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A distributed network of neurons regulates wake, non‐rapid eye movement (NREM) sleep, and REM sleep. However, there are also glia in the brain, and there is growing evidence that neurons and astroglia communicate intimately to regulate behaviour. To identify the effect of optogenetic stimulation of astrocytes on sleep, the promoter for the astrocyte‐specific cytoskeletal protein, glial fibrillary acidic protein (GFAP) was used to direct the expression of channelrhodopsin‐2 (ChR2) and the linked reporter gene, enhanced yellow fluorescent protein (EYFP), in astrocytes. rAAV‐GFAP‐ChR2 (H134R)‐EYFP or rAAV‐GFAP‐EYFP was microinjected (750 nL) into the posterior hypothalamus (bilateral) of mice. Three weeks later baseline sleep was recorded (0 Hz) and 24 h later optogenetic stimulation applied during the first 6 h of the lights‐off period. Mice with ChR2 were given 5, 10 or 30 Hz stimulation for 6 h (10‐ms pulses; 1 mW; 1 min on 4 min off). At least 36 h elapsed between the stimulation periods (5, 10, 30 Hz) and although 0 Hz was always first, the order of the other three stimulation rates was randomised. In mice with ChR2 (n = 7), 10 Hz, but not 5 or 30 Hz stimulation increased both NREM and REM sleep during the 6‐h period of stimulation. Delta power did not increase. In control mice (no ChR2; n = 5), 10 Hz stimulation had no effect. This study demonstrates that direct stimulation of astrocytes powerfully induces sleep during the active phase of the sleep–wake cycle and underlines the inclusion of astrocytes in network models of sleep–wake regulation.

We have previously demonstrated that interleukin-17A (IL-17) producing T helper 17 cells are significantly elevated in blood and bone marrow (BM) in multiple myeloma (MM) and IL-17A promotes MM cell growth via the expression of IL-17 receptor. In this study, we evaluated anti-human IL-17A human monoclonal antibody (mAb), AIN457 in MM. We observe significant inhibition of MM cell growth by AIN457 both in the presence and the absence of BM stromal cells (BMSCs). Although IL-17A induces IL-6 production, AIN457 significantly downregulated IL-6 production and MM cell adhesion in MM-BMSC co-culture. AIN457 also significantly inhibited osteoclast cell differentiation. More importantly, in the SCIDhu model of human myeloma administration of AIN457 weekly for 4 weeks after the first detection of tumor in mice led to a significant inhibition of tumor growth and reduced bone damage compared with isotype control mice. To understand the mechanism of action of anti-IL-17A mAb, we report, here, that MM cells express IL-17A. We also observed that IL-17A knockdown inhibited MM cell growth and their ability to induce IL-6 production in co-cultures with BMSC. These pre-clinical observations suggest efficacy of AIN457 in myeloma and provide the rationale for its clinical evaluation for anti-myeloma effects and for improvement of bone disease.

Multiple myeloma (MM) is a plasma cell neoplasm that proceeds through a premalignant state of monoclonal gammopathy of unknown significance; however, the molecular events responsible for myelomagenesis remain uncharacterized. To identify cellular pathways deregulated in MM, we addressed that sumoylation is homologous to ubiquitination and results in the attachment of the ubiquitin-like protein Sumo onto target proteins. Sumoylation was markedly enhanced in MM patient lysates compared with normal plasma cells and expression profiling indicated a relative induction of sumoylation pathway genes. The Sumo-conjugating enzyme Ube2I, the Sumo-ligase PIAS1, and the Sumo-inducer ARF were elevated in MM patient samples and cell lines. Survival correlated with expression because 80% of patients with low UBE2I and PIAS1 were living 6 years after transplantation, whereas only 45% of patients with high expression survived 6 years. UBE2I encodes the sole Sumo-conjugating enzyme in mammalian cells and cells transfected with a dominant-negative sumoylation-deficient UBE2I mutant exhibited decreased survival after radiation exposure, impaired adhesion to bone marrow stroma cell and decreased bone marrow stroma cell–induced proliferation. UBE2I confers cells with multiple advantages to promote tumorigenesis and predicts decreased survival when combined with PIAS1. The sumoylation pathway is a novel therapeutic target with implications for existing proteasomal-based treatment strategies.

