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p38 mitogen-activated protein kinase (MAPK), which is constitutively activated in human myeloma, has been implicated in bone destruction by this cancer, but the processes it recruits are obscure. In this study, we show that p38 activity in myeloma inhibits osteoblast differentiation and bone formation, but also enhances osteoclast maturation and bone resorption. p38 regulated the expression and secretion of the Wnt pathway antagonist DKK-1 and the monocyte chemoattractant MCP-1. Attenuating p38, DKK-1, or MCP-1 were each sufficient to reduce bone lesions in vivo. Although it is well known that DKK-1 inhibits osteoblast differentiation, we found that together with MCP-1, it could also promote osteoclast differentiation and bone resorption. The latter effects were mediated by enhancing expression of RANK in osteoclast progenitor cells and by upregulating secretion of its ligand RANKL from stromal cells and mature osteoblasts. In summary, our study defined the mechanisms by which p38 signaling in myeloma cells regulates osteoblastogenesis, osteoclastogenesis, and bone destruction. Our findings, which may have implications for bone invasion by other cancers where p38 is elevated, strongly suggests that targeting p38 for inhibition may offer an effective therapeutic approach to treat osteolytic bone lesions in patients with myeloma. Cancer Res; 72(24); 6393–402. ©2012 AACR.

PDF file - 96K, Supplemental experimental procedures, including immunohistochemical analysis, western blotting analysis, Real-time PCR, cytokine array, ELISA, in vitro osteoclast and osteoblast formation and activity analysis and statistical analysis

PDF file - 81K, Schematic representation of the mechanism underlying myeloma cell p38-induced osteoclastogenesis activation and osteoblastogenesis inhibition

Few studies implicate immunoregulatory gene expression in tumor cells in arbitrating brain tumor progression. Here we show that fibrinogen-like protein 2 (FGL2) is highly expressed in glioma stem cells and primary glioblastoma (GBM) cells. FGL2 knockout in tumor cells did not affect tumor-cell proliferation in vitro or tumor progression in immunodeficient mice but completely impaired GBM progression in immune-competent mice. This impairment was reversed in mice with a defect in dendritic cells (DCs) or CD103+ DC differentiation in the brain and in tumor-draining lymph nodes. The presence of FGL2 in tumor cells inhibited granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced CD103+ DC differentiation by suppressing NF-κB, STAT1/5, and p38 activation. These findings are relevant to GBM patients because a low level of FGL2 expression with concurrent high GM-CSF expression is associated with higher CD8B expression and longer survival. These data provide a rationale for therapeutic inhibition of FGL2 in brain tumors., Fibrinogen-like protein 2 (FGL2) mediates immune suppression in glioblastoma (GBM). Here, the authors show that FGL-2 expressed by GBM cancer cells acts by suppressing the differentiation of CD103+ DC cells required to activate the anti-tumor CD8+ T cell response via blocking GM-CSF signalling at NFKB, STAT1/5 and p38 level.

// Mingjun Zhang 1, 2 , Jin He 1 , Zhiqiang Liu 1 , Yong Lu 1, 2 , Yuhuan Zheng 1, 2 , Haiyan Li 1, 2 , Jingda Xu 1 , Huan Liu 1 , Jianfei Qian 1, 2 , Robert Z. Orlowski 1 , Larry W. Kwak 1 , Qing Yi 1, 2 , Jing Yang 1, 3 1 Department of Lymphoma/Myeloma, Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA 2 Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA 3 Cancer Research Institute and Cancer Hospital, Guangzhou Medical University, Guangzhou, China Correspondence to: Jing Yang, e-mail: jiyang@mdanderson.org Qing Yi, e-mail: yiq@ccf.org Keywords: multiple myeloma, anti-β 2 M monoclonal antibody, bortezomib, autophagy, NF-κ p65 Received: November 17, 2014 Accepted: January 29, 2015 Published: March 31, 2015 ABSTRACT Our previous studies showed that anti-β 2 M monoclonal antibodies (mAbs) have strong and direct apoptotic effects on multiple myeloma (MM) cells, suggesting that anti-β 2 M mAbs might be developed as a novel therapeutic agent. In this study, we investigated the anti-MM effects of combination treatment with anti-β 2 M mAbs and bortezomib (BTZ). Our results showed that anti-β 2 M mAbs enhanced BTZ-induced apoptosis of MM cell lines and primary MM cells. Combination treatment could also induce apoptosis of BTZ-resistant MM cells, and the enhanced effect depended on the surface expression of β 2 M on MM cells. BTZ up-regulated the expression of autophagy proteins, whereas combination with anti-β 2 M mAbs inhibited autophagy. Sequence analysis of the promoter region of beclin 1 identified 3 putative NF-κB-binding sites from –615 to –789 bp. BTZ treatment increased, whereas combination with anti-β 2 M mAbs reduced, NF-κB transcription activities in MM cells, and combination treatment inhibited NF-κB p65 binding to the beclin 1 promoter. Furthermore, anti-β 2 M mAbs and BTZ combination treatment had anti-MM activities in an established MM mouse model. Thus, our studies provide new insight and support for the clinical development of an anti-β 2 M mAb and BTZ combination treatment to overcome BTZ drug resistance and improve MM patient survival.

Osteoclasts (OCs) are bone resorbing cells whose activity can be regulated by activated T cells and their cytokines. However, the immune function of OCs is largely unknown. In this study, we found that as bystanders, human OCs effectively suppressed T-cell proliferation induced by allogeneic, microbial antigenic, and T-cell receptor stimuli in vitro. Mechanism studies revealed that T cell-derived IFN-γ and CD40 ligand (CD40L) induced the expression of indoleamine 2,3-dioxygenase (IDO) in OCs, which mediated the immunosuppressive function on T-cell proliferation through depleting tryptophan. Neutralizing IFN-γ and blocking CD40L, or silencing or inhibiting IDO in OCs restored T-cell proliferation in the presence of OCs. Our data reveal a novel function of human OCs as inducible immunosuppressive cells, and a feedback loop between OCs and activated T cells. Thus, this study provides new insight into the mechanism of the immunosuppressive function of OCs, and may be helpful for developing novel therapeutic strategies for human diseases involving both the bone and immune systems.

