Bone Marrow Stromal Cell Line Research Articles

Pyruvate kinase isoform M2 (PKM2) participates in multiple myeloma cell proliferation, adhesion and chemoresistance

Cell adhesion mediated drug resistance (CAM-DR) remains the major barrier in human multiple myeloma (MM) therapy. In the present study, we aimed at investigating the role of pyruvate kinase isoform M2 (PKM2) in MM CAM-DR. We determined that PKM2 expression was positively correlated with cell proliferation and knockdown of PKM2 contributed to the increased cell adhesion rate in MM. The enhancement in the adhesion of MM cells to fibronectin 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. Taken together, our data shed new light on the molecular mechanism of CAM-DR in MM, and targeting PKM2 may be a novel therapeutic approach for improving the effectiveness of chemotherapy in MM.

The AP-1 Transcription Factor JunB Promotes Multiple Myeloma (MM) Cell Proliferation, Survival and Drug Resistance in the Bone Marrow Microenvironment

MEK/ERK and NF-kB signaling pathways have been reported to play a key role in multiple myeloma (MM) survival, proliferation and drug resistance. These pathways regulate the activity of numerous transcription factors. For example, the activator protein-1 (AP-1) transcription factor has been implicated in a multitude of physiologic processes, but also tumorigenesis. However, the function of AP-1 in MM is largely unknown. Our data show a vast variety of AP-1 (c-Jun, JunB, JunD, c-Maf and c-Fos) expression levels in MM cells. Importantly, co-culture of MM cells with bone marrow stromal cells (BMSCs), i.e. isotypic primary BMSCs as well as BMSC lines KM-105 and HS-27A, rapidly and strongly induces expression of JunB, but not other AP-1 members. Previous studies have shown that JunB exerts opposite functions depending on the cellular origin and the physiopathological context. For example, it serves as a gatekeeper in acute and chronic myeloid leukemia, but as a positive regulator in Hodgkin9s lymphomas and anaplastic large cell lymphomas. The relevance of JunB activity in MM growth, survival and drug resistance is elusive. First, our data demonstrate that induction of JunB is predominantly mediated by soluble factors secreted by BMSCs rather than direct MM-BMSC contact. Indeed, using cytokine arrays, we identified IL-6 among the most potent factors that trigger JunB expression. Mechanistically, JunB upregulation occurs at both transcriptional as well as translational level. Pharmacologic inhibition was used next in order to identify upstream signaling pathways, which mediate BMSC- induced JunB upregulation in MM cells. Our data show that activation of MEK/ERK or NF-kB is required for induction of JunB expression and AP-1 transcriptional activity. To delineate the specific functional role of JunB in MM pathogenesis, we transduced MM cells with pLKO.1-JunB shRNA or pLKO.1-scrambled shRNA (SCR). After puromycin- selection, effects of JunB knockdown on MM proliferation, survival and drug resistance were analyzed by 3H-thymidine incorporation, flow cytometry and western blot. Indeed, we observed significant inhibition of proliferation in MM/ JunB shRNA (decreased to ~ 25 – 40 %, p Furthermore, 4-hydroxytamoxifen (4-OHT) treatment of MM cell lines stably transduced with pMSCV-JunB-ER-IRES-GFP but not pMSCV-IRES-GFP induced significant AP-1 luciferase activity (~ 3.3 fold, p 1000 MM patient samples of different prognostic groups were compared to samples from healthy donors using the gene set enrichment analysis (GSEA). Our results further support a key role for JunB in MM pathogenesis. In summary, our data demonstrate for the first time an important role of JunB/AP-1 in MM tumorigenesis and strongly propose it as a novel therapeutic target in MM. Disclosures No relevant conflicts of interest to declare.

Abstract 2372: Inflammatory cytokine induced TSLP from bone marrow niches contributes to relapse of high risk TSLPR overexpressing acute lymphoblastic leukemia

Abstract Although 5 year survival rates are near 90%, leukemia is the most common cause of death from cancer in children with treatment failure primarily caused by relapse. A subset of acute lymphoblastic leukemia (ALL) patients that have an increased risk of relapse and consequently poorer overall survival have leukemia that overexpresses thymic stromal lymphopoietin receptor (TSLPR/CRLF2). However, it remains unclear what role Thymic Stromal Lymphopoietin (TLSP) signaling may play in increasing the risk of relapse. We hypothesize that TSLP/TSLPR axis may support progression of ALL by providing a signal through overexpressed TSLPR, which would sensitize the leukemia to low levels of the cytokine in bone marrow (BM) niches and promote the survival of the leukemia. We have created a high TSLPR expressing leukemia model (TSLPRhigh) through retroviral transduction of a transplantable syngeneic mouse ALL model (TSLPRlow) in which leukemia progression can be examined. This TSLPRhigh leukemia has expression of TSLPR comparable to what is found on patient derived ALL that naturally overexpress TSLPR and the receptor is functional as we see increased phosphorylation of STAT5 protein in response to TSLP stimulation. When ALL lines were injected into mice, we observed an 8 fold difference in the percentage of TSLPRhigh vs. TSLPRlow ALL in the BM 5 days after injection, where leukemia accounted for less than 4% of the BM corresponding to an early stage of ALL progression, whereas overall leukemia progression was not significantly different. This implies that the TSLP axis is most critical at low leukemic burden when bone marrow niches are intact and not disrupted by high levels of ALL in the BM. TSLPRlow and TSLPRhigh expressing cells show no significant difference in growth rate in vitro or in vivo, however when we treated ALL cells with chemotherapy in the absence or presence of TSLP we found that TSLP signaling significantly decreased the number of apoptotic cells in TSLPRhigh but not TSLPRlow ALL. These data suggest that high TSLPR expression sensitizes leukemia cells to TSLP to promote survival and not growth of ALL. This is significant because we believe that TSLP is likely to be expressed at low levels in the BM under normal circumstances. Interestingly, we have evidence using a nestin positive murine BM stromal cell line and stromal cells derived from fresh murine BM that TSLP mRNA and protein is induced by IL-1α and TNF-α stimulation. We further have found that cytarabine treatment of TSLPRhigh cells leads to induction of both IL-1α and TNF-α suggesting that chemotherapeutic treatment of ALL could potentially provide an unintended advantage to TSLPR overexpressing ALL. Based on this data, we postulate that therapies targeting the TSLP signaling axis in ALL would decrease the risk of relapse in TSLPR overexpressing ALL particularly in the context of standard therapy. Citation Format: Christopher D. Chien, Sang Nguyen, Haiying Qin, Terry J. Fry. Inflammatory cytokine induced TSLP from bone marrow niches contributes to relapse of high risk TSLPR overexpressing acute lymphoblastic leukemia. [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 2372. doi:10.1158/1538-7445.AM2015-2372