Neurons containing melanin-concentrating hormone (MCH) are located in the hypothalamus. In mice, optogenetic activation of the MCH neurons induces both non-rapid eye movement (NREM) and rapid eye movement (REM) sleep at night, the normal wake-active period for nocturnal rodents [R. R. Konadhode et al. (2013) J. Neurosci., 33, 10257-10263]. Here we selectively activate these neurons in rats to test the validity of the sleep network hypothesis in another species. Channelrhodopsin-2 (ChR2) driven by the MCH promoter was selectively expressed by MCH neurons after injection of rAAV-MCHp-ChR2-EYFP into the hypothalamus of Long-Evans rats. An in vitro study confirmed that the optogenetic activation of MCH neurons faithfully triggered action potentials. In the second study, in Long-Evans rats, rAAV-MCH-ChR2, or the control vector, rAAV-MCH-EYFP, were delivered into the hypothalamus. Three weeks later, baseline sleep was recorded for 48 h without optogenetic stimulation (0 Hz). Subsequently, at the start of the lights-off cycle, the MCH neurons were stimulated at 5, 10, or 30 Hz (1 mW at tip; 1 min on - 4 min off) for 24 h. Sleep was recorded during the 24-h stimulation period. Optogenetic activation of MCH neurons increased both REM and NREM sleep at night, whereas during the day cycle, only REM sleep was increased. Delta power, an indicator of sleep intensity, was also increased. In control rats without ChR2, optogenetic stimulation did not increase sleep or delta power. These results lend further support to the view that sleep-active MCH neurons contribute to drive sleep in mammals.

Cataplexy, a sudden unexpected muscle paralysis, is a debilitating symptom of the neurodegenerative sleep disorder, narcolepsy. During these attacks, the person is paralyzed, but fully conscious and aware of their surroundings. To identify potential neurons that might serve as surrogate orexin neurons to suppress such attacks, the gene for orexin (hypocretin), a peptide lost in most human narcoleptics, was delivered into the brains of the orexin-ataxin-3 transgenic mouse model of human narcolepsy. Three weeks after the recombinant adenoassociated virus (rAAV)-mediated orexin gene transfer, sleep–wake behavior was assessed. rAAV-orexin gene delivery into neurons of the zona incerta (ZI), or the lateral hypothalamus (LH) blocked cataplexy. Orexin gene transfer into the striatum or in the melanin-concentrating hormone neurons in the ZI or LH had no such effect, indicating site specificity. In transgenic mice lacking orexin neurons but given rAAV-orexin, detectable levels of orexin-A were evident in the CSF, indicating release of the peptide from the surrogate neurons. Retrograde tracer studies showed that the amygdala innervates the ZI consistent with evidence that strong emotions trigger cataplexy. In turn, the ZI projects to the locus ceruleus, indicating that the ZI is part of a circuit that stabilizes motor tone. Our results indicate that these neurons might also be recruited to block the muscle paralysis in narcolepsy.

While inflammation has been associated with the development and progression of colorectal cancer, the exact role of the inflammatory Th17 pathway remains unclear. In this study, we aimed to determine the relative importance of IL-17A and the master regulator of the Th17 pathway, the transcription factor RORγt, in the sporadic intestinal neoplasia of APC(MIN/+) mice and in human colorectal cancer. We show that levels of IL-17A are increased in human colon cancer as compared to adjacent uninvolved colon. Similarly, naive helper T cells from colorectal cancer patients are more inducible into the Th17 pathway. Furthermore, IL-17A, IL-21, IL-22, and IL-23 are all demonstrated to be directly mitogenic to human colorectal cancer cell lines. Nevertheless, deficiency of IL-17A but not RORγt is associated with decreased spontaneous intestinal tumorigenesis in the APC(MIN/+) mouse model, despite the fact that helper T cells from RORγt-deficient APC(MIN/+) mice do not secrete IL-17A when subjected to Th17-polarizing conditions and that Il17a expression is decreased in the intestine of RORγt-deficient APC(MIN/+) mice. Differential expression of Th17-associated cytokines between IL-17A-deficient and RORγt-deficient APC(MIN/+) mice may explain the difference in adenoma development.