Breast cancer cells frequently metastasize to bone and induce osteolytic bone destruction in patients. These metastases cause severe bone pain, high risk of fractures and hypercalcemia, and are essentially incurable and fatal. Recent studies show that breast cancer cells in bone activate osteoclastogenesis and bone resorption. However the underlying mechanism is poorly understood. This study shows that the p38 MAPK (p38) isoform MAPK11 (p38β) is expressed in breast cancer cells. By using specific small hairpin RNAs for MAPK11, we demonstrated that p38β-mediated p38 activity in breast cancer cells is responsible for breast cancer-induced osteolytic bone destruction. The addition of conditioned media from breast cancer cell lines MDA-MB-231 and MDA-MB-468, which have high expression of p38β, induced osteoclast differentiation and bone resorption. In contrast, knockdown of p38β in breast cancer cells reduced osteoclast differentiation in vitro and reduced bone destruction in severe combined immunodeficiency (SCID) mouse models. The knockdown of p38β did not affect tumor growth or survival or the ability of cancer cells to home to bone. Furthermore, our results showed that p38β upregulated the expression and secretion of monocyte chemotactic protein-1 (MCP-1) in breast cancer cells, and upregulated MCP-1 activates osteoclast differentiation and activity. This study elucidates a novel molecular mechanism of breast cancer cell-induced osteolytic bone destruction. This study also indicates that targeting breast cancer cell p38β and its product MCP-1 may be a viable approach to treat or prevent bone destruction in patients with bone-metastatic breast cancer.

p38 MAPK signaling controls cell growth, proliferation and the cell cycle under stress conditions. However, the function of p38 activation in tumor metastasis is still not well understood. We report that p38 activation in breast cancer cells inhibits tumor metastasis but does not substantially modulate primary tumor growth. Stable p38 knockdown in breast cancer cells suppressed NF-κB p65 activation, inhibiting miR-365 expression and resulting in increased IL-6 secretion. The inhibitory effect of p38 signaling on metastasis was mediated by suppression of mesenchymal stem cell (MSC) migration to the primary tumor and sites of metastasis, where MSCs can differentiate into cancer-associated fibroblasts to promote tumor metastasis. The migration of MSCs to these sites relies on CXCR4-SDF1 signaling in the tumor microenvironment. Analysis of human primary and metastatic breast cancer tumors showed that p38 activation was inversely associated with IL-6 and vimentin expression. This study suggests that combination analysis of p38 MAPK and IL-6 signaling in patients with breast cancer may improve prognosis and treatment of metastatic breast cancer.

Although many studies link brain tumor progression to oncogene activation and tumor-suppressor gene inactivation in tumor cells, few studies implicate immune regulatory gene expression in tumor cells in arbitrating brain tumor progression. Here we show that fibrinogen-like protein 2 (FGL2) is highly expressed in glioma stem cells and primary glioblastoma (GBM) cells. FGL2 knockout (FGL2KO) in GL261, DBT, and LLC tumor cells did not affect tumor cell proliferation in vitro or tumor progression in immunodeficient NSG mice, but completely impaired GBM progression in immune-competent C57bl/6 mice. This impairment was reversed in mice with a defect in Batf3 (a key transcription factor for CD103+ DCs differentiation). Mechanistic studies revealed that FGL2KO in tumor cells induces CD103+ DCs differentiation in both the central nervous system (CNS) and in tumor draining lymph nodes (TDLN). The increased CD103+ DCs population in the CNS and TDLNs induce CD8+ T cells priming and activation and thereby gliomas regress. More specifically, the presence of FGL2 in tumor cells inhibited granulocyte-macrophage colony-stimulating factor (GM-CSF)–induced CD103+ DC differentiation by suppressing NF-κB, STAT1/5, and p38 activation. These findings are relevant to GBM patients because a low level of FGL2 expression with concurrent high GM-CSF expression is associated with higher CD8B expression and longer survival. These data provide a rationale for therapeutic inhibition of FGL2 in brain tumors.

We used clustered regularly interspaced short palindromic repeats/Cas9-mediated genomic modification to investigate B-cell receptor (BCR) signaling in cell lines of diffuse large B-cell lymphoma (DLBCL). Three manipulations that altered BCR genes without affecting surface BCR levels showed that BCR signaling differs between the germinal center B-cell (GCB) subtype, which is insensitive to Bruton tyrosine kinase inhibition by ibrutinib, and the activated B-cell (ABC) subtype. Replacing antigen-binding BCR regions had no effect on BCR signaling in GCB-DLBCL lines, reflecting this subtype's exclusive use of tonic BCR signaling. Conversely, Y188F mutation in the immunoreceptor tyrosine-based activation motif of CD79A inhibited tonic BCR signaling in GCB-DLBCL lines but did not affect their calcium flux after BCR cross-linking or the proliferation of otherwise-unmodified ABC-DLBCL lines. CD79A-GFP fusion showed BCR clustering or diffuse distribution, respectively, in lines of ABC and GCB subtypes. Tonic BCR signaling acts principally to activate AKT, and forced activation of AKT rescued GCB-DLBCL lines from knockout (KO) of the BCR or 2 mediators of tonic BCR signaling, SYK and CD19. The magnitude and importance of tonic BCR signaling to proliferation and size of GCB-DLBCL lines, shown by the effect of BCR KO, was highly variable; in contrast, pan-AKT KO was uniformly toxic. This discrepancy was explained by finding that BCR KO-induced changes in AKT activity (measured by gene expression, CXCR4 level, and a fluorescent reporter) correlated with changes in proliferation and with baseline BCR surface density. PTEN protein expression and BCR surface density may influence clinical response to therapeutic inhibition of tonic BCR signaling in DLBCL.

Tumor stem cells, postulated to be the source cells for malignancies, have been identified in several cancers using cell-surface expression of markers including CD133, a pentaspan membrane protein. CD133+ve cells form neurospheres, exhibit self-renewal and differentiation, and are tumorigenic. However, despite its association with stem cells, a causal relationship of CD133 to tumorigenesis remains to be defined. Hypothesizing that specific epigenetic and transcription factors implicated in driving the stem cell state may concurrently regulate CD133 expression in stem cells, we analyzed the structure and regulation of CD133 promoter in glioma stem cells and glioma cell lines. Initially, a minimal promoter region was identified by analyzing the activity of CD133 promoter-driven luciferase-expressing 5'-and 3'-deletion-constructs upstream of the transcription start site. This region contained a CpG island that was hypermethylated in CD133-ve glioma stem cells (GSC) and glioma cells but unmethylated in CD133+ve ones. Of several predicted TF-binding sites in this region, the role of tandem Sp1 (-242 and -221) and two Myc (-541 and -25)-binding sites were examined. Overexpression of Sp1 or Myc increased CD133 minimal promoter-driven luciferase activity and CD133 levels in GSC and in glioma cell line. Mithramycin, a Sp1 inhibitor, decreased minimal promoter activity and downregulated CD133 levels in GSC. Gel-shift assays demonstrated direct binding of Sp1 to their predicted sites that was competitively inhibited by oligonucleotide-binding-site sequences and supershifted by anti-Sp1 confirming the interaction. Sp1 and Myc-antibody chromatin immunoprecipitation (ChIP) analysis in GSC showed enrichment of regions with Sp1 and Myc-binding sites. In CD133-ve cells, ChIP analysis showed binding of the methyl-DNA-binding proteins, MBD1, MBD2 and MeCP2 to the methylated CpG island and repression of transcription. These results demonstrate that Sp1 and Myc regulate CD133 transcription in GSC and that promoter methylation and methyl-DNA-binding proteins cause repression of CD133 by excluding transcription-factor binding.