Stromal CYR61 Confers Resistance to Mitoxantrone via Spleen Tyrosine Kinase Activation in Human Acute Myeloid Leukaemia

Approximately 50% of children with acute myeloid leukaemia (AML) relapse, despite aggressive chemotherapy. The bone marrow stromal environment protects leukaemia cells from chemotherapy (i.e., stroma-induced chemoresistance), eventually leading to recurrence. Our goal is to delineate the mechanisms underlying stroma-mediated chemoresistance in AML. We used two human bone marrow stromal cell lines, HS-5 and HS-27A, which are equally effective in protecting AML cells from chemotherapy-induced apoptosis in AML-stromal co-cultures. We found that CYR61 was highly expressed by stromal cells, and was upregulated in AML cells by both stromal cell lines. CYR61 is a secreted matricellular protein and is associated with cell-intrinsic chemoresistance in other malignancies. Here, we show that blocking stromal CYR61 activity, by neutralization or RNAi, increased mitoxantrone-induced apoptosis in AML cells in AML-stromal co-cultures, providing functional evidence for its role in stroma-mediated chemoresistance. Further, we found that spleen tyrosine kinase (SYK) mediates CYR61 signalling. Exposure to stroma increased SYK expression and activation in AML cells, and this increase required CYR61. SYK inhibition reduced stroma-dependent mitoxantrone resistance in the presence of CYR61, but not in its absence. Therefore, SYK is downstream of CYR61 and contributes to CYR61-mediated mitoxantrone resistance. The CYR61-SYK pathway represents a potential target for reducing stroma-induced chemoresistance.

Efficacy of phosphatidylinositol-3 kinase inhibitors with diverse isoform selectivity profiles for inhibiting the survival of chronic lymphocytic leukemia cells.

Pharmacological inhibition of phosphatiylinositide-3-kinase (PI3K)-mediated signaling holds great promise for treating chronic lymphocytic leukemia (CLL). Therefore we assessed three structurally related PI3K inhibitors targeting the PI3K-δ isoform for their ability to inhibit the survival of freshly isolated CLL cells. The purely PI3K-δ-selective inhibitor idelalisib was compared to copanlisib (BAY 80-6946) and duvelisib (IPI-145), with isoform target profiles that additionally include PI3K-α or PI3K-γ, respectively. The concentrations leading to half-maximal reduction of the survival of CLL cells were more than ten-fold lower for copanlisib than for idelalisib and duvelisib. At concentrations reflecting the biological availability of the different inhibitors, high levels of apoptotic response among CLL samples were attained more consistently with copanlisib than with idelalisib. Copanlisib selectively reduced the survival of CLL cells compared to T cells and to B cells from healthy donors. In addition copanlisib and duvelisib impaired the migration of CLL cells towards CXCL12 to a greater extent than equimolar idelalisib. Similarly copanlisib and duvelisib reduced the survival of CLL cells in co-cultures with the bone marrow stroma cell line HS-5 more strongly than idelalisib. Survival inhibition by copanlisib and idelalisib was enhanced by the monoclonal CD20 antibodies rituximab and obinutuzumab (GA101), while antibody-dependent cellular cytotoxicity mediated by alemtuzumab and peripheral blood mononuclear cells was not substantially impaired by both PI3K inhibitors for the CLL-derived JVM-3 cell line as target cells. Taken together, targeting the α- and δ- p110 isoforms with copanlisib may be a useful strategy for the treatment of CLL and warrants further clinical investigation.

Quantitative analysis of glycans, related genes, and proteins in two human bone marrow stromal cell lines using an integrated strategy

Altered expression of glycans is associated with cell-cell signal transduction and regulation of cell functions in the bone marrow micro-environment. Studies of this micro-environment often use two human bone marrow stromal cell lines, HS5 and HS27a, co-cultured with myeloid cells. We hypothesized that differential protein glycosylation between these two cell lines may contribute to functional differences in in vitro co-culture models. In this study, we applied an integrated strategy using genomic, proteomic, and functional glycomic techniques for global expression profiling of N-glycans and their related genes and enzymes in HS5 cells versus HS27a cells. HS5 cells had significantly enhanced levels of bisecting N-glycans (catalyzed by MGAT3 [β-1,4-mannosyl-glycoprotein 4-β-N-acetylglucosaminyltransferase]), whereas HS27a cells had enhanced levels of Galβ1,4GlcNAc (catalyzed by β4GalT1 [β4-galactosyltransferase I]). This integrated strategy provides useful information regarding the functional roles of glycans and their related glycogenes and glycosyltransferases in the bone marrow microenvironment, and a basis for future studies of crosstalk among stromal cells and myeloma cells in co-culture.

An Electrochemical Assay for Monitoring Differentiation of the Osteoblastic Cell Line (MBA‐15) on the Sensor Chip

AbstractAn electrochemical assay for the indication of the activity of the cell bound differentiation marker alkaline phosphatase (ALP) is proposed using voltammetry on an in‐vitro cell culture. The basis of the assay is cultivation of cells on gold microelectrodes in wells of a microplate, catalytic hydrolysis of p‐aminophenyl phosphate by ALP and indication of p‐aminophenol oxidation by square wave voltammetry (SWV) with the sensors onto which the cells attached. The morphology of the bone marrow stromal cell line (MBA‐15) on the electrode surface was investigated and it exhibited in vitro osteogenic characteristics. Since ALP is expressed on the cell surface in early differentiation stage of osteoblastic cells, its activity was followed after different culture times over a period of 144 h by recording repetitive voltammograms at different time points upon addition of the substrate p‐aminophenyl phosphate. The ALP activity was estimated from the signal increase related to formation rate of p‐aminophenol and the number of cells. The highest value was measured at 120 h, when the cells reached confluence. The results of the electrochemical activity assay are consistent with the colorimetric acquired value from p‐nitrophenol formation rate.