The early expression of Epap-1 (early pregnancy associated protein), a 90 kDa anti-HIV-1 active glycoprotein, in the first trimester placental tissue suggests that it is one of the innate immune factors/proteins protecting the fetus from HIV infections. In the present investigation, we have cloned and expressed Epap-1 in bacterial and baculovirus expression systems. The recombinant Epap-1 as well as native Epap-1 shows a conserved molecular mode of action. These proteins exhibit significant antiviral activity and inhibit the cell fusion reaction between gp120 expressing HeLa (HL2/3) cells and T cell line (SupT1). Further, the rhodamine labeled Epap-1 specifically bound to gp120 expressed on the surface of HL2/3 cells during fusion reaction thereby inhibiting viral entry. Analysis of the interacting gp120 epitopes revealed that Epap-1 binds specifically to epitopes of gp120, recognizing constant-5 (C5) region and the variable-3 (V3) epitope of gp120 expressed on HL2/3 cells; It exhibits specific interaction with C5 region of cell-free virus in four HIV-1 isolates suggesting that the molecular interaction of Epap-1 is specific and is highly conserved in binding to gp120 leading to inhibition of viral entry. Epap-1 can thus be a very efficient natural protection mechanism against cell-free and cell-associated viral infections during early pregnancy.

A response to Fraigne JJ, Peever JH, Jones BE, Hassani OK, McGinty D, Alam N, Luppi PH, Peyron C, and Fort P. Critical Topics Forum. Sleep 2013;36:1767–1776.

Melanin concentrating hormone (MCH) is a cyclic neuropeptide present in the hypothalamus of all vertebrates. MCH is implicated in a number of behaviors but direct evidence is lacking. To selectively stimulate the MCH neurons the gene for the light-sensitive cation channel, channelrhodopsin-2, was inserted into the MCH neurons of wild-type mice. Three weeks later MCH neurons were stimulated for 1 min every 5 min for 24 h. A 10 Hz stimulation at the start of the night hastened sleep onset, reduced length of wake bouts by 50%, increased total time in non-REM and REM sleep at night, and increased sleep intensity during the day cycle. Sleep induction at a circadian time when all of the arousal neurons are active indicates that MCH stimulation can powerfully counteract the combined wake-promoting signal of the arousal neurons. This could be potentially useful in treatment of insomnia.

STUDY OBJECTIVES Narcolepsy is a sleep disorder characterized by loss of orexin neurons. Previously, our group demonstrated that transfer of the orexin gene into surrogate neurons in the lateral hypothalamus and the zona incerta significantly reduced cataplexy bouts in the orexin-ataxin-3 mice model of narcolepsy. The current study determined the effects of orexin gene transfer into the dorsolateral pontine neurons in the orexin knockout (KO) mice model of narcolepsy. The dorsolateral pons was chosen because it plays a critical role in regulating muscle tone and thus it is conceivable to be involved in cataplexy as well. Cataplexy is the pathognomonic symptom in narcolepsy. DESIGN Independent groups of orexin KO mice were given bilateral microinjections (0.75 μL each side) of either recombinant adenoassociated virus-orexin (rAAV-orexin; n = 7), or rAAV-green fluorescent protein (rAAV-GFP; n = 7) into the dorsolateral pons. A group of orexin KO mice that did not receive rAAV (n = 7) and a group of wild-type mice (C57BL/J6; n = 5) were used as controls. Three weeks after rAAV-mediated gene transfer narcolepsy symptoms were examined using sleep and behavioral recordings. Number, location of the orexin-immunoreactive neurons, and relative density of orexin immunoreactive fibers were determined. MEASUREMENTS AND RESULTS Orexin gene transfer into the dorsolateral pons significantly decreased cataplexy and modestly improved wake maintenance compared to the orexin KO mice that did not receive rAAV. In contrast, GFP gene transfer worsened narcoleptic symptoms compared to the no-rAAV orexin KO group. CONCLUSION Orexin gene transfer into the dorsolateral pontine neurons can control cataplexy attacks and modestly improve wake maintenance.