The original version of this Article contained errors in the author affiliations. Qingnan Zhao, Xueqing Xia, Longfei Huo and Shulin Li were incorrectly associated with Beijing Institute for Brain Disorders, 100069, Beijing, China.This has now been corrected in both the PDF and HTML versions of the Article.

A major problem in patients with multiple myeloma is chemotherapy resistance, which develops in myeloma cells upon interaction with bone marrow stromal cells. However, few studies have determined the role of bone marrow adipocytes, a major component of stromal cells in the bone marrow, in myeloma chemotherapy resistance. We reveal that mature human adipocytes activate autophagy and upregulate the expression of autophagic proteins, thereby suppressing chemotherapy-induced caspase cleavage and apoptosis in myeloma cells. We found that adipocytes secreted known and novel adipokines, such as leptin and adipsin. The addition of these adipokines enhanced the expression of autophagic proteins and reduced apoptosis in myeloma cells. In vivo studies further demonstrated the importance of bone marrow-derived adipocytes in the reduced response of myeloma cells to chemotherapy. Our findings suggest that adipocytes, adipocyte-secreted adipokines, and adipocyte-activated autophagy are novel targets for combatting chemotherapy resistance and enhancing treatment efficacy in myeloma patients.

Gliomas are the most common type of brain tumor in both children and adults. Several low-grade gliomas (LGG) have the ability to progress into more aggressive tumors -high-grade gliomas (HGG) including glioblastoma (GB). Although patients harboring a LGG may survive for years, after the tumor transforms to HGG, life expectancy rapidly declines to 12 to 15 months in adults and 40 months in children. Thus, inhibiting this process of malignant transformation (MT) is an attractive therapeutic strategy because of the more indolent course associated with LGGs. Immune response plays a critical role in surveillance against malignant transformation. Our previous study shows that fibrinogen-like protein 2 (FGL2) is a key hub of tumor-mediated immune suppression. Hence, we investigated the role of FGL2 in promoting tumor progression from LGG to HGG in glioma. Analysis of TCGA expression data showed that increased FGL2 expression is associated with poorer survival in LGG and GB patients. And there is a positive correlation of expression level between FGL2 and mesenchymal glioma marker CD44, and a negative correlation between FGL2 and proneural glioma marker OLIG2. Engineered expression of FGL2 in a PDGFB-dependent mouse model of oligodendroglioma, a common glioma subtype, yielded a significantly higher rate of HGGs (72% vs 29%, p=0.034) and poorer-symptom free survival (63 vs 90 days, p=0.003) than PDGFB expression alone. And HGGs from FGL2 + PDGFB expressing mice exhibited a distinct mesenchymal phenotype validating TCGA data. Further, FGL2 induced high numbers of CD4+FoxP3+ cells from an early time point of tumor formation underscoring its role in tumor progression. And FGL2 overexpression educated M2 skew in the tumors characterized by high expression of Iba1 and Arginase1 in macrophages. Finally, treatment with anti-FGL2 antibody significantly improves survival in mice, shifts the phenotype from mesenchymal HGG to proneural LGG, and rescues M2 macrophage skewing. Our results show that FGL2 is critical for malignant progression of glioma by inducing immunosuppression in tumor microenvironment, and raise the potential of FGL2 to be a promising target to suppress/reverse glioma progression and provide survival benefit in clinical. Citation Format: Khatri Latha, Jun Yan, Yuhui Yang, Loyola V. Gressot, Lingyuan Kong, Ganiraju Manyam, Ravesanker Ezhilarasan, Qianghu Wang, Erik P. Sulman, Jingda Xu, Richard E. Davis, Suyun Huang, Gregory N. Fuller, Arvind Rao, Amy B. Heimberger, Shulin Li, Ganesh Rao. Fibrinogen-like protein 2 drives malignant tumor progression in glioma [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 5606. doi:10.1158/1538-7445.AM2017-5606

Hedgehog (Hh) signaling plays an important role in the oncogenesis of B-cell malignancies such as multiple myeloma (MM). However, the source of Hh ligand sonic hedgehog (SHH) and its target cells remains controversial. Previous studies showed that stromally induced Hh signaling is essential for the tumor cells and that CD19(+)CD138(-) MM stem cells are the target cells of Hh signaling. Here we demonstrate that SHH was mainly secreted by human myeloma cells but not by stromal cells in MM bone marrow. Autocrine SHH enhanced CD138(+) myeloma cell proliferation and protected myeloma cells from spontaneous and stress-induced apoptosis. More importantly, autocrine SHH protected myeloma cells against chemotherapy-induced apoptosis in vitro and in vivo. Combinational treatment with chemotherapy and SHH-neutralizing antibody displayed synergistic antimyeloma effects. Mechanistic studies showed that SHH signaling activated the SHH/GLI1/BCL-2 axis, leading to the inhibition of myeloma cell apoptosis. Thus, this study identifies the myeloma autocrine Hh signaling pathway as a potential target for the treatment of MM. Targeting this pathway may improve the efficacy of chemotherapy in MM patients.

Osteoclasts (OCs) are bone resorbing cells whose activity can be regulated by activated T cells and their cytokines. However, the immune function of OCs is largely unknown. In this study, we found that as bystanders, human OCs effectively suppressed T-cell proliferation induced by allogeneic, microbial antigenic and T-cell receptor stimuli in vitro. Mechanistic studies revealed that T cell-derived IFN-γ and CD40 ligand (CD40L) induced the expression of indoleamine 2,3-dioxygenase (IDO) in OCs, which mediated the immunosuppressive function on T-cell proliferation through depleting tryptophan. Neutralizing IFN-γ and blocking CD40L, and silencing or inhibiting IDO in OCs restored T-cell proliferation in the presence of OCs. Our data reveal a novel function of human OCs as inducible immunosuppressive cells, and a feedback loop between OCs and activated T cells. Thus, this study provides new insight into the mechanism of the immunosuppressive function of OCs, and may be helpful for developing novel therapeutic strategies for human diseases involving both the bone and immune systems.