Abstract B15: Bone marrow stromal cell-secreted inflammatory cytokines promote treatment resistance and survival of prostate cancer cells

Abstract It is well established that prostate cancer (PCa) cells hone to, adapt, and thrive in the bone. Importantly, bone metastases are found in over 80% of PCa deaths. Therefore, it is imperative to elucidate and characterize the molecular mechanisms that enable PCa cells to thrive in the bone and resist conventional PCa therapies, such as hormone ablation or anti-androgens. We found that the HS-5 bone marrow stromal cell (BMSC) line, a secretory BMSC line that can support hematopoietic stem cell growth, secretes paracrine factors that induce apoptosis in PCa cells. However a subpopulation of the PCa cells can survive the BMSC-induced death. The surviving PCa subpopulation differentiates into a neuronal morphology and looses androgen receptor (AR) expression, reminiscent of treatment-resistant neuroendocrine PCa. Ideally, the loss of AR would induce PCa cell death; however we observe a concomitant increase in the autophagy-related cell survival protein, p62/SQSTM1, and induction of cytoprotective autophagy. Furthermore, we find that interleukin-1 beta (IL-1β) and IL-6, inflammatory cytokine secreted by HS-5 BMSCs, are sufficient to promote PCa neuronal morphology, reduce AR accumulation, upregulate p62/SQSTM1 levels, and induce autophagy. In addition, genetic silencing of p62/SQSTM1 causes PCa cell death and pharmacological inhibition of cytokine or autophagy signaling attenuates PCa neuroendocrine differentiation. Thus, our PCa-bone marrow stromal cell model suggests that PCa cells can co-opt the immune system in the bone microenvironment leading to PCa androgen independence, treatment resistance, and survival in the bone. Furthermore, our data suggests that inhibiting inflammatory cytokine signaling and/or the autophagy process are rational strategies to combat PCa bone metastasis. Citation Format: Megan Chang, Micaela Morgado, Viral Patel, Michael Gwede, Mary Cindy Farach-Carson, Nikki Delk. Bone marrow stromal cell-secreted inflammatory cytokines promote treatment resistance and survival of prostate cancer cells. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B15. doi:10.1158/1538-7445.CHTME14-B15

SHH Inhibitor PF-0449913 in Vitro Remodels Leukemic Niche into HSC Supportive Niche

BackgroundAcute myelogenous leukemia (AML) is a highly lethal disease with only 20% of 5 years survival. Most AML patients experience relapse usually leading to death. Progression to therapy resistant AML is driven by leukemia stem cells (LSC) harboring enhanced survival, dormancy and self-renewal capacity in supportive niches. Growing evidence indicates that in the development of myeloid malignancies deregulation of stem cells activity is as important as deregulation of the BM microenvironment. Sonic hedgehog (SHH) pathway molecules in general and SMO in particular are well known to maintain the growth of cancer cells. However, it’s role in leukemia niche-stem cell interaction is not well defined. Therefore, here we investigate: 1/ how the small molecule, SMO antagonist, PF-0449913 impacts the AML BM microenvironment and 2/ how in turn, changes in the activity of AML BM niche cells contribute to its remodeling into HSC supportive niche.MethodsCD34- cell from bone marrow patients undergoing hip replacement surgery (normal BM) as well as AML bone marrow were utilized for the development of the primary human stromal monolayers. We investigated stromal cultures of primary normal BM (NBM, n=6) and AML (n=6). As a control human normal bone marrow stromal cell line HS-5 was used. Then human CD34+ cells were selected from AML primary samples (n=6). As a normal control, CD34+ cells from cord blood (CB, n=5), or NBM (n=5) were utilized for the co-culture experiments for up to 9 weeks and then plated in survival and self-renewal assays. PF-0449913 was added to stromal co-cultures or, as a pre-treatment, directly to stromal monolayers.ResultsIn order to examine the role of SMO regulation in LSC generation and maintenance, primary AML (n=6) or cord blood (n=3) CD34+ cells were co-cultures with normal BM (n=3) or AML (n=3) stroma in the presence of novel small molecule inhibitor (PF-0449913) for 2 weeks. It significantly reduced LSC survival and self-renewal, while spared HSC. These effects were not observed in the absence of stroma. Importantly, pre-treatment of the AML stroma with PF-0449913 for 1 week prior- to co-culture also resulted in LSC’s inhibition of survival and self-renewal.AML- and normal BM-derived stroma differ in their ability to support HSC and LSC: LSC (n=6) were capable to self-renew after 9 weeks of co-culture with both normal and AML stroma, while cord blood (n=4) as well as normal BM (n=5) HSC lost their self-renewal potential after only 2 weeks of co-culture with the AML stroma. PF-0449913 induced changes in AML stroma. which was previously unsupportive to HSC survival, it became compliant : self-renewal of HSC increased more than 3 times. It reversed also the inhibitory effect of AML stroma-derived conditioned media (CM).Pre-treatment of the HS-5 cells with CM from AML stroma for 4 weeks prior to co-culture experiments led to significant inhibition of the cord blood (n=3) and NBM (n=3) HSC survival and self-renewal. However, in CM obtained from AML stroma treated with PF-0449913 that effect was reversed.Combined treatment of AML cells (n=6) with PF-0449913 and de-methylating agent Vidaza (5-azacytodine) or CXCR4 antagonist AMD3100 resulted in even greater inhibition of LSC survival and self-renewal. However, Vidaza did not mediate changes in AML stroma.ConclusionsTogether these data indicate that, while both conditioned media and co-culture with AML stroma impaired HSC survival and self-renewal, co-culture conditions resulted in a greater reduction in survival and self-renewal capacity, implicating that cell-cell contact or unstable secreted factors exacerbate the effects. That suggests that microenvironmental cues play a key role in regulating normal HSC versus LSC survival and maintenance, and leukemic stroma exibit severely compromised ability to maintain normal HSCs, but effectively support LSCs. PF-0449913treatment of AML induces not only eradication of LSC, but also mediate changes in AML stroma, supporting HSC generation and maintenance.Simultaneous targeting of both AML niche and LSC could represent a novel avenue for treatment of AML patients. DisclosuresNo relevant conflicts of interest to declare.