Research over the past 50 years has determined that specific neurons in the brain are responsible for generating waking, non-REM sleep, and REM sleep. Some of the neurons responsible for keeping us awake are also involved in regulating energy metabolism. One such arousal neuronal population contains the neuropeptide hypocretin, also known as orexin. The HCRT neurons are located in the hypothalamus, an area that also contains other neurons regulating energy metabolism. The hypocretin neurons are most active during waking and silent during sleep, and their activity has been shown to regulate brown adipose tissue (BAT) thermogenesis. The hypocretin neurons are also activated by low glucose levels and shut off when the glucose levels increase. Thus, the activity of the hypocretin neurons is linked to energy metabolism. Based on this relationship, it is easy to see how inadequate sleep or even frequent arousals during sleep, as occurs in obstructive sleep apnea, will affect energy metabolism and adiposity.

INTRODUCTION: The incidence of Barrett esophageal adenocarcinoma (BEAC) has been increasing at an alarming rate in western countries. In this study, we have evaluated the therapeutic potential of sulforaphane (SFN), an antioxidant derived from broccoli, in BEAC. METHODS: BEAC cells were treated with SFN, alone or in combination with chemotherapeutic, paclitaxel, or telomerase-inhibiting agents (MST-312, GRN163L), and live cell number determined at various time points. The effect on drug resistance/chemosensitivity was evaluated by rhodamine efflux assay. Apoptosis was detected by annexin V labeling and Western blot analysis of poly(ADP-ribose) polymerase cleavage. Effects on genes implicated in cell cycle and apoptosis were determined by Western blot analyses. To evaluate the efficacy in vivo, BEAC cells were injected subcutaneously in severe combined immunodeficient mice, and after the appearance of palpable tumors, mice were treated with SFN. RESULTS: SFN induced both time- and dose-dependent decline in cell survival, cell cycle arrest, and apoptosis. The treatment with SFN also suppressed the expression of multidrug resistance protein, reduced drug efflux, and increased anticancer activity of other antiproliferative agents including paclitaxel. A significant reduction in tumor volume was also observed by SFN in a subcutaneous tumor model of BEAC. Anticancer activity could be attributed to the induction of caspase 8 and p21 and down-regulation of hsp90, a molecular chaperon required for activity of several proliferation-associated proteins. CONCLUSIONS: These data indicate that a natural product with antioxidant properties from broccoli has great potential to be used in chemoprevention and treatment of BEAC.

Elevated cytokines in bone marrow (BM) micro-environment (interleukin-6 [IL-6], transforming growth factor-beta [TGF-β], and IL-1β) may play an important role in observed immune dysfunction in multiple myeloma (MM). As IL-6 and TGF-β are important for the generation of T-helper 17 (TH17) cells, we evaluated and observed a significantly elevated baseline and induced frequency of Th17 cells in peripheral blood mononuclear cells (PBMCs) and BM mononuclear cells (BMMCs) from MM patients compared with healthy donors. We observed significant increase in levels of serum IL-17, IL-21, IL-22, and IL-23 in blood and BM in MM compared with healthy donors. We also observed that myeloma PBMCs after TH17 polarization significantly induced IL-1α, IL-13, IL-17, and IL-23 production compared with healthy donor PBMCs. We next observed that IL-17 promotes myeloma cell growth and colony formation via IL-17 receptor, adhesion to bone marrow stromal cells (BMSCs) as well as increased growth in vivo in murine xenograft model of human MM. Additionally, we have observed that combination of IL-17 and IL-22 significantly inhibited the production of TH1-mediated cytokines, including interferon-γ (IFN-γ), by healthy donor PBMCs. In conclusion, IL-17–producing Th17 cells play an important role in MM pathobiology and may be an important therapeutic target for anti-MM activity and to improve immune function.

