIONS: GMI-1271 and GMI-1359 disrupt the interaction between MM cells and bone marrow microenvironment as determined by reduction of adhesion, chemotaxis and microenvironment-induced drug resistance in vitro. However, the dual inhibition of E-selectin and CXCR4 produced more profound effects compared to E-selectin alone. Moreover, GMI-1271 sensitized MM cells to lenalidomide by alleviating chemoresistance and delayed tumor growth in vivo. Citation Format: Barbara Muz, Henna Y. Bazai, Anita Sekula, William E. Fogler, Ted Smith, John L. Magnani, Abdel Kareem Azab. Inhibition of E-selectin or E-selectin together with CXCR4 resensitizes multiple myeloma to treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5005. doi:10.1158/1538-7445.AM2017-5005

The multi-faceted potential of CD38 antibody targeting in multiple myeloma.

CD38, an adenine dinucleotide phosphate (ADP) ribose cyclase and a cyclic ADP ribose hydrolase, is widely expressed on the surface of multiple myeloma (MM) cells. It is known to play a pivotal role in the downstream pathways that mediate MM cell growth, signal transduction, and adhesion. The clinical use of CD38 monoclonal antibodies (MoAbs), such as daratumumab, either as monotherapy or in combination with other anti-MM agents, has produced impressive results in patients who have failed standard MM therapy. CD38 MoAbs exhibit several cytotoxic mechanisms on MM cells. In addition to the classical effector mechanisms associated with antibody therapy, CD38 MoAbs induce MM apoptosis and clonal T-cell expansion. Here, we summarize the results of some pivotal clinical studies using a human CD38 MoAb, daratumumab, in patients with MM, discuss the anti-MM effector mechanisms induced by CD38 MoAbs, and review the potential tumor antigens that may be suitable targets for immunotherapy of MM. Finally, we present a paradigm of immunotherapy for MM patients using CD38 MoAbs followed by GM-CSF and an immune checkpoint inhibitor in patients who have undergone high dose chemotherapy and autologous stem cell transplant. CD38 MoAbs have emerged as a novel and ultimately very promising immunotherapeutic agent for MM because of its ability to induce MM cytotoxicity through both arms of the adaptive immune responses.

EphA4 promotes cell proliferation and cell adhesion-mediated drug resistance via the AKT pathway in multiple myeloma.

Eph receptor A4 (EphA4), a member of the erythropoietin-producing hepatocellular (Eph) family, has been reported to upregulate in several tumors. However, the role of EphA4 in multiple myeloma has not been clarified yet. In this study, we found that EphA4 promoted proliferation of multiple myeloma cells via the regulation of cell cycle. Besides, EphA4 was closely related to cell adhesion of multiple myeloma cells and promoted cell adhesion-mediated drug resistance by enhancing the phosphorylation levels of Akt (p-AKT) expression in multiple myeloma. More interestingly, we discovered that EphA4 can interact with cyclin-dependent kinase 5 (CDK5) and regulate its expression in multiple myeloma. CDK5 has been reported to be overexpressed in multiple myeloma which mediated bortezomib resistance and also participated in AKT pathway. And we have also proved the fact. So, we supposed that EphA4 interacted with CDK5 and promoted its expression which in turn enhanced p-AKT expression and promoted cell adhesion-mediated drug resistance in multiple myeloma. Therefore, this study clarifies the molecular mechanism of cell adhesion-mediated drug resistance and may be useful in identifying potential target for treatment of multiple myeloma.