Stromal CYR61 Confers Resistance to Mitoxantrone Via Spleen Tyrosine Kinase Activation in Human Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a life-threatening bone marrow malignancy with a relapse rate near 50% in children, despite aggressive chemotherapy. Accumulating evidence shows that the bone marrow stromal environment protects a subset of leukemia cells and allows them to survive chemotherapy, eventually leading to recurrence. The factors that contribute to stroma-induced chemotherapy resistance are largely undetermined in AML. Our goal is to delineate the mechanisms underlying stroma-mediated chemotherapy resistance in human AML cells.We used two human bone marrow stromal cell lines, HS-5 and HS-27A, to study stroma-induced chemotherapy resistance. Both stromal cell lines are equally effective in protecting AML cell lines and primary samples from apoptosis induced by chemotherapy agents, including mitoxantrone, etoposide, and cytarabine. By gene expression profiling using the Affymetrix U133Plus 2 platform, we previously found that CYR61 was among the genes that were commonly upregulated in AML cells by both stromal cell lines. CYR61 is a secreted matricellular protein that is expressed at relatively low levels by AML cells, and at higher levels by stromal cells. CYR61 binds and activates integrins and enhances growth factor signaling in AML cells, and it has been associated with chemoresistance in other malignancies. Our current data provide functional evidence for a role for this protein in stroma-mediated chemoresistance in AML. First, we added anti-CYR61 neutralizing immunoglobulin (Ig), or control IgG, to AML-stromal co-cultures, treated with chemotherapy for 24 hours, and measured apoptosis with Annexin V staining and flow cytometry. In THP-1+HS-27A co-cultures treated with 50 nM mitoxantrone, the apoptosis rate was 33.0 ± 3.7% with anti-CYR61 Ig v. 16.3 ± 4.2% with control IgG; p=0.0015). Next, we knocked down CYR61 in the HS-5 and HS-27A stromal cell lines by lentiviral transduction of two individual shRNA constructs, and confirmed knockdown (KD) at the gene and protein levels for both cell lines. These CYR61-KD stromal cells provided significantly less protection for co-cultured AML cells treated with mitoxantrone, compared to stromal cells transduced with the non-silencing control. For example, the apoptosis rate for THP-1 cells co-cultured with CYR61-KD HS-27A cells was 10.8 ± 0.8%, compared to 6.8 ± 1.1% for THP-1 cells co-cultured with control HS-27A cells (p=0.02). Similar results were obtained with NB-4 AML cells. These results demonstrate that CYR61 contributes to stroma-mediated chemoresistance.CYR61 binds to integrin αvβ3 (Kireeva, et al, J. Biol. Chem., 1998, 273:3090), and this integrin activates spleen tyrosine kinase (Syk) (Miller, et al, Cancer Cell, 2013, 24:45). Using intracellular flow cytometry, we found that activated Syk (pSyk) increased in THP-1 and NB-4 cell lines, and in primary AML patient samples, upon exposure to control HS-27A cells. In primary samples, the mean fluorescence intensity (MFI) for pSyk averaged 11.7 ± 1.3 in co-culture v. 6.6 ± 0.6 for cells cultured alone (p=0.004, n=10). In contrast, pSyk did not significantly increase in AML cells co-cultured with CYR61-KD HS-27A cells (MFI for primary patient samples: 8.6 ± 0.8). This result implicates Syk as a downstream signaling mediator of CYR61. To determine the role of CYR61-induced Syk signaling in chemotherapy resistance, we treated AML-stromal cell co-cultures with 3 uM R406, a potent Syk inhibitor, or DMSO, then added 300 nM mitoxantrone, and measured apoptosis after 24 hours. In AML cells co-cultured with control HS-27A cells, mitoxantrone-induced apoptosis was significantly increased by Syk inhibition (THP-1 cells: 13.7 ± 0.7% with R406 v. 10.0 ± 0.3% with DMSO, p<0.05), consistent with reduced chemoresistance. Notably, R406 did not further increase mitoxantrone-induced apoptosis in AML cells co-cultured with CYR61-KD HS-27A stromal cells (THP-1 cells: 15.7 ± 0.2% with R406 v. 16.9 ± 0.4% with DMSO). Similar results were seen with NB-4 cells, as well. These results support the notion that CYR61 signals through the integrin-Syk pathway to protect AML cells from chemotherapy. Therefore, the CYR61 - integrin - Syk pathway may be a potential therapeutic target for overcoming stroma-induced chemotherapy resistance in AML. DisclosuresNo relevant conflicts of interest to declare.

Tracking Human Myeloma Cell Therapeutic Resistance Using a Novel Diagnostic Assay for Detection of RNA Editing Biomarkers of Cancer Stem Cell Generation

INTRODUCTIONMultiple myeloma is a heterogeneous hematopoietic malignancy that represents 10% of all blood cancers and is characterized by malignant plasma cell expansion in the bone marrow, which is rich in pro-inflammatory cytokines such as interleukin-6 (IL-6). The emergence of therapeutically recalcitrant disease even following intensive treatment suggests the existence of a drug-resistant myeloma-initiating population. These myeloma-initiating cells are thought to co-opt stem cell pathways that enhance their capacity to self-renew and become dormant in protective niches. Recently, we and others showed that aberrant RNA editing plays a key role in malignant transformation through activation of the inflammation-responsive RNA editase ADAR1. In human leukemia stem cells, ADAR1 activation is associated with enhanced self-renewal of dormant progenitors, while lentiviral-shRNA ADAR1 knockdown reduces malignant stem cell self-renewal in vivo. Notably, in 30% of multiple myeloma cases copy number amplification of the ADAR locus on chromosome 1q21, which portends a poor prognosis. Thus, the goal of this study was to investigate whether ADAR1-mediated RNA editing in multiple myeloma occurs as a result of chromosome amplification and pro-inflammatory signaling, and to evaluate the effects of prolonged immunomodulatory therapy on ADAR1 activation leading to the emergence of drug-resistant myeloma-initiating cells.METHODS AND RESULTSIncreased ADAR1 expression in myeloma patient samplesTo determine whether gene expression of ADAR1 correlates with chromosome 1q21 amplification, we evaluated existing microarray datasets (Multiple Myeloma Genomics Initiative) from well-characterized patient samples harboring 2 to ≥4 copies of the CKS1B gene, which is located within 500kB adjacent to ADAR as well as the interleukin-6 (IL-6) receptor locus IL6R on chromosome 1q21. Notably, gain of 1q21 copy number is associated with more proliferative disease and poor-risk cytogenetics, which could also be related to ADAR activation. Interestingly, our analysis of ADAR1 gene expression showed increased ADAR1 levels in patients with a CKS1B score of 4 or greater (n=6) compared with patients with a score of 2 (n=18).IL-6 mediated induction of RNA editing activityTo evaluate the role of pro-inflammatory cytokine stimulation of ADAR1 in the chromosome 1q21-amplified human myeloma cell line H929, cells were exposed to increasing doses (5-20ng/mL) of recombinant human IL-6. ADAR1 activity, evaluated using a novel diagnostic RNA editing site-specific qPCR (RESSq-PCR) assay to detect cancer stem cell generation, showed that IL-6-treated H929 harbored increased ADAR1 activity.ADAR1 activation after prolonged lenalidomide exposureTo investigate the effects of immunomodulatory drug treatment on ADAR1-dependent RNA editing, drug-resistant human myeloma cell lines were experimentally derived by low-dose treatment (1 mM) of H929 cells over 10 weeks. Further incubation with lenalidomide at 10mM selected a robustly drug-resistant population. These cells showed a marked increase in ADAR1 expression and RNA editing activity, as measured by direct sequencing and RESSq-PCR. Long-term lenalidomide treatment of H929 also expanded the CD138-negative fraction, suggesting the emergence of a previously-identified myeloma-initiating cell population. Stromal co-culture of H929 cells with a 1:1 mixture of previously inactivated human bone marrow stromal cell lines (HS-5 and HS-27a) secreting IL-6 and other cytokines also enriched for CD138-negative and CD138-dim populations.CONCLUSIONConsidering the recently described role for ADAR1 in malignant transformation and cancer stem cell generation, myeloma cells harboring 1q21 amplification could gain a self-renewal advantage through ADAR1 activation. Using RESSq-PCR to detect aberrant RNA editing, we observed increased ADAR1 activity during the evolution of 1q21-amplified human myeloma cells under prolonged drug treatment, coupled with an expansion of the CD138-negative fraction, which was also observed following bone marrow stromal co-culture. Together these data suggest that ADAR1 represents a novel diagnostic and therapeutic target for multiple myeloma. Moreover, this multiple myeloma niche model represents a valuable tool for evaluating novel methods to inhibit aberrant ADAR1 activation in drug-resistant malignancies. DisclosuresJamieson:Sanofi: Honoraria; Roche: Honoraria.