Introduction Narcolepsy is now considered a neurodegenerative sleep disorder characterized by a massive loss of neurons containing the neuropeptide, orexin, also known as hypocretin. Since the orexin neurons have degenerated it is necessary to identify surrogate neurons that can release orexin and thereby ameliorate symptoms of narcolepsy. Can these surrogate orexin neurons be activated to reduce narcolepsy symtpoms? The neurons containing melanin concentrating hormone are particularly interesting as these neurons are silent during waking, which is when cataplexy is triggered. These neurons also project to the same targets as the orexin neurons. Can they be stimulated and does it change sleep- wake time? Materials and methods To answer this question the gene for the light-sensitive cation channel, channelrhodopsin-2, was delivered into the brains of wild-type or the mice models of human narcolepsy. Three weeks after recombinant adeno-associated virus (rAAV)-mediated gene transfer sleep-wake behavior was assessed. Mice were tethered to light- weight swivels and allowed 10 days to acclimate to the EEG cables. Sleep-wake behavior was recorded by polysomnogram and video. Results In-vitro slice electrophysiology studies confirmed that the ChR2 containing neurons were responsive to blue light. In-vivo studies consisted of recording sleep-wake behavior in freely-behaving WT mice ( n = 14) for 48 h. It was found that 10 and 30 Hz optogenetic stimulation of the MCH neurons induced sleep. The effects were seen at night but not the day. At night and day MCH stimulation reduced the length of wake bouts. In the next experiment, the arousal peptide, orexin, was inserted into MCH neurons and light stimulation was used to release it during waking. Research will be presented to show the effects of optogenetic stimulation of this pathway in controlling cataplexy. Conclusion These studies identify novel ways of using emerging new methods to treat sleep disorders. Narcolepsy is especially useful as animal models exist and specific pathways can be selectively manipulated. Acknowledgements Supported by the NIH and the Veterans Administration. We thank Dr. Anthony van den Pol for the MCH promoter.

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

Abstract 446 Waldenstrom's macroglobulinemia (WM), similar to multiple myeloma (MM), is associated with immune dysfunction. Both T and B cell dysfunctions are reported with suppressed uninvolved immunoglobulin, and inadequate vaccine and T cell responses. Although some mechanisms mediating immune dysregulation in WM have been studied, its molecular and cellular basis remains ill defined. Similarly, number of inflammatory cytokines and chemokines has been implicated in this process, but their effect on WM cell growth and immune function has not been well characterized. Recently, TH17 cells, a new CD4 cell population, has been identified by the presence of IL-17. TH17 cells play an important role in auto-immunity and in the development of anti-tumor immunity. As TH17 cells support MM cell growth and induce immune dysfunction in MM, we have evaluated the the role of TH17 cells and associated pro-inflammatory cytokines in WM. We first analyzed T helper cell subsets (TH1, TH2, and TH17) in freshly isolated PBMC from WM, and observed that all three cell types were decreased in WM compared with normal donors. Particularly, the IFN-γ producing TH1 cells from patients with WM were significantly reduced compared to normal donors (11±2% vs 30±3% respectively, P Disclosures: Treon: Millennium Pharmaceuticals, Genentech BiOncology, Biogen IDEC, Celgene, Novartis, Cephalon: Consultancy, Honoraria, Research Funding; Celgene Corporation: Research Funding; Novartis Corporation: Research Funding; Genentech: Consultancy, Research Funding. Munshi:Millennium Pharmaceuticals: Honoraria, Speakers Bureau.

Abstract 456 We have previously demonstrated that IL-17 producing TH17cells, a new subset of T helper cells, are significantly elevated in peripheral blood and bone marrow (BM) from patients with multiple myeloma (MM) and IL-17 produced by these cells promotes MM cell growth and survival, suppresses immune responses and induces osteoclast differentiation both in vitro and in vivo. Based on these observations we have investigated the effects of human anti-IL-17A monoclonal antibody (mAb), AIN-457, in MM. We observed that whereas IL-17A induced proliferation of MM cells (+30.7+2.7%) compared to control; anti-IL-17A mAb AIN-457 significantly inhibited MM cell growth both in presence and absence of BM stromal cells, as measured by thymidine incorporation (−18.7+1.5% and −22.7+2.6% respectively). We have further confirmed these inhibitory effects of anti-IL-17A antibody using MM cell colony forming assay with MethoCult agar plates. While presence of IL-17A increased the colony number from 80 in control plates to 188, presence of AIN-457 reduced the colonies to 60%. We next evaluated the efficacy of AIN-457 in vivo in the murine models of human myeloma; in the subcutaneous MM xenograft model, we observed significant reduction in tumor volumes by pre-treatment with AIN457 compared to control (142+77 mm versus 355+56 mm, p=0.019) while IL-17A significantly increased MM cell growth (727+135 mm, p=0.01). More importantly in the SCIDhu model of human myeloma where MM cells grow within the human microenvironment in the mice, administration of AIN-457 weekly for 4 weeks after the first detection of tumor in mice led to a significant inhibition of tumor growth as measured by human sIL-6 receptor compared to control mice (5.9±2.2 ng/ml versus 23.2±6.3 ng/ml; n=7; P Disclosures: No relevant conflicts of interest to declare.