PI3K\u03b4 and PI3K\u03b3 isoforms have distinct functions in regulating pro-tumoural signalling in the multiple myeloma microenvironment

Phosphoinositide-3-kinase and protein kinase B (PI3K-AKT) is upregulated in multiple myeloma (MM). Using a combination of short hairpin RNA (shRNA) lentivirus-mediated knockdown and pharmacologic isoform-specific inhibition we investigated the role of the PI3K p110γ (PI3Kγ) subunit in regulating MM proliferation and bone marrow microenvironment-induced MM interactions. We compared this with inhibition of the PI3K p110δ (PI3kδ) subunit and with combined PI3kδ/γ dual inhibition. We found that MM cell adhesion and migration were PI3Kγ-specific functions, with PI3kδ inhibition having no effect in MM adhesion or migration assays. At concentration of the dual PI3Kδ/γ inhibitor duvelisib, which can be achieved in vivo we saw a decrease in AKT phosphorylation at s473 after tumour activation by bone marrow stromal cells (BMSC) and interleukin-6. Moreover, after drug treatment of BMSC/tumour co-culture activation assays only dual PI3kδ/γ inhibition was able to induce MM apoptosis. shRNA lentiviral-mediated targeting of either PI3Kδ or PI3Kγ alone, or both in combination, increased survival of NSG mice xeno-transplanted with MM cells. Moreover, treatment with duvelisib reduced MM tumour burden in vivo. We report that PI3Kδ and PI3Kγ isoforms have distinct functions in MM and that combined PI3kδ/γ isoform inhibition has anti-MM activity. Here we provide a scientific rationale for trials of dual PI3kδ/γ inhibition in patients with MM.

ADP-ribosylation factor 1 (ARF1) takes part in cell proliferation and cell adhesion-mediated drug resistance (CAM-DR).

Cell adhesion-mediated drug resistance (CAM-DR) remains the primary obstacle in human multiple myeloma (MM) therapy. In this study, we aimed at investigating the expression and biologic function of ARF1 in MM. We determined that ARF1 expression was positively correlated with cell proliferation and knockdown of ARF1 contributed to CAM-DR. The enhancement in the adhesion of MM cells to fibronectin (FN) or the bone marrow stroma cell line HS-5 cells translated to an increased CAM-DR phenotype. Importantly, we showed that this CAM-DR phenotype was correlated with the phosphorylation of Akt and ERK in MM cells. Moreover, we sought to determine whether ARF1 could interact with p27 in RPMI8226 cells. Knockdown of ARF1 also significantly decreased pT157-p27 protein expression in RPMI8226 cells. Our research shows ARF1 may reverse CAM-DR by regulating phosphorylation of p27 at T157 in MM. Taken together, our data shed new light on the molecular mechanism of CAM-DR in MM, and targeting ARF1 may be a novel therapeutic approach for improving the effectiveness of chemotherapy in MM.

Myeloma Cells Induce High Level of TAF12 Expression in Bone Marrow Stromal Cells, Resulting in Increased Osteoclastogenesis and Myeloma Cell Growth in Response to 1,25(OH)2D3