The CXCR4/CXCL12 Axis Mediates Chemotaxis, Survival, and Chemoresistance in T-Cell Acute Lymphoblastic Leukemia

Background: While multi-agent chemotherapy has led to remarkable improvements in survival for children with T-cell acute lymphoblastic leukemia (T-ALL), outcomes remain dismal for the ~ 20% of patients that fail therapy. The goal of this study was to elucidate biological mechanisms contributing to chemoresistance in T-ALL. We hypothesized that chemoresistance owes not only to the aberrant signaling pathways intrinsic to the leukemia cells but also to contributions from an abnormal microenvironment in which they reside. The chemokine receptor ligand pair CXCR4/CXCL12 is important for normal leukocyte trafficking and deregulation is frequently observed in several hematologic malignancies. However, the role this axis plays in T-ALL is largely unknown. Here, we test the hypothesis that CXCR4/CXCL12 creates a sanctuary microenvironment that promotes T-ALL survival and provides protection from chemotherapy.Methods: A panel of T-ALL cell lines and primary patient samples expanded in NOD/SCID/c null (NSG) mice were cultured in the presence or absence of the CXCR4 antagonist, AMD3100, with or without chemotherapy. The human bone marrow stromal cell line, HS27a, which constitutively expresses the CXCR4 ligand CXCL12, was used to recapitulate the tumor microenvironment ex vivo. Transwell migration and modified pseudo-emperipolesis assays were used to examine CXCL12-mediated chemotaxis. Multi-parameter flow cytometry was used to evaluate the impact of activation of the CXCR4/CXCL12 axis on signaling networks, cell survival, and chemotherapy resistance.Results: We found CXCR4 membrane expression on all T-ALL cell lines and xenografts samples tested. Incubation with the CXCR4 ligand CXCL12 resulted in activation of survival signaling cascades (PI3K/AKT and MAPK), an effect blocked with AMD3100. Using a transwell system, we found dose dependent chemotaxis of T-ALL lymphoblasts to CXCL12 that was prevented by AMD3100. T-ALL cells also migrated into HS27a stromal cells in a CXCR4/CXCL12 dependent fashion. In addition, co-culturing T-ALL xenografts cells with HS27a imparted a survival advantage that was promptly eliminated by AMD3100 exposure. To test whether the CXCR4/CXCL12 microenvironment contributed to chemotherapy resistance, T-ALL xenografts cells were co-cultured ex vivo in the presence or absence of HS27a stromal cells and bortezomib or dexamethasone. Stromal cells conferred a marked chemoprotective effect that was specifically blocked by AMD3100. This highlights that stroma-mediated chemoresistance in xenograft samples is in part due to stromal-cell mediated activation of the CXCR4/CXCL12 axis.Conclusions: This study provides evidence for a T-ALL microenvironment that exploits the CXCR4/CXCL12 axis for leukemic cell recruitment, enhanced cell survival, and chemotherapy protection. Our findings also implicate the stroma as a major contributor to chemotherapy resistance in primary expanded patient samples partially due to the activation of the CXCR4/CXCL12 axis. These results further suggest that targeting the stroma through inhibition of this axis may be of therapeutic benefit in patients with chemotherapy resistant T-ALL. DisclosuresNo relevant conflicts of interest to declare.

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.

Th17 Cells Are Increased in the Bone Marrow but Not the Peripheral Blood in Multiple Myeloma and Demonstrate Defective Functionality and Aberrant Phenotypes

IntroductionImmune dysfunction in multiple myeloma (MM) is now a well accepted, if not yet completely understood, phenomenon contributing to both an increased incidence of infection and suboptimal responses to immunotherapies. The recently described Th17 subset of T helper cells is expanded in a range of cancers, and may contribute either to the evolution or control of tumours, but its role in myeloma pathogenesis remains unclear. We therefore investigated the presence, phenotype and function of this novel immune subset in a cohort of MM patients and healthy donors.MethodsPeripheral blood (PB) samples were taken from healthy donors (HC, n=27) and MM patients (n=31); mononuclear cells (PBMC) were isolated by density centrifugation. Bone marrow aspirate (BM) samples were taken from MM patients (n=7) and HC (n=3); mononuclear cells were isolated by red cell lysis (BioLegend). For phenotyping, cells were treated with phorbol 12-myristate 13-acetate, ionomycin and brefeldin A and assessed by flow cytometry. Where stated, cells were stimulated with candidal wall mannoprotein MP65 (Peptivator, Miltenyi Biotec). For coculture experiments, CD4+T cells were isolated from PBMC using magnetic bead separation (MACS, Miltenyi Biotec) and cultured with human myeloma cell lines (HMCLs: U266B, JIM3, KMS11) with or without human bone marrow stromal cell lines (BMSC: HS-5, HS-27). Statistical analysis was performed using GraphPad Prism v6.ResultsAs noted in previous studies, patients with myeloma have a marked CD4+ T-cell lymphopenia. Within the CD4+ population, MM patients also had a reduced frequency of IL-17+ cells (Th17) compared to HC: 0.41% (95% CI 0.27-0.56) vs. 0.81% (95% CI 0.47-1.15), p=0.0241 (student’s t-test). Using the mean fluorescence intensity for IL-17 as a surrogate indicator of cytokine production, IL-17 production in Th17 from MM patients was significantly reduced compared to HC (p=0.0195). Advancing disease stage correlated significantly with a reduction in PB Th17 frequency (p<0.0001, one-way ANOVA), with those with relapsed disease having the lowest levels (0.10%). In contrast, Th17 cells were expanded in the BM of MM patients compared to HC: 0.9% (95% CI 0.22-1.58) vs. 0.2% (95% CI -0.1198-0.5192), p=0.15). Th17 frequency was not significantly related to serum paraprotein concentration, prior lines of therapy, previous exposure to immunomodulatory novel agents (thalidomide, lenalidomide, bortezomib) or high-dose therapy, frequency of residual normal plasma cells in BM, serum creatinine, or high risk cytogenetic or molecular markers (del13 by FISH or IGHrearrangement). Th17 frequency was, however, lower in patients with IgG isotype disease compared to IgA (p=0.036) or light chain disease (p=0.0264).PB Th17 cells from myeloma patients showed a marked reduction in expression of CD161 compared with HC: 25.5% vs. 54.2%; this was again a stage-related phenomena (p=0.0019, one-way ANOVA). CCR6 expression on Th17s was also reduced in MM compared to controls in a stage-related manner (p=0.0036). Furthermore, PB Th17 cells in MM had impaired capacity to produce IL-17 in response to stimulation with a fungal antigen: 7% responded cf. 21% in HC (p=NS).To determine what, if any, effect MM tumour cells exerted on Th17 homeostasis, we employed a coculture model. CD4+ T cells from HC were cultured with mitomycin C-treated HMCLs for 7 days. Presence of HMCLs resulted in an expanded Th17 population compared to control cultures of CD4+ alone: 2.4% (95% CI: 0.13-1.30) vs..0.7% (95% CI: -0.20-5.07), p=0.09. The expansion of Th17 cells was augmented when BMSC were included (p=0.0476). We observed higher CD161 expression on coculture-derived Th17s when stromal cells were present.ConclusionsMultiple abnormalities are apparent in Th17 immunity in MM: their frequency is decreased in PB and increased in BM, in a stage-related manner suggestive of either a homing defect or a tumour microenvironmental expansion. Our in vitro co-culture data suggest tumour-derived Th17 expansion may be responsible. The Th17 phenotype in MM is aberrant, with the relative absence of key lineage markers and attenuated functional responses to fungal antigens. Since IL-17 has been reported to be a growth factor for MM cells, these abnormalities may contribute both to the propagation of the malignant clone and the impaired anti-infective immunity seen in this disease. DisclosuresNo relevant conflicts of interest to declare.