Introduction. Targeting antigen-driven B-cell receptor (BCR) signaling with the BTK inhibitor ibrutinib is clinically effective against most B-cell lymphomas, including activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL), but not germinal center B-cell (GCB) DLBCL. We have formally confirmed that GCB-DLBCL cell lines utilize tonic BCR signaling, by showing: 1) sensitivity (variable) to knockout (KO) of the BCR, SYK, and CD19; 2) dependence on CD79A ITAM phosphorylation; and 3) independence from BCR antigen specificity. However, uncertainty remains about molecular events in upstream parts of tonic BCR signaling, why dependence of GCB-DLBCL cells on tonic BCR signaling is variable, and their clinical relevance. Methods. We used CRISPR/Cas9 methods to modify selected genes by KO and/or knock-in (KI) of the cDNA of a fluorescent protein (FP; e.g., GFP), with the FP serving as a marker of cells with gene KO or modification, or as a gene-fused tag for localization or quantitation. Cells expressing a membrane-targeted Forster resonance energy transfer (FRET) based AKT activity reporter (Lyn-AktAR2) were used to measure AKT activity directly by flow cytometry (FCM). Results. The effect of KI of CD79A Y188F mutation alone was similar to complete BCR KO, implying that CD79A Y188 phosphorylation is essential for tonic BCR signal transduction. Western blot analysis of GCB-DLBCL cell lines after BCR KO showed variable decreases of AKT S473 phosphorylation (frequently used as surrogate measure of AKT activity), but these did not correlate well with the variable decreases in proliferation of GCB-DLBCL cell lines caused by BCR KO. Measuring AKT activity directly (Fig. 1), or by another indirect approach (surface expression of CXCR4, a target gene of FOXO1 inhibited by AKT activity), showed high correlation between decreases in AKT activity and proliferation after BCR KO. In contrast to the variable effect of BCR KO on growth, pan-AKT KO was uniformly growth-slowing in GCB-DLBCL lines (Fig. 2). Interestingly, baseline surface density of BCR units in GCB lines, quantified by FCM using CD79A-GFP KI cells or anti-CD79B staining, correlated highly with reduction in growth or AKT activity caused by BCR KO (Fig. 3). These findings lead us to conclude that the BCR contributes to AKT activation in GCB-DLBCL cell lines, to a variable degree determined by BCR surface density. We also conclude that BCR surface density is determined by cell line-specific factors, as well as immunoglobulin heavy (IgH) and light (IgL) hypervariable region (HVR) sequences, based on measurements of BCR surface levels after exchanging endogenous HVR sequences in OCI-Ly19 and OCI-Ly7 cell lines for HVRs derived from other GCB and ABC-DLBCL cell lines. Reduction of AKT activity after BCR KO (measured by FRET reporter) and baseline BCR surface density in GCB-DLBCL cell lines also correlated well with the sensitivity of GCB-DLBCL lines to the clinically-tested SYK inhibitor (P505-15, PRT062607) or FDA-approved PI3K p110d isoform specific inhibitor (idelalisib). Interestingly, isogenic GCB-DLBCL cell lines with KO of PTEN, a negative regulator of AKT activation, were substantially more resistant to both inhibitors. A crucial role of PTEN deletion in overcoming dependence on tonic BCR signaling in GCB-DLBCL is supported by evidence from two naturally PTEN-deficient cell lines: SUDHL10, which adjusts to BCR KO and resumes normal growth, and HT, which lacks BCR expression, due to a frameshifting deletion in its IgH HVR. Re-expression of the BCR in HT, by KI to correct the IgH sequence, does not affect HT cell line growth. Conclusion. Our findings suggest a biomarker-guided therapeutic strategy in GCB-DLBCL: targeting tonic BCR signaling in BCR-high patients, by inhibiting CD79A phosphorylation, SYK, or PI3K, and downstream targeting of AKT in BCR-low and/or PTEN-deficient patients. Figure 1. Correlation of relative proliferation after BCR KO with decrease of AKT activity (as measured by FRET efficiency of AKT activity reporter) in GCB-DLBCL cell lines. Figure 1. Correlation of relative proliferation after BCR KO with decrease of AKT activity (as measured by FRET efficiency of AKT activity reporter) in GCB-DLBCL cell lines. Figure 2. Effect of BCR KO or pan-AKT KO in GCB-DLBCL cell lines. Figure 2. Effect of BCR KO or pan-AKT KO in GCB-DLBCL cell lines. Figure 3. Correlation of relative proliferation after BCR KO with baseline BCR surface density (as measured by flow cytometry of cells with CD79A-GFP fusion) in GCB-DLBCL cell lines. Figure 3. Correlation of relative proliferation after BCR KO with baseline BCR surface density (as measured by flow cytometry of cells with CD79A-GFP fusion) in GCB-DLBCL cell lines. Disclosures Burger: Pharmacyclics: Research Funding. Westin:Chugai: Membership on an entity's Board of Directors or advisory committees; Spectrum: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; ProNAi: Membership on an entity's Board of Directors or advisory committees.

p38 mitogen-activated protein kinase (MAPK), which is constitutively activated in human myeloma, has been implicated in bone destruction by this cancer, but the processes it recruits are obscure. In this study, we show that p38 activity in myeloma inhibits osteoblast differentiation and bone formation, but also enhances osteoclast maturation and bone resorption. p38 regulated the expression and secretion of the Wnt pathway antagonist DKK-1 and the monocyte chemoattractant MCP-1. Attenuating p38, DKK-1, or MCP-1 were each sufficient to reduce bone lesions in vivo. Although it is well known that DKK-1 inhibits osteoblast differentiation, we found that together with MCP-1, it could also promote osteoclast differentiation and bone resorption. The latter effects were mediated by enhancing expression of RANK in osteoclast progenitor cells and by upregulating secretion of its ligand RANKL from stromal cells and mature osteoblasts. In summary, our study defined the mechanisms by which p38 signaling in myeloma cells regulates osteoblastogenesis, osteoclastogenesis, and bone destruction. Our findings, which may have implications for bone invasion by other cancers where p38 is elevated, strongly suggests that targeting p38 for inhibition may offer an effective therapeutic approach to treat osteolytic bone lesions in patients with myeloma. Cancer Res; 72(24); 6393–402. ©2012 AACR.

Th9 cells are a subset of CD4+ Th cells that produce the pleiotropic cytokine IL-9. IL-9/Th9 can function as both positive and negative regulators of immune response, but the role of IL-9/Th9 in tumor immunity is unknown. We examined the role of IL-9/Th9 in a model of pulmonary melanoma in mice. Lack of IL-9 enhanced tumor growth, while tumor-specific Th9 cell treatment promoted stronger antitumor responses in both prophylactic and therapeutic models. Th9 cells also elicited strong host antitumor CD8+ CTL responses by promoting Ccl20/Ccr6-dependent recruitment of DCs to the tumor tissues. Subsequent tumor antigen delivery to the draining LN resulted in CD8+ T cell priming. In agreement with this model, Ccr6 deficiency abrogated the Th9 cell-mediated antitumor response. Our data suggest a distinct role for tumor-specific Th9 cells in provoking CD8+ CTL-mediated antitumor immunity and indicate that Th9 cell-based cancer immunotherapy may be a promising therapeutic approach.