Multiple Myeloma (MM) is a fatal neoplasm of B-cell origin characterized by the clonal proliferation and accumulation of malignant plasma cells in the bone marrow. While recent advances in mechanistic understanding and treatment modalities have extended median survival to >6 years and 10% of patients survive >10 years, the vast majority of MM remains incurable with conventional, high dose therapy or stem cell transplantation. Bortezomib is a selective, reversible inhibitor of the 26S proteasome that inhibits protein degradation and is now FDA-approved for the treatment of newly diagnosed, relapsed and refractory MM. Though the catabolism of ubiquitinated substrates has been targeted therapeutically with significantly improved prognosis, patient response to bortezomib remains highly variable and cannot be predicted accurately. E3 ligases confer specificity on target selection for Ub+proteasome degradation. We therefore analyzed the expression of individual E3's using a microarray dataset obtained from MM patient tumor samples and found a striking variability in the expression level of individual E3 ligases between normal plasma cells and patients MM cells. RNF4, an E3 specific for poly-sumoylated proteins, was induced in MM patients and correlated with decreased patient response to the proteasome inhibitor bortezomib. Expression profiling of pretreatment tumor samples obtained from MM patients in independent clinical trials were used to generate a signature that correlated expression of SUMO+Ub+Proteasome pathway components with clinical outcome to predict patient response to bortezomib. Experimental validation by overexpression of RNF4-wt rendered myeloma cell lines relatively resistant to bortezomib while RNF4 depletion by shRNA enhanced sensitivity to bortezomib. Transfection of HA-tagged SUMO followed by bortezomib exposure led to the accumulation of HA-SUMO∼conjugates that were immunoreactive with Ub and proteasome components to further link the pathways. In summary, RNF4 an E3 ligase specific for SUMO-conjugates and induced in myeloma, modulates the cellular response to proteasome-based therapy and promotes bortezomib resistance. Our results support regulators of the sumoylation pathway as biomarkers to predict clinical response to bortezomib and provide evidence for targeting SUMO pathway to improve therapeutic outcome in myeloma in general and bortezomib specifically. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1978.

Abstract 1841 Poster Board I-867 Sp1 is a transcription factor with an important role in regulating expression of cell cycle, cell differentiation and apoptosis related genes containing proximal GC/GT-rich promoter elements. It affects growth and metastatic potential of tumor cells. In multiple myeloma (MM), key growth and survival genes such as NF-kB p65, IGF-IR, VEGF, and IL-6 contains proximal GC-rich promoter sequences that interact with Sp proteins to control their expression. Based on this we have evaluated role of Sp1 in MM. We have observed high Sp1 expression along with increased Sp1 activity in MM cells compared to normal peripheral blood mononuclear cells. We have confirmed, by immunohistochemistry, higher nuclear localization of Sp1 in MM cells compared to other normal elements in bone marrow biopsy specimens. Moreover, we have also observed further induction of Sp1 activity shama(≥2 fold increase) after adhesion to BMSC. We next evaluated Sp1 function by analyzing the effect of Sp1 knock down in MM. Interference of Sp1 activity using siRNA showed inhibition of MM cell proliferation (≥75% inhibition) induction of apoptosis and G1 arrest. We have also used three lentiviral shRNA constructs confirming similar effects of Sp1 knock down on MM cells. We have further investigated effects of Sp1 inhibitor Terameprocol on MM cells. Terameprocol is clinical applicable small molecule that specifically competes with Sp1 for specific DNA binding domains within promoter regions. Terameprocol significantly inhibited MM cell growth in a dose-dependent fashion (IC50 beetween 5–20 μM at 24 hours) and was able to overcome the protective effects of BMSCs, inducing apoptosis, via induction of caspases activation and reduction in survivin expression at both gene and protein levels. Based on the data suggesting that both dexamethasone and Velcade increase Sp1 activity, we have combined Terameprocol with these agents and observed synergistic activity. Finally, Terameprocol treatment of nude mice bearing human myeloma xenograft resulted in tumor regression by triggering apoptosis and improving survival of mice. In conclusion, our results confirm significant biological role of Sp1 transcription factor in myeloma that can be therapeutically targeted for clinical development. Disclosures: Mohrland: Erimos Pharmaceuticals: Employment.