Combination Therapy with Reovirus and Immunomodulatory Drugs Induces Direct Oncolytic and Immune-Mediated Killing of Multiple Myeloma Cells and Overcomes Stromal-Mediated Microenvironmental Protection

IntroductionDespite considerable advances in therapy over recent years, multiple myeloma (MM) remains largely incurable with a clinical course characterised by ever-shortening periods of treatment and relapse; new therapies with novel mechanisms of action are needed. The immunomodulatory agents (IMiDs) in current clinical practice can augment immune effector function and combination with other immunologically active agents is a rational avenue of investigation – indeed trials of IMiDs with agents such as monoclonal antibody therapies are already underway. The naturally occurring oncolytic virus reovirus serotype 3 Dearing (Reolysin, Oncolytics Biotech Inc) has been shown to be safe in the context of advanced epithelial-derived cancers, and more recently in patients with MM. In this study we demonstrate the potential of reovirus in combination with IMiDs and steroids to induce both direct tumour cell killing and an anti-tumour immune response, particularly in the context of the cytoprotective effect of stromal cell interactions.MethodsHuman myeloma cell lines (HMCLs) were tested for JAM1 expression (the high affinity receptor for reovirus) by flow cytometry (FACS). HMCLs, alone or in co-culture with human stromal cell lines, were treated with reovirus at a range of multiplicities of infection (MOIs), alone or in combination with lenalidomide and/or dexamethasone and examined by flow cytometry to determine time- and concentration- dependent cell death. Peripheral blood mononuclear cells (PBMC) from healthy donors and patients were treated with reovirus/drug combinations and immune effector cell activation determined by surface expression of CD69. Treated PBMC and untreated HMCL were co-cultured and NK cell degranulation and NK cell-mediated HMCL cell death was determined. Bone marrow aspirate samples (BM) were collected from patients with MM, treated with reovirus, lenalidomide and dexamethasone combinations, and examined by FACS for killing of CD138+ cells and immune effector cell activation.ResultsJAM1 was expressed on the HMCLs H929, JIM3, KMS11, KMS18, JJN3 and U266B, but not on OPM2. All lines except OPM2 (which was resistant) were susceptible to reovirus-induced oncolysis in a time- and MOI-dependent manner. Addition of dexamethasone and lenalidomide to these treatments further increased the killing of tumour cells. Reovirus treatment induced upregulation of CD69 on NK cells, CD4+ T cells, CD8+ T cells and NKT cells isolated from the peripheral blood of both healthy donors and patients with MM. Healthy donor NK cells activated ex vivo with reovirus showed augmented degranulation (by surface CD107a expression) and killing of HMCL tumour targets. Importantly, in the case of OPM2, which was resistant to direct viral-induced cell death, reovirus-activated NK cells killed the HMCL. NK cells from MM patients showed enhanced degranulation against autologous tumour cells. Cell contact-independent killing of HMCL by reovirus-treated PBMC was also noted. Coculture of HMCLs with human bone marrow stromal cell lines (HS-5 and HS-27) protected against lenalidomide- and dexamethasone-induced HMCL cell death. However, treatment with reovirus overcame this cytoprotection. In keeping with this, when BM from MM patients was treated with lenalidomide and/or dexamethasone, the addition of reovirus augmented killing of CD138+MM cells.ConclusionsOur data indicate reovirus has dual modes of action against MM – not only can the virus mediate direct oncolytic cytotoxicity against the malignant clone, but in addition it can activate immune effector cells and thus induce immunologically-mediated tumour killing. Importantly, this immune-mediated action was noted even against HMCLs resistant to the direct viral effects. Furthermore, the cytoprotection and immune evasion afforded by stromal cells was mitigated by reovirus therapy and thus addition of reovirus to established immunomodulatory myeloma therapies augments their efficacy. Reovirus-based immunomodulatory combination therapies are a rational therapeutic approach for patients with relapsed MM and warrant further investigation in the pre-clinical and early phase clinical trial setting. DisclosuresCoffey:Oncolytics Biotech Inc.: Employment.

Stroma-Derived Factors Significantly Impact the Drug Response Profiles of Patient-Derived Primary AML Cells: Implications for Drug Sensitivity Testing