Angiogenesis is thought to depend on a perfectly coordinated balance between endogenous-positive and negative regulatory factors. Of these factors, the vascular endothelial growth factor (VEGF) and angiopoietins (Angs) seem to play an essential role. Recently, we reported the expression of the Ang-natural receptor, Tie2, in neoplastic astrocytic cells within gliomas. Because of the VEGF/Ang2 functional partnership together with the presence of Tie2 in gliomas, we hypothesized a role of Ang2 on the modulation of VEGF levels in these tumors. We examined the effect of Ang2 on VEGF expression in a panel of glioma cells, which showed that Ang2 inhibited VEGF expression at both mRNA and protein levels in Tie2-expressing cells, but not in Tie2-negative cells. VEGF promoter analysis showed that Ang2 regulated VEGF expression at the transcriptional level in relation to a decrease in HIF-1alpha expression and HIF-DNA-binding activity. Tie2 silencing by siRNA rescued the Ang2-mediated downmodulation of VEGF, suggesting an essential role for Tie2 in this regulatory loop. To our knowledge, this is the first report on the role of Ang2/Tie2 in the regulation of HIF-1alpha/VEGF expression, providing additional evidence of the intrinsic coordination that occurs among these factors during angiogenesis.

Introduction. Targeting BCR signaling with the BTK inhibitor ibrutinib is clinically effective against most B-cell lymphomas, including the activated B-cell (ABC) subtype of diffuse large B-cell lymphoma (DLBCL), but not the germinal center B-cell (GCB) subtype. Active BCR signaling in GCB-DLBCL was suggested by studies with a Syk inhibitor and our previous studies using BCR knockout (KO). We addressed these questions: why is the BCR active in DLBCL, and how does it signal in GCB-DLBCL? Methods. We used CRISPR/Cas9 technology to modify selected genes by KO or homologous recombination-mediated knock-in (KI). For some genes KI was used to express a fluorescent protein (FP; e.g., GFP) instead of the targeted gene (KI/KO), or to modify the targeted gene together with KI of an FP, for detection of modified cells. Results. In GCB lines (OCI-Ly7 and OCI-Ly19) and ABC lines (U2932 and HBL-1), we simultaneously replaced the hypervariable region (HVR) exons of both immunoglobulin heavy (IgH) and light chains (IgL) with HVR sequences from normal B cells recognizing tetanus toxoid (TT). GFP and CFP respectively marked KI of IgH and IgL HVRs, and KI of the endogenous HVR sequences in each line served as controls. In CFP+/GFP+ cells, the TT specific BCR (TT-BCR) was expressed at similar or higher levels than the endogenous BCR (endo-BCR) and was functional, as shown by calcium flux in response to TT. The TT-BCR maintained growth of GCB lines (Fig. 1), indicating that they use "tonic", antigen-independent BCR signaling. Other features of tonic signaling were confirmed in more GCB lines: 1) the toxicity of BCR KO, which eliminates AKT S473 phosphorylation, was rescued by PTEN KO or expression of constitutively active AKT (mAKT), showing that BCR signaling serves principally to activate PI3K/AKT; and 2) KO of SYK or CD19, or truncation or ITAM mutation of the cytoplasmic tail of CD79A, none of which affect surface BCR levels, were as toxic as BCR KO but were non-toxic in BCR/PTEN double-KO cells. In contrast, the TT-BCR was as growth-slowing as BCR KO to the ABC line U2932 (Fig. 1), and substantially toxic to HBL-1, indicating that BCR signaling is self antigen-dependent in ABC-DLBCL. Reversion of somatic hypermutations in the U2932 HVRs was also as growth-slowing as BCR KO (Fig. 1), suggesting that self-antigen reactivity developed during BCR affinity maturation. Tonic signaling by the TT-BCR provided a detectable benefit (as compared to BCR KO) in PTEN-expressing HBL-1, whereas there was no difference between TT-HVR BCR and BCR KO in PTEN-deficient U2932. The surface TT-BCR level was higher than the endo-BCR level in ABC lines, and dropped with TT stimulation, suggesting that endo-BCRs in ABC lines undergo constant antigen stimulation with BCR internalization. The presumed self-antigen in ABC lines seems to be cell line-specific, since HVRs from ABC lines TMD8 and HBL-1 did not rescue growth of U2932. BCR KO in ABC lines was also not rescued by PTEN KO or mAKT. In cells whose BCRs were labeled by KI to fuse GFP to CD79A, super-resolution microscopy showed macro-clustering of BCR complexes at the surface of ABC line HBL-1, not seen in GCB lines (Fig. 2). Several findings suggested the clinical potential of targeting tonic BCR signaling in DLBCL: 1) clinical trial-stage inhibitors of SYK (P505-15) and PI3K (idelalisib) were toxic to GCB lines (less so with PTEN KO); 2) GCB lines (6/8) were sensitized by BCR KO to an in vitro CHOP-like regimen; 3) P505-15 or idelalisib sensitized GCB lines (3/3) to CHOP in vitro; and 4) evidence of tonic signaling in ABC line HBL-1 after removing antigen-driven signaling by HVR replacement. Conclusion. The BCR provides antigen-independent tonic signals to activate PI3K/AKT in GCB-DLBCL and antigen-dependent signaling in ABC-DLBCL. Targeting of B-cell specific tonic signling alone or in combination could be clinically effective in both types of DLBCL. Figure 1. Effect of BCR KO or HVR replacement in OCI-LY19 (A) and U2932 (B) cell lines. Endogenous IgH and IgL HVRs were replaced with HVR pairs (TT3 and/or TT6) recognizing tetanus toxoid, reverted to undo the effect of SHM, or restored with original HVRs. Figure 1. Effect of BCR KO or HVR replacement in OCI-LY19 (A) and U2932 (B) cell lines. Endogenous IgH and IgL HVRs were replaced with HVR pairs (TT3 and/or TT6) recognizing tetanus toxoid, reverted to undo the effect of SHM, or restored with original HVRs. Figure 2. Representative super-resolution images of BCR localization in live DLBCL cells. BCR labeled by CD79A-GFP fusion, surface membrane by CellMask staining. (bars = 5 µ m) Figure 2. Representative super-resolution images of BCR localization in live DLBCL cells. BCR labeled by CD79A-GFP fusion, surface membrane by CellMask staining. (bars = 5 µ m) Disclosures Westin: Spectrum: Research Funding.