Abstract 1852 Poster Board I-878 Multiple myeloma patients suffer from infection related complications. Abnormalities in both cellular and humoral immune responses have been considered responsible. Patients have been routinely immunized with vaccinations to prevent infection related problems, however, efficacy of such vaccination in early or stable myeloma remains unclear. Previously, we have shown immunomodulatory and T cell co-stimulatory effects of lenalidomide, which can up-regulate cellular immune responses in myeloma. Based on these results we initiated a study to evaluate the efficacy of lenalidomide compared to placebo on the effect of Hepatitis B (HepB) vaccination in patients with monoclonal gamopathy of undetermined significance (MGUS), smoldering myeloma or stable multiple myeloma (MM) not requiring any therapy. Patients were randomized to lenalidomide or placebo for 14 days with HepB vaccination on day 8. They were given option for 2nd and 3rd HepB vaccinations at 1 month and 6 month. Primary objective was to evaluate antibody response to Hepatitis Surface antigen (HepBSAg) at 1 month after vaccination. We also measured HepBSAg-specific cellular immune responses using HepBSAg protein and HLA-A2 peptide. At the time of data analysis, the study remains blinded. Thirty two patients have completed their initial vaccination (25 MGUS and 7 MM), while 22 patients (16 MGUS, 6 MM) have completed 3 vaccinations with 6 months follow up. None of the 32 patients, with MGUS or MM, had antibody response to vaccination at 1 month; while after 3 vaccination only 30% patients (7 of 24) demonstrated antibody response to HepBSAg (titer values 128.4±36.4). This is significantly below responses reported in literature in healthy individuals (90%). Responses in patients with MGUS (4 of 16) were not significantly different than in patients with MM (3 of 6). No base line patient characteristics predicts responders vs. non-responders. We have further analyzed HepBSAg-specific T cell immune response by detecting the presence of pentamer-positive CD8 cells with HepB surface antigen-peptide in HLA-A2+ samples. Five of seven responders were HLA-A2 positive, and none of them showed T cell response to HbSAg following vaccination as detected by change in pentamer positive cells. Three patients showed T cell-proliferative responses to HepBsAg; one of which had long term response. None of the non-responders tested demonstrated proliferative response to HepBSAg. The randomization remains blinded at the moment and hence effect of lenalidomide on immune response is not available at the present time. These results have very high clinical significance. It suggests that even in MGUS there is significant and profound functional immune suppression. Strategies to prevent infection and improve immune responses needs to be developed for both preventative purposes as well as for anti-MM vaccinations. Disclosures: Laubach: Novartis: . Richardson:Keryx Biopharmaceuticals: Honoraria. Anderson:Millenium: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding.