Background:Bone marrow (BM) microenvironment plays an important role in development of drug resistance in acute myeloid leukemia (AML) by supporting survival of therapy resistant leukemic cells that eventually may lead to disease relapse. Consideration of tumor stroma factors is therefore critically important when assessing the efficacy of drugs in the ex vivo drug testing of primary AML cells. To study the effects of tumor stroma factors on the response of AML patient cells ex vivo to a panel of cancer drugs, we evaluated the effects of stroma-derived conditioned medium (CM) on the sensitivity of genomically defined primary AML cells.Methods:Primary AML cells were isolated by Ficoll gradient separation from BM aspirates or peripheral blood of AML patients (n=13). For drug testing, 303 small molecule inhibitors were plated on 384-well plates in 5 different concentrations over a 10,000-fold concentration range. The stromal-cell conditioned medium (CM) was made by culturing human bone marrow (BM) stromal cell line HS-5 (American Type Culture Collection) in RPMI 1640 medium for 3 days. AML cells were added to the plates in either CM diluted with RPMI 1640 medium (25% CM) or in mononuclear cell medium (MCM, Promocell), which was used as the standard medium comparison. Cell viability was measured after 72 h and dose response curves generated for each drug. Drug sensitivity scores (DSS) were calculated as described previously (Yadav et al, 2014). Phosphorylation profiles of 43 proteins were analyzed with a human phospho-kinase array (R&D Systems). In addition, somatic mutations were identified by exome sequencing using DNA from the leukemia cells and matched skin biopsies, while expressed fusion genes were identified by transcriptome sequencing.Results:AML samples with activating mutations to kinases such as FLT3 or PDGFRB exhibited more sensitive ex vivo drug response profiles, particularly to broad-spectrum kinase inhibitors, compared to samples driven by other types of mutations. When the same AML samples were compared between the two conditions, CM or MCM, the drug sensitivities were different for many classes of drugs (Table 1). In CM, samples typically lost sensitivity to many of the tested drugs, such as topoisomerase II inhibitors, BCL2 inhibitors and several other classes of tyrosine kinase inhibitors (TKIs). The loss of TKI sensitivity in CM was particularly striking in the FLT3 and PDGFRB mutated cases. Cluster analysis of overall drug responses for AML samples tested in MCM resulted in a tight group of most TKIs, reflecting their overlapping target profiles. However, when the analysis was applied to responses from the same cells tested in CM, the TKI grouping was more dispersed. Thus, these results indicate that tyrosine kinase signaling is stringently regulated in standard medium, whereas CM helps to support cell survival resulting in lower responses to a range of TKIs. To test this hypothesis, phosphorylation of 43 different kinases was measured with AML samples incubated in either CM or MCM. CM induced phosphorylation of multiple proteins including p38α, HSP27, Src, Lyn, Hck and STAT6 proving the activation of other signaling pathways.Conclusions:Our dataindicate that stromal cell conditioned medium may have prominent effects on ex vivo drug responses of AML cells. BM factors likely provide survival cues that make primary patient-derived AML cells resistant to several targeted agents, such as topoisomerases and TKIs. This underscores the need to develop drug testing methods that take into account tumor-microenvironment interactions. [Display omitted] DisclosuresGjertsen:BerGenBio AS: Membership on an entity’s Board of Directors or advisory committees; Boehringer Ingelheim : Equity Ownership, Membership on an entity’s Board of Directors or advisory committees; Kinn Therapeutics AS: Equity Ownership. Porkka:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Kallioniemi:Medisapiens: Consultancy, Membership on an entity’s Board of Directors or advisory committees. Wennerberg:Pfizer: Research Funding. Heckman:Celgene: Research Funding.

The AP-1 Transcription Factor JunB Promotes Multiple Myeloma (MM) Cell Proliferation, Survival and Drug Resistance in the Bone Marrow Microenvironment

MEK/ERK and NF-kB signaling pathways have been reported to play a key role in multiple myeloma (MM) survival, proliferation and drug resistance. These pathways regulate the activity of numerous transcription factors. For example, the activator protein-1 (AP-1) transcription factor has been implicated in a multitude of physiologic processes, but also tumorigenesis. However, the function of AP-1 in MM is largely unknown.Our data show a vast variety of AP-1 (c-Jun, JunB, JunD, c-Maf and c-Fos) expression levels in MM cells. Importantly, co-culture of MM cells with bone marrow stromal cells (BMSCs), i.e. isotypic primary BMSCs as well as BMSC lines KM-105 and HS-27A, rapidly and strongly induces expression of JunB, but not other AP-1 members. Previous studies have shown that JunB exerts opposite functions depending on the cellular origin and the physiopathological context. For example, it serves as a gatekeeper in acute and chronic myeloid leukemia, but as a positive regulator in Hodgkin’s lymphomas and anaplastic large cell lymphomas. The relevance of JunB activity in MM growth, survival and drug resistance is elusive.First, our data demonstrate that induction of JunB is predominantly mediated by soluble factors secreted by BMSCs rather than direct MM-BMSC contact. Indeed, using cytokine arrays, we identified IL-6 among the most potent factors that trigger JunB expression. Mechanistically, JunB upregulation occurs at both transcriptional as well as translational level. Pharmacologic inhibition was used next in order to identify upstream signaling pathways, which mediate BMSC- induced JunB upregulation in MM cells. Our data show that activation of MEK/ERK or NF-kB is required for induction of JunB expression and AP-1 transcriptional activity.To delineate the specific functional role of JunB in MM pathogenesis, we transduced MM cells with pLKO.1-JunB shRNA or pLKO.1-scrambled shRNA (SCR). After puromycin- selection, effects of JunB knockdown on MM proliferation, survival and drug resistance were analyzed by 3H-thymidine incorporation, flow cytometry and western blot. Indeed, we observed significant inhibition of proliferation in MM/ JunB shRNA (decreased to ~ 25 – 40 %, p < 0.01) compared with MM/ SCR control cells, when co-cultured with BMSCs in particular. Moreover, our preliminary data show that knockdown of JunB overcomes resistance of MM cells against doxorubicin as well as melphalan.Furthermore, 4-hydroxytamoxifen (4-OHT) treatment of MM cell lines stably transduced with pMSCV-JunB-ER-IRES-GFP but not pMSCV-IRES-GFP induced significant AP-1 luciferase activity (~ 3.3 fold, p < 0.01) as well as MM cell proliferation. In ongoing experiments, the in vivo relevance of our in vitro data is evaluated in a xenograft mouse model inoculated with MM /JunB-ER-IRES-GFP and MM/ IRES-GFP cells. Finally, gene expression profiles on > 1000 MM patient samples of different prognostic groups were compared to samples from healthy donors using the gene set enrichment analysis (GSEA). Our results further support a key role for JunB in MM pathogenesis.In summary, our data demonstrate for the first time an important role of JunB/AP-1 in MM tumorigenesis and strongly propose it as a novel therapeutic target in MM. DisclosuresNo relevant conflicts of interest to declare.