Introduction. An essential role for the B-cell receptor (BCR) has been shown in multiple types of B-cell lymphoma by studies of cell lines and clinical responses to inhibitors of SYK or BTK. Diffuse large B-cell lymphoma (DLBCL) lines of the germinal center B-cell (GCB) type express a BCR, which can signal after crosslinking, but are unaffected by BCR pathway targeting toxic to lines of the activated B-cell (ABC) DLBCL subtype: knockdown of BCR signaling mediators (BTK, CD79A, and CD79B) by shRNA, and small-molecule inhibition of BTK by ibrutinib. GCB-DLBCL lines (and primary samples) also lack constitutive NF-kB activity and mutations in ITAM domains of CD79A or CD79B, BCR-related features of ABC-DLBCL. Most GCB-DLBCL patients resist BTK inhibition by ibrutinib, further suggesting that BCR signaling is not a feature of GCB-DLBCL. Methods. In 8 GCB-DLBCL lines (OCI-Ly7, OCI-Ly19, SUDHL-4, SUDHL-6, SUDHL-10, DB, BJAB, and HT) and one ABC-DLBCL line (HBL-1), we used electroporation to deliver a plasmid expressing Cas9 protein and a guide RNA (gRNA) targeting one of these: constant exons of IGHM, IGHG, or Igκ; the cell line-specific IgH hypervariable region (HVR); or CXCR4. Knock-in (KI) of mouse CD8a (mCD8a), after the HVR V segment leader sequence and followed by a polyA signal, was used as a positive marker of BCR knockout (KO) in HBL-1 and OCI-Ly19 cell lines. Surface BCR, CXCR4, and mCD8a were detected by flow cytometry (FACS). BCR KO cells were viably sorted 4-6 days after electroporation, cultured 1-3 days more, and studied by whole-genome gene expression profiling (GEP) on Illumina HT12v4 arrays and Western blotting. Results. Only 2 days after electroporation, FACS showed cells with correlated loss of surface BCR proteins (IgH, Igκ or Igl, and CD79B), which eventually declined to undetectable levels. Forward and side scatter showed that BCR KO cells were smaller. The proportion of BCR KO (or mCD8a KI/KO) cells declined over time, steadily after complete BCR elimination (Fig. 1A). BCR KO cells in GCB-DLBCL lines grew more slowly than BCR-replete cells but variably, from almost no difference in BJAB to growth cessation in SUDHL-4, SUDHL-10 and HBL-1 (Fig. 1B). CXCR4 KO cells were a stable proportion (Fig. 1A) with a normal growth rate (Fig. 1B), indicating that growth reduction by BCR KO is specific. Continued expression of mCD8a indicated viability and sustained IgH transcription in BCR KO cells. Cell cycle analysis showed lower proportions of S and G2/M phases in BCR KO cells, proportional to growth retardation, and sub-G1 cells in OCI-Ly7 (Fig. 2), SUDHL-4 and SUDHL-10. Apoptosis in OCI-Ly7 BCR KO cells was confirmed with a caspase-3 fluorogenic substrate. Igκ KO similarly caused complete BCR loss and growth retardation, in OCI-Ly7 cells even more than with IgH KO. In the HT cell line, which lacked BCR expression due to a single-nucleotide deletion in its IgH HVR, KI repaired the HVR and caused expression of surface BCR (IgM with Igκ and CD79B) but no change in growth rate, suggesting BCR-proximal activators of BCR signaling pathways. Targeted BCR KO is not currently a therapeutic option, but BCR KO cells were relatively more sensitive to an in vitro regimen modeling the non-prednisone drugs of CHOP. No change in drug sensitivity was observed with BCR KO in BJAB, or in CXCR4 KO cells. GEP showed that BCR KO downregulated several genes characteristically expressed by GCB-DLBCL, and genes associated with negative regulation of BCR signaling. Pathway analysis with Gene Set Enrichment Analysis (GSEA) showed that BCR KO reduced expression of proliferation-related signatures, and produced changes associated with B-cell differentiation stages lacking a mature BCR, either early (pre-B cells) or late (plasma cells). GSEA implicated loss of MAPK/ERK and PI3K/AKT signaling pathways as mediators of BCR KO-induced changes, confirmed by Western blotting showing loss of phosphorylation of SYK, AKT and ERK after BCR KO. Conclusions. Complete BCR KO by Cas9/gRNA showed that GCB-DLBCL lines require the BCR for optimal viability, cell growth, and chemotherapy resistance. BCR KO-induced changes are mediated by MAPK/ERK and PI3K/AKT signaling pathways. Table A. B. Figure 1. Figure 1A. BCR KO cells (distinguished from BCR-replete cells by FACS), but not CXCR4 KO cells, show relative decline (A) and slower absolute growth (B) in mixed cultures. Figure 1A. BCR KO cells (distinguished from BCR-replete cells by FACS), but not CXCR4 KO cells, show relative decline (A) and slower absolute growth (B) in mixed cultures. Figure 1B Figure 1B. Figure 2 Cell cycle changes with BCR KO in OCI-Ly7. Figure 2. Cell cycle changes with BCR KO in OCI-Ly7. Disclosures No relevant conflicts of interest to declare.

Introduction: XPO1 (CRM1, Exportin 1) is the sole transporter of many tumor suppressor proteins (including MYC, BCL2, BCL6, BTK, IkB) and is elevated in non-Hodgkin Lymphoma. Selinexor (Sel, KPT-330) is an oral covalent inhibitor of XPO1, the first clinical molecule of the Selective Inhibitors of Nuclear Export drug class. The phase I clinical trial of Sel in hematologic malignancies showed promising early single-agent efficacy with modest toxicity in relapsed Diffuse Large B-cell Lymphoma (DLBCL, Gutierrez et al, ASCO 2014). DLBCL, the most common lymphoid malignancy, is currently cured in only 10% of relapsed patients, and consists of 2 subtypes based on putative cell of origin (COO): activated B-cell (ABC) and germinal center B-cell (GCB). We performed preclinical studies of Sel, modeling its single-agent efficacy in frontline and relapsed DLBCL and its potential synergy with other clinically relevant therapeutics. Methods: To model drug resistant DLBCL, resistant subpopulations of 12 patient-derived DLBCL cell lines were created by in vitro intermittent exposure to active congeners of cyclophosphamide, doxorubicin, and vincristine (ivCHOP), approximating clinical practice. To determine if CHOP-resistant DLBCL is also resistant to other agents, we determined single-agent dose response curves and IC50 values for both parental and ivCHOP resistant (CHOP-res) subclones of 4 of these lines at submission (HBL1 & TMD8 of ABC subtype, OCI-Ly7 & HT of GCB subtype, with 8 lines in progress) with Sel, chemotherapy (CT, ivCHOP, DHAP, and ICE), and targeted therapy (TT, ibrutinib, ABT-199, idelalisib, everolimus, MLN0128, alisertib, lenalidomide, bortezomib, I-BET151, and ONC201). Viability was assessed with CellTiter-Glo (Promega) after a 3 day cell culture. IC50 values were determined using GraphPad Prism. Based on these results, we evaluated the ability of Sel to synergize with other agents or restore sensitivity in CHOP-res with a combination “checkerboard” (orthogonal dose titration for each drug). The Combination Index (CI) for pairs at all concentrations was calculated with ComboSyn, with CI values

Four new triterpenoid saponins, named as cirenshenosides S(1), T(2), U(3) and V(4), were isolated from the leaves of Oplopanax elatus Nakai. Their structures were elucidated to be 28-O-α-L-rhamnopyranosyl(1 → 4)- β- D-glucopyranosyl (1 → 6)- β- D-glucopyranosyl esters of 3 β ,23-dihydroxylup-20(29)-en-28-oicacid3-O- 13- β- -D- glucopyranoside(1), hederagenin3-O- β -D-glucopyranoside(2), 3 β -hydroxyolean-9(11), 12-dien-28-oic acid 3-O-β -D-glucopyranoside(3), 3 α-hydroxyolean- 12-en-23,28-dioic acid(4), respectively.