1,25 dihydroxy vitamin D3 (1,25D) plays multiple roles in normal and malignant cell functions, including cell differentiation and proliferation, and is a regulator of bone metastasis. However, the role of 1,25D in multiple myeloma (MM) is poorly characterized. We previously showed that TAF12, a member of the TFIID transcription complex, acts as a co-activator for vitamin D receptor (VDR) mediated transcription to increase 1,25D responsivity. Further, we reported that IL-6 can increase TAF12 levels in bone marrow stromal cells (BMSC). Since IL-6 is upregulated in the MM microenvironment, we assessed TAF12 levels in BMSC from 13 MM patients and 10 normals. TAF12 protein levels were significantly increased (25x) in MM BMSC compared to normal. Further, co-culture of BMSC from 3 normals with primary human MM cells significantly increased TAF12 levels in the normal BMSC. Similarly, when 5TGM-1 murine MM cells were inoculated intratibially into C57BL/KaLwRij mice and bone marrow cells collected 3 weeks later, TAF12 protein levels in MM exposed BMSC were significantly increased compared to PBS injected control mice. We then assessed if increased TAF12 levels persisted in normal BMSC (SAKA-T cells) after they were co-cultured with JJN3 MM cells and subsequently cultured in the absence of MM cells. We found that increased TAF12 levels persisted for at least 2 weeks after removal of MM cells, consistent with our findings that BMSC from MM patients express elevated TAF12 levels in the absence of MM cells. To determine if IL-6 contributes to the increased TAF12 levels induced by MM cells, we co-cultured SAKA-T cells with JJN3 MM cells in the presence or absence of anti-IL-6. Anti-IL-6 blocked the increase in TAF12 levels in these co-cultures.We then tested if the increased TAF12 levels enhance the 1,25D responsivity of BMSC. Addition of 1,25D (10-10 - 10-8 M) greatly increased expression of RANKL mRNA and VCAM-1 protein in MM BMSC compared to normals. The increased RANKL and VCAM-1 in MM BMSC treated with 1,25D correlated with increased osteoclast (OCL) formation, MM cell growth and adhesion of MM cells. When human OCL precursors and MM or normal BMSC were co-cultured with 1,25D with/without osteoprotegerin (OPG), OCL numbers were significantly increased in co-cultures of MM BMSC treated with 1,25D compared to normal, and the increase was blocked by OPG. Further, co-culture of JJN3 cells with normal or MM BMSC and 1,25D for 6 hours followed by removal of non-adherent MM cells, demonstrated that the number of MM cells adherent to BMSC was significantly increased with 1,25D treatment of MM BMSC co-cultures compared to normal BMSC. In addition, when JJN3 cells were co-cultured with normal or MM BMSC and 1,25D, JJN3 cells numbers were significantly increased in co-cultures with MM BMSC compared to normal BMSC. Finally, we determined if high levels of TAF12 in MM BMSC increased their 1,25D responsivity. We knocked-down (k/d) TAF12 with a siRNA in normal and MM BMSC, and found that TAF12 k/d in MM or normal BMSC decreased the levels of VCAM 1 induced by 1,25D compared to control siRNA transduced cells. To further investigate this effect, we assessed if TAF12 levels affected the stability of VDR in the presence of 1,25D, using TAF12 heterozygous or WT mouse BMSC. VDR half-life was decreased approximately 50% in TAF12 heterozygous BMSC compared with WT.These results show that MM cells increase expression of TAF12 in BMSC that is mediated by IL-6. BMSCs expressing high TAF12 levels have greater responsivity to 1,25D that enhances RANKL and VCAM-1 expression to induce osteoclastogenesis, MM cell growth and MM cell adhesion. These results further suggest that blocking TAF12 -VDR interactions may be a potential therapeutic target for MM bone disease. DisclosuresRoodman:Amgen: Consultancy.

Daratumumab Impairs Myeloma Cell Adhesion Mediated Drug Resistance through CD38 Internalization