Abstract 3383: JunB/AP-1 controls MM cell proliferation, survival and drug resistance in the bone marrow microenvironment

Abstract The activator protein-1 (AP-1) transcription factor is a dimeric complex that comprises members of the JUN, FOS, activating transcription factor (ATF) and musculoaponeurotic fibrosarcoma (MAF) protein families. Resultant heterodimers and homodimers determine the genes that are regulated by AP-1. Functionally, AP-1 has been implicated in a multitude of biological processes but also tumorigenesis, dependent on the cell type and the pathophysiologic context. In multiple myeloma (MM), the role of AP-1 is largely unknown. The present study focuses on the impact of JunB, a central member of the AP-1 family, in MM growth, survival and drug resistance. First, our data demonstrate rapid and strong induction of AP-1/JunB expression in MM cell lines and primary tumor cells upon co-culture with bone marrow stromal cells (BMSCs), i.e. isotypic primary BMSCs as well as BMSC lines KM-105 and HS-27A. Importantly, utilizing a non-contact co-culture system, we show that this effect is predominantly mediated by soluble factors secreted by BMSCs rather than direct MM-BMSC contact. Consistent with gene expression profiling data, JunB mRNA levels detected by quantitative real-time PCR are increased in MM cells when co-cultured with BMSCs. Specifically, we found that upregulation of JunB occurs predominantly at the translational level, since cycloheximide, but not actinomycin D or MG-132 significantly blocked BMSC- induced JunB expression. Pharmacologic inhibition was used next in order to identify upstream signaling pathways, which mediate BMSC- induced JunB upregulation in MM cells. Our data demonstrate that activation of MEK/MAPK or NF-kB, but not PI3K/Akt is required for induction of JunB expression and AP-1 transcriptional activity. To delineate the specific functional role of JunB in MM pathogenesis, we subsequently generated stable MM cell lines carrying the pLKO.1-puro-JunB shRNA vector or the pLKO.1-puro control vector, respectively. Our preliminary data show significantly decreased proliferation and resistance to chemotherapy in MM/ pLKO.1-puro-JunB shRNA but not MM/ pLKO.1-puro control cells when co-cultured with BMSCs. Consistently, 4-hydroxytamoxifen (4-HT) treatment of MM cells stably transduced with pBabe-puro-JunB-ER-IRES-GFP but not of MM cells transduced with pBabe-puro-IRES-GFP promotes tumor cell proliferation, survival and drug resistance, independent of BMSC co-culture. Ongoing studies aim to identify downstream effectors of JunB, using gene array profiling of MM/ pLKO.1-puro-JunB shRNA cells versus MM/ pLKO.1-puro control cells. Furthermore, we are conducting a survey of published and unpublished datasets to uncover the clinical and prognostic relevance of JunB expression in MM patient samples. In summary, our data demonstrate for the first time an important role of AP-1/JunB in MM tumorigenesis and strongly propose it as a novel therapeutic target in MM. Citation Format: Fengjuan Fan, Sonia Vallet, Martin Sattler, Giovanni Tonon, Muhammad Hasan Bashari, Latifa Bakiri, Hartmut Goldschmidt, Erwin F. Wagner, Dirk Jaeger, Klaus Podar. JunB/AP-1 controls MM cell proliferation, survival and drug resistance in the bone marrow microenvironment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3383. doi:10.1158/1538-7445.AM2014-3383

Reduced Radiation Pulmonary Fibrosis in Toll-Like Receptor-4 (TLR4) Deletion Recombinant Negative Mice

The inflammatory mediator receptor, TLR4 has been demonstrated to be upregulated in the lungs of thoracic irradiated C57BL/6J/NHsd mice. We investigated whether pulmonary irradiation of TLR4 -/- mice (B6 background) led to reduced lung fibrosis (organizing alveolitis). Since lung fibrosis involves pulmonary migration of marrow-origin fibroblasts, we compared bone marrow hematopoiesis in TLR4 -/- mice compared to TLR4 +/+ C57BL/6NHsd mouse long term marrow cultures (LTBMCS) LTBMC were established from femur bone marrow of TLR4-/- or wild type C57BL/6J into T25 flasks and hematopoiesis was measured over 30 weeks. Bone marrow stromal and IL-3 dependent hematopoietic progenitor cells lines were established from each genotype. TLR4 -/- and wild type 6-8 week old C57BL/6J mice were thoracic irradiated to 20 Gy, and pulmonary fibrosis was quantitated at 200 days after irradiation. Homozygous deletion of TLR4 resulted in improvement of multiple parameters of hematopoiesis in LTBMC including: Increased formation of cobblestone islands from week 1 (297.8±80.6 vs 136.0±36.6 for wild type, p = 0.0107) and week 7 (2228.5±165.5 vs 1994.5±47.2, p = 0.0346). Non-adherent cell production, another indicator of stem cell activity was significantly higher in TLR4-/- LTBMCS compared to wild type LTBMCS from week 1 (35.7±2.0 x105 vs 21.4±3.2 x105, p = 0.0003) through week 17 (8.2±2.2 x105 vs 3.3±1.7 x105 p = 0.0194). LTBMC from TLR4-/- continued to produce cobblestone islands and non-adherent hematopoietic cells until week 31,while those from wild type mice ceased at week 21. Production of day 14 colony forming CFU-GEMM cells was higher in TLR4-/- LTBMCS from week 3 (349.7±15.2 vs 194.3±8.0, p = 0.0001) to week 20 (164.0±7.0 vs 11.7±2.1, p = <0.0001). Lungs from 20GY thoracic irradiated TLR4-/- mice showed reduced irradiation pulmonary fibrosis and collagen compared to those from wild type mice (1.0 +/- 0.5 and 20.83 +/- 3.4, p < 0.0001). Absence of TLR4 decreases irradiation pulmonary fibrosis in C57BL/6 mice and improves hematopoiesis in LTBMCS suggesting a common role of bone marrow stromal cells in both processes.

Resolving the titer of murine cytomegalovirus by plaque assay using the M2-10B4 cell line and a low viscosity overlay

BackgroundMurine cytomegalovirus (MCMV) is increasingly used as an infectious model to investigate host-pathogen interactions in mice. Detailed methods have been published for using primary murine embryonic fibroblasts (MEFs) for preparing stocks and determining viral titers of MCMV. For determining the titer of MCMV by plaque assay, these methods rely on a high viscosity media that restricts viral spreading through the supernatant of the culture, but is also usually too viscous to pipet. Moreover, MEFs must be repeatedly generated and can vary widely from batch-to-batch in purity, proliferation rates, and the development of senescence. In contrast, the M2-10B4 bone marrow stromal cell line (ATCC # CRL-1972), which is also permissive for MCMV, has been reported to produce high-titer stocks of MCMV and has the considerable advantages of growing rapidly and consistently. However, detailed methods using these cells have not been published.MethodsWe modified existing protocols to use M2-10B4 cells for measuring MCMV titers by plaque assay.ResultsWe found that MCMV plaques could be easily resolved on monolayers of M2-10B4 cells. Moreover, plaques formed normally even when cultures of M2-10B4 cells were less than 50% confluent on the day of infection, as long as we also used a reduced viscosity overlay.ConclusionsOverall, our protocol enabled us to use a consistent cell line to assess viral titers, rather than repeatedly producing primary MEFs. It also allowed us to start the assay with 4-fold fewer cells than would be required to generate a confluent monolayer, reducing the lead-time prior to the start of the assay. Finally, the reduced viscosity CMC could be handled by pipet and did not need to be pre-mixed with media, thus increasing its shelf-life and ease-of-use. We describe our results here, along with detailed protocols for the use of the M2-10B4 cell lines to determine the titer and grow stocks of MCMV.