Currently, chemotherapy is the most effective treatment for multiple myeloma (MM). Although some new drugs have been shown to prolong survival in MM patients, these patients are prone to rapid relapse after high-dose treatment. Recent studies show that several bone marrow (BM) stromal cells are potentially involved in drug resistance. However, the role of other stromal cells is unclear. Adipocytes (ADs) are a major component of BM stromal cells. ADs have been shown to be involved in tumor rapid growth, metastasis, and apoptosis. Clinical studies suggest that BM ADs are associated with an increased risk of MM. Moreover, ADs isolated from patient BM biopsies were shown to support MM proliferation and migration. However, no published study has examined the importance of ADs in MM drug resistance. In addition, autophagy activation has been shown to induce drug resistance in cancer patients. We hypothesized that BM ADs protect MM cells from chemotherapy drug-induced apoptosis by autophagy activation. To examine the role of ADs in MM drug resistance, MM cells were cocultured with ADs at a ratio of 1:5 for 24 hours in medium with melphalan, dexamethasone, or bortezomib, the commonly used drugs for the treatment of MM. MM cells included primary MM cells isolated from BM aspirates of 5 MM patients and 6 MM cell lines. Human ADs were generated from mesenchymal stem cells derived from the BM mononuclear cells of healthy human fetal bones or BM aspirates of MM patients or healthy adult donors, cultured in AD medium for 2 weeks. ADs generated in vitro contained cytoplasmic Oil red O+ lipid droplets and produced triglycerol. Our results showed less drug-induced MM apoptosis in cocultures of MM cells and ADs compared with cultures of MM cells alone. Western blot analysis showed that treatment with melphalan upregulated the levels of cleaved caspase-9 and -3, but not -8, and PARP in MM cells. Compared with cultures alone, cocultures with ADs showed significantly lower levels of cleaved caspase-9, -3, and PARP in melphalan-treated MM cells. Mechanistic studies further showed that cocultures of ADs, compared with cultures alone, significantly upregulated the expression of autophagy proteins LC3B, Atg3, Atg5, and LAMP-1, but not Beclin-1. The addition of autophagy inhibitors 3-methyl adenine and chloroquine diphosphate to the cocultures remarkably enhanced apoptosis and caspase activation. Furthermore, we observed that cocultures of MM cells and ADs with either cell-cell contact or those separated by transwell inserts conferred similar protection from drug-induced apoptosis. We identified that AD-produced adipokines such as adiponection, leptin, adipsin, IL-6, MCP-1, TNF-a, and IGF-1, but not VEGF and CRP, were abundant in all examined ADs. Among these adipokines, adiponection, leptin, and adipsin were mainly produced from ADs and not from BM stromal cells, whereas other adipokines were produced from both cells. The addition of antibodies against these adipokines to the cocultures enhanced apoptosis and reduced autophagy, whereas addition of these adipokines to the cultures alone inhibited apoptosis and enhanced autophagy. In vivo studies validated these findings that injection of BM-derived ADs into the implanted human bones of SCID-hu mice bearing primary MM cells reduced response to treatment with melphalan and induced autophagy activation. Taken together, our findings elucidate a novel mechanism of MM drug resistance, through BM ADs. Our studies also provide evidence that targeting BM ADs may be a new approach to improve the efficacy of chemotherapy for the treatment of MM. Disclosures: No relevant conflicts of interest to declare.

Chemotherapy is the most effective treatment for multiple myeloma (MM). Several new drugs have been developed to prolong MM patient survival. However, application of these drugs, such as bortezomib (BTZ), usually induces drug resistance, and patients are prone to quick relapse. It is known that cell death or proliferation is regulated by the crosstalk between apoptosis and autophagy, and autophagy activation inhibits apoptosis by reducing caspase cleavage. Recent studies have shown that autophagy activation plays a role in chemotherapy drug resistance in cancer patients, in particular, BTZ treatment activates autophagy in MM, indicating that inhibition of autophagy could overcome BTZ-induced drug resistance. We have recently reported the generation of monoclonal antibodies (mAbs) against human beta2 microglobuline (b2M), and have found that high dose mAbs have strong apoptotic effects on MM. In this study, using a BTZ treatment-setting, we hypothesized that anti-β2M mAbs could overcome drug resistance and enhance chemotherapy efficacy in MM by inhibiting autophagy. To investigate the combined effects of mAbs and BTZ, MM cells were cultured with addition of BTZ (5 nM) or mAbs (clone D1; 10 mg/ml) or both for 24 hours. Results from Annexin-V binding assay indicated that combined treatment significantly enhanced apoptosis in MM cell lines, such as ARK, ARP-1, MM.1S, and U266, and in primary MM cells as compared with individual treatment, respectively. Next, BTZ-sensitive or -resistant MM cells KAS-6 WT/V10R or OPM-2 WT/V10R were used to determine the importance of BTZ in the synergistic effects, and such synergistic effects was only shown in apoptosis of BTZ-sensitive cells, but not in BTZ-resistant cells. Furthermore, combined treatment significantly increased apoptosis in β2M-overexpressing, but decreased in β2M-knocking down MM cells, which indicated that the synergistic effects is dependent on the surface β2M expression on MM cells. Mechanistic studies further showed that BTZ treatment resulted in an accumulation of cleaved-caspase 9/3 and PARP cleavage, suggesting that combined treatment enhanced caspase activation. In addition, Western blot analysis showed that BTZ treatment upregulated the expression of autophagy proteins, such as LAMP-1, Beclin 1, and LC3B, in a dose dependent manner, whereas combined treatment decreased their expression. Beclin 1, which is a key protein required for autophagy, has been reported to have a potential NF-kB p65 binding site on its promoter. Therefore, we checked if mAbs inhibited BTZ-induced autophagy via NF-kB signaling pathway. Our data showed that BTZ treatment increased p65 nuclear translocation and the levels of phosphorylated p65 in MM cells, while combined treatment significantly reduced both. ChIP assay has further verified that mAb treatment inhibited p65 binding to Beclin 1 promoter. To examine our hypothesis in vivo, MM cells ARP-1 or MM.1S were subcutaneously injected into SCID mice. The mice were then subcutaneously injected with mAbs (0.6 mg/kg) or intraperitoneally injected with BTZ (0.1 mg/kg) individually or in combination. Tumor burdens were assessed by measuring tumor volumes and serum M-protein levels by ELISA. The combination of anti-β2M mAb and BTZ treatment repressed tumor growth and prolonged the survival of tumor-bearing mice as compared with individual treatment. In conclusion, our study for the first time demonstrated that anti-β2M mAbs prevent BTZ resistance and enhance its anti-MM efficacy by reducing the expression of autophagy proteins via NF-kB signaling. Thus, our studies provide a new insight into clinical development of anti-β2M mAbs to overcome chemotherapy drug resistance and improve MM patient survival. Disclosures: No relevant conflicts of interest to declare.