Introduction: Daratumumab (HuMax-CD38, Dara) is an immunoglobulin G1 kappa (IgG1k) human monoclonal antibody that binds a CD38 epitope that has been recently approved by the Food and Drugs Administration as a single agent for the treatment of Multiple Myeloma (MM). Multiple myeloma (MM) is a plasma cell disorder affecting approximately 83,000 US citizens with 30,330 new cases per year in the US. The discovery of intra-clonal heterogeneity strengthens the scientific rationale of using novel therapy combinations to overcome mechanisms of resistance.While CD38 participates in NAD+ hydrolysis generating adenosine and influences intracellular calcium homeostasis through cADPR and NAADP synthesis, CD38 facilitates bone marrow (BM) homing of plasma cells through interaction with CD31 which is highly expressed in MM BM stromal cells (BMSCs) and macrophages (BM-M). Since adhesion of MM cells to stromal cells induces cell adhesion mediated drug resistance (CAM-DR), in this work, tested whether CD38 internalization after Daratumumab treatment impairs stromal cell adhesion, sensitizing MM cells to other drugs including proteasome inhibitors.Methods:Flow cytometry analysis and single cell flow analysis was done to measure the extent of surface CD38 internalization into MM cells (MM cell lines and primary cells) in vitro and ex-vivo. Adhesion assays were performed using MM cell lines treated with Daratumumab and co-cultured with BMSCs and BM-M. Apoptotic assays including cell proliferation and Annexin-V/PI staining were done to assess proteasome inhibitor induced apoptosis (bortezomib, BTZ) of MM cells pretreated with Daratumumab in the presence or absence of BM stroma. Chou-Talalay synergy analysis was used to assess the ability of Daratumumab to synergize with BTZ.Results:Single cell flow analysis revealed surface CD38 internalization into MM cell lines (MM1.S, H929, L363, RPMI-8226) and in primary myeloma cells upon incubation with increasing doses of Daratumumab. Our data show that MM cell lines and primary CD138+ MM plasma cells (MM-PCs) revealed loss of adhesion in a dose and time dependent fashion in co-culture experiments with BMSc. Moreover our data indicate that both BMSCs and BM-M protect MM cells to BTZ treatments. In order to investigate whether loss of adhesion of MM cells deprives them of protection, MM cell lines and primary cells were treated with Daratumumab and co-cultured with BM stroma and then treated with BTZ. Interestingly, it was observed that although stromal cells could protect MM cells from induced apoptosis, it failed to do so when MM cells were pretreated with Daratumumab. A more than two-fold increase in MM cell apoptosis was observed with Daratumumab-BTZ combination compared to the single agent treatments. This indicates that Daratumumab potentiates BTZ killing of MM cells.Conclusion:Daratumumab in combination with both proteasome inhibitors and immune modulators (IMiDs) are synergistic as evidenced by the results of CASTOR and POLLUX trials respectively, but the mechanisms of killing and resistance will likely be different. The main anti-MM effect of Daratumumab has so far been attributed to its antibody-dependent cellular cytotoxicity, complement dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis activities and in promoting T cell expansion in relapsed/refractary MM patients enrolled in Daratumumab monotherapy trials. Our data indicate that Daratumumab potentiates the BTZ killing of MM cells via CD38 internalization, providing rationale to further explore CD38 targeting using other drugs or cell therapies. DisclosuresHofmeister:Celgene: Research Funding; Karyopharm Therapeutics: Research Funding; Arno Therapeutics, Inc.: Research Funding; Incyte, Corp: Membership on an entity’s Board of Directors or advisory committees; Signal Genetics, Inc.: Membership on an entity’s Board of Directors or advisory committees; Janssen: Pharmaceutical Companies of Johnson & Johnson: Research Funding; Takeda Pharmaceutical Company: Research Funding; Teva: Membership on an entity’s Board of Directors or advisory committees.

The role of ubiquitin-specific protease 14 (USP14) in cell adhesion-mediated drug resistance (CAM-DR) of multiple myeloma cells.

Cell adhesion-mediated drug resistance (CAM-DR) is one of the mechanisms underlying the drug resistance in multiple myeloma (MM). Ubiquitin-specific protease 14 (USP14) is downregulated in the apoptotic model and upregulated in the adhesive model of MM. This study was undertaken to determine the role of USP14 in CAM-DR of MM cells. We examined the expression of USP14 in the apoptotic model of MM. The mechanism of USP14 in the process of apoptosis was further explored by flow cytometry assay and co-immunoprecipitation. We then performed the cell co-culture and adhesion assay and cell viability assay to investigate the effect of USP14 on adhesive rate and drug resistance in MM. We discovered that USP14 played a negative role in cell apoptosis, which is correlated with Bcl-xl. Moreover, overexpression of USP14 in MM cell adhesion model could enhance the ability of cell adhesion by regulating Wnt-signaling pathways, thereby promoting the CAM-DR in MM. USP14 participates in CAM-DR of MM through acting as a bridge between Bcl-xl apoptotic pathway and Wnt-signaling pathways and may be represented as a good candidate for pursuing clinical trials in MM.