Abstract 566 Bone destruction is a hallmark of multiple myeloma (MM). More than 80% of MM patients have osteolysis, which is characterized by pathological fractures, severe bone pain, spinal cord compression, and hypercalcemia. These symptoms can severely compromise a patient's quality of life and performance status. It has been proposed that MM cells activate osteoclast (OC)-mediated bone resorption and inhibit osteoblast (OB)-mediated bone formation. However, the mechanism underlying the association of MM cells with development of bone lesions remains poorly elucidated. Our previous studies showed that p38 mitogen-activated protein kinase (MAPK), which is constitutively activated in MM cells, is a master regulator of MM-mediated bone destruction. Knocking down or inhibiting p38 MAPK activity in MM cells prevented MM-induced bone destruction in vivo. In the present study, we further investigated the mechanism of MM cell p38 MAPK-induced bone destruction. We hypothesized that p38 MAPK activity in MM cells can regulate OB and OC differentiation and activity by upregulating cytokine production by MM cells. In a cytokine array analysis, we examined the expression and secretion of MM-derived cytokines that regulate OB and OC differentiation. Our results showed for the first time that either knockdown or inhibition of p38 MAPK activity by p38 MAPK short hairpin RNAs or inhibitors significantly downregulated the production of dickkopf-1 (DKK-1) and monocyte chemotactic protein-1 (MCP-1) by MM cells. Real-time PCR and ELISA quantified and confirmed the array analysis results. To determine the role of p38 MAPK-upregulated DKK-1 and MCP-1 production in bone destruction, we administered treatment with neutralizing antibodies to SCID mice injected intravenously with ARP-1 or MM.1S cells. Our results showed that neutralization of DKK-1 and MCP-1 led to fewer bone lesions in these mice. Furthermore, we examined the impact of MM cell p38 MAPK activity on OB and/or OC differentiation. Our results showed that knockdown or inhibition of MM cell p38 MAPK significantly downregulated osteoclastogenesis but upregulated osteoblastogenesis in vitro and in vivo. Although DKK-1 is well known to inhibit OB differentiation, we found that DKK-1, together with MCP-1, promoted OC differentiation and bone resorption. Mechanistic studies further showed that MCP-1 upregulated RANK expression in OC precursors and that DKK-1 increased RANKL secretion from stromal cells and mature OBs, all of which led to activation of the NF-kB and MAPK signaling pathways in OCs. Thus, our study uncovered a novel mechanism by which p38 MAPK signaling in MM cells regulates osteoblastogenesis, osteoclastogenesis, and bone destruction in patients with this disease. These findings strongly suggest that disrupting and targeting MM cell p38 signaling are effective approaches to treating osteolytic bone lesions in MM patients. Disclosures: No relevant conflicts of interest to declare.

Abstract 439 In multiple myeloma (MM), an incurable bone malignancy characterized by plasma cell accumulation in the bone marrow (BM), patients have increased osteoclast (OC) activity. As a result, more than 80% of MM patients develop osteolytic bone lesions during the course of the disease. Osteolytic bone lesions cause morbidity, such as pathological fractures, bone pain, and hypercalcemia, and therefore, severely affect the patients' quality of life. A better understanding of the mechanism of MM cell-induced OC activation could lead to a novel approach to treating MM bone disease. It is commonly accepted that MM cells are responsible for OC activation. Cocultures of MM cells with monocyte-derived OC precursors (preOCs) induce OC formation and bone resorption. In addition, previous studies have demonstrated the critical roles of receptor activator of nuclear factor κB ligand (RANKL) and receptor activator of nuclear factor κB (RANK) in aberrant OC activity upregulation in MM BM. Moreover, MM-derived cytokines, such as IL-3, IL-7, and MIP-1d, have been shown to enhance OC formation in a RANKL-dependent or -independent manner. When we cocultured MM cells with monocytes, but not preOCs, and treated them with RANKL, we made the novel observation that MM cells inhibited RANKL-induced OC differentiation. Specifically, human monocytes, isolated from five different healthy donor PBMCs or murine monocytic/macrophage cell line RAW264.7 cells, were transwell-cocultured with MM cells, either human MM cell lines or primary MM cells isolated from patients, in mediums with or without RANKL (50 mg/mL) for 14 days. Mature OCs, characterized as TRAP+ multinuclear cells, were detected by TRAP staining and further confirmed by quantitative RT-PCR for the expressions of mature OC marker genes, such as CTSK, CALCA, and TRAP. Our results showed that coculture with MM cells inhibited the development of mature OCs from monocytes, and suppressed RANKL-induced NFκB and JNK activation. By ELISA, we analyzed the levels of soluble cytokines in the conditioning medium of MM cells and found that MM cells produced large amounts of IL-10. Adding neutralizing antibody against IL-10 significantly abrogated MM inhibition of osteoclastogenesis, whereas adding IL-10 inhibited OC differentiation and downregulated the mRNA and protein levels of RANK on monocytes/preOCs. As MM cells grow in the BM, we wondered whether bone marrow stromal cells (BMSCs) could regulate the MM cell inhibitory effect on osteoclastogenesis. Our results showed that although BMSC itself did not affect osteoclastogenesis, cocultures of monocytes with both MM cells and BMSCs significantly restored RANKL-induced osteoclastogenesis. Analysis of soluble cytokines by ELISA showed that the levels of MCP-1, but not IL-10, were significantly upregulated in medium from MM/BMSC cocultures. Adding neutralizing antibody against MCP-1 highly inhibited OC activation, while adding MCP-1 enhanced OC differentiation and upregulated the expression of RANK on monocytes/preOCs. Overall, our findings are the first to elucidate a novel mechanism by which MM cells inhibit osteoclastogenesis by producing IL-10 and the presence of BMSCs suppresses MM inhibitory effects by up-regulating MCP-1. Regulation of RANK expression on monocytes/preOCs by MCP-1 and IL-10 determines osteoclastogenesis. Our studies provide evidence that targeting BM microenvironmental cells and/or factors may be a new approach to treating MM bone lesions. Disclosures: No relevant conflicts of interest to declare.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in malignant cells, including gliomas, and is currently in anticancer clinical trials. However, the full-length and tagged forms of TRAIL, unlike the untagged ligand (soluble TRAIL (sTRAIL)), exhibits toxicity against normal cells. Here, we report the generation and testing of an adenovirus (AdsTRAIL) that expresses untagged sTRAIL in an intracranial xenograft model and a human glioma organotypic slice culture model. AdsTRAIL efficiently induced apoptosis in glioma cell lines, including those resistant to sTRAIL, but not in normal human astrocytes (NHAs). It inhibited anchorage-independent glioma growth and exerted a bystander effect in transwell assays. Intratumoral injections of AdsTRAIL in a rodent intracranial glioma model resulted in reduced tumor growth and improved survival compared with Ad-enhanced green fluorescent protein (EGFP)- or vehicle-treated controls without toxicity. Human glioma organotypic slices treated with AdsTRAIL demonstrated apoptosis induction and caspase activation.