Abstract 3283: APRIL/BCMA activation promotes human multiple myeloma progression and further induces immunosuppressive bone marrow microenvironment

Abstract Elevated B cell maturation antigen (BCMA) and its ligand a proliferation-inducing ligand (APRIL) may directly advance human multiple myeloma (MM) malignancy in vivo. Here, we first show that BCMA downregulation potently diminishes viability and colony formation of MM cells while BCMA overexpression augments MM cell growth and survival via induction of AKT, MAPK, and NFκB signaling cascades and molecules essential for proliferation and anti-apoptosis. Importantly, BCMA itself forces accelerated tumor growth of xenografted MM cells harboring p53 mutation in mice. BCMA-overexpressing tumors exhibit significantly enhanced CD31/microvessel density and VEGF than paired control tumors. Concurrently, these tumors express increased factors crucial for osteoclast activation, adhesion, and angiogenesis/metastasis, as well as immune inhibition including PD-L1, TGFβ and IL-10 which further triggers 38 IL-10 signaling molecules. In parallel, these identified target genes are induced by paracrine APRIL binding to BCMA in MM cells and they are potently blocked by an antagonistic anti-APRIL monoclonal antibody hAPRIL01A (01A). 01A is cytotoxic against MM cells protected by abnormal bone marrow (BM) myeloid cells, i.e., osteoclasts, macrophages, and plasmacytoid dendritic cells. It further decreases APRIL-induced adhesion and migration of MM cells via blockade of canonical and non-canonical NFκB pathways. It prevents in vivo MM cell growth within implanted human bone chips in SCID mice. 01A-inhibited MM cell viability induced by osteoclasts is further enhanced by lenalidomide and bortezomib. Taken together, these results further characterize new molecular mechanisms of APRIL/BCMA-induced in vivo MM progression and immunosuppressive BM microenvironment, strongly supporting targeting this prominent pathway in MM. Citation Format: Yu-Tzu Tai, Chirag Acharya, Gang An, Michele Moschetta, Mike Y. Zhong, Xiaoyan Feng, Michele Cea, Antonia Cagnetta, Kenneth Wen, Hans van Eenennaam, Andrea van Elsas, Nikhil Munshi, Kenneth Anderson. APRIL/BCMA activation promotes human multiple myeloma progression and further induces immunosuppressive bone marrow microenvironment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3283.

Role of Myeloma-Derived MIF in Myeloma Cell Adhesion to Bone Marrow and Chemotherapy Response.

Multiple myeloma (MM) remains an incurable cancer characterized by accumulation of malignant plasma cells in the bone marrow (BM). The mechanism underlying MM homing to BM is poorly elucidated. The clinical significance of migration inhibitory factor (MIF) expression was examined by analyzing six independent gene expression profile databases of primary MM cells using the Student's t test and Kaplan-Meier test. Enzyme-linked immunosorbent assay was used to examine MIF expression. In vivo bioluminescent imaging was used to determine MM cell localization and treatment efficacy in human MM xenograft mouse models, with three to four mice per group. MM cell attachment to BM stromal cells (BMSCs) was monitored by cell adhesion assay. MIF regulation of the expression of adhesion molecules was determined by chromatin immunoprecipitation (ChIP) assay. Statistical tests were two-sided. High levels of MIF were detected in MM BM (MIF level in BM plasma: healthy = 10.72 ± 5.788 ng/mL, n = 5; MM = 1811 ± 248.7 ng/mL, n = 10; P < .001) and associated with poor survival of patients (Kaplan-Meier test for MM OS: 87 MIF(high) patients, 86 MIF(low) patients, P = .02). Knocking down MIF impaired MM cell adhesion to BMSCs in vitro and led to formation of extramedullary tumors in SCID mice. MIF acted through surface receptor CXCR4 and adaptor COPS5 to regulate the expression of adhesion molecules ALCAM, ITGAV, and ITGB5 on MM cells. More importantly, MIF-deficient MM cells were sensitive to chemotherapy in vitro when cocultured with BMSCs and in vivo. MIF inhibitor 4-IPP sensitized MM cells to chemotherapy. MIF is an important player and a novel therapeutic target in MM. Inhibiting MIF activity will sensitize MM cells to chemotherapy.