Abstract 1105 Poster Board I-127 Genetic recombination plays a critical role in telomere maintenance, chromosomal translocation, and gene amplification, and may therefore underlie the chromosomal aberrations observed with high frequency in number of malignancies. The molecular mechanism/s inducing genomic instability remains ill-defined and their elucidation may provide methods to prevent tumor progression and development of drug resistance. Our earlier work has demonstrated that homologous recombination (HR) activity is elevated in multiple myeloma (MM) cells and leads to increased rate of mutation and progressive accumulation of genetic variation over time. We have further demonstrated that the inhibition of HR activity in MM cells by siRNAs targeting recombinase leads to significant reduction in the acquisition of new genetic changes in the genome; and conversely, induction of HR activity leads to significant elevation in the number of new mutations over time, and development of drug resistance in MM cells. Here we have evaluated a PI3K inhibitor Wortmaninin which has significant inhibitory activity against both HR and non-HR (nHR) pathways. Exposure of MM cells (OPM1, ARP and RPMI 8226) to wortmannin (WM) led to reduced expression of recombinase (hsRAD51) and nearly complete inhibition of HR activity, within 24 hrs as determined by a plasmid based assay in which generation of active gene product by recombination is measured. Similarly nHR was evaluated by measuring generation of intact gene product from a linearized plasmid. We evaluated effect of WM on nHR by 3 hours preincubation before transfecting the plasmid followed by cell culture for 72 hrs at 37° C. Cells were harvested and analyzed for nHR as previously described. Treatment with WM led to >40% reduction in nHR, indicating that WM affects both HR and NHR pathways. Downregulation of these pathways by wortmannin was also associated with a reduced growth rate of myeloma cells in culture by 20-25% at 48 hours. Importantly, WM treatment markedly decreased the acquisition of new genomic changes in MM as measured by genome-wide loss of heterozygosity assay as an indicator of genomic stability. To evaluate the impact of WM on in vivo tumor growth, OPM1 cells were injected subcutaneously in SCID mice and following appearance of palpable tumors, mice were treated with WM at 0.75 mg/kg, injecting daily intraperitoneally. Treatment with WM was associated with almost complete inhibition of tumor growth in vivo. Long term exposure of myeloma cells to WM was consistently associated with reduced telomere length, probably by blocking HR dependent ALT pathway. These data identifies dysregulated recombination activity as a key mediator of DNA instability and progression of MM, and WM as a potential therapeutic agent for prevention of myeloma progression and possibly drug resistance. Disclosures Anderson: Millenium: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Munshi:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis : Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Then focused on the RTK’s which were shown to be upregulated in WM and tested the effect of TKI-258 effect on their activity. Moreover, we tested the effect of TKI-258 survival, proliferation, and apoptosis of WM cells, and its effect of different signaling pathways in WM. Results: We found that epidermal growth factor receptor (EGF-R), ephrin receptor (Eph-R) and receptor tyrosine kinase–like orphan receptor 1 (ROR-1) were highly activated in WM patients compared to normal subjects (5, 10, and 14-fold increase, respectively), and were found to be upregulated also in BCWM1. TKI-258 reduced the activity of EGF-R and Eph-R, but not ROR1. Moreover, TKI-258 induced cytotoxicity, prevented proliferation and induced apoptosis in WM cells in a dose dependent manner with IC50 of 500-800 nM. Moreover, it was shown to induce cleavage of PARP, caspase-3 and caspase-9, and also reduced phosphorylations of ERK1/2. Conclusion: We identified two novel therapeutic targets for WM; EGF-R and Eph-R, and identified Eph-R as a novel target of TKI-258. Moreover, TKI-258 reduced WM progression in vitro, and we are now testing the effect of TKI-258 on WM progression in vitro and alone and in combination with other drugs.

TH17 cells are T helper cells generated from naïve CD4 population in the presence of TGF-beta and IL-6 or IL-1-beta; and expanded in presence of IL-23. TH17 cells predominantly produce IL-17 and/or IL-22 and participate in both development of immunity and autoimmunity. Since TH17 cells are involved in modulation of immune response and we haveobserved both B and T cell immune dysfunction in multiple myeloma (MM), we have further evaluated the role of TH17 cells and associated cytokines in tumor cell growth, as well as, the imbalance of immune homeostasis in myeloma. We first observed that the baseline frequency of TH17 cells was significantly higher in mononuclear cells from MM patient peripheral blood (n=11) (2 folds) and bone marrow (n=4) (50%) compared to normal donor samples. Furthermore, under polarizing conditions with cytokine cocktail, significantly higher number of TH17 cells were induced in presence of TGF-beta in mononuclear cells from MM patients in both blood (4 fold) and bone-marrow (9 fold) compared to normal donor samples. These results indicate that the cytokines present in the BM microenvironment favor the development of TH17 cell subset. We next evaluated the serum levels of TH17-associated cytokines in sera from patients with MM, as well as, normal donors. We observed significant increase in levels of IL-17 (2 fold), IL-21 (5 fold), IL-22 (8 fold) and IL-23 (6 fold) in myeloma (n=17) compared with normal donor (n=6). Importantly, IL-23 levels were 10 fold higher in myeloma BM samples compared with matching blood samples. Next, we evaluated the effect of TH17-associated pro-inflammatory cytokines on MM cell growth and survival. We observed induction of proliferation of 6 MM cell lines, as measured by thymidine incorporation, by IL-17 (30%), IL-21 (35%) and IL-22 (25%). Additionally, IL-17 significantly induced growth of MM cells in clonogenic assay. These growth promoting effects are not observed in the presence of anti-IL-17 antibody. In an effort to evaluate the cellular basis for this growth promoting effect of these cytokines, we investigated production of MM growth promoting cytokines in the BM milieu. We observed induction of IL-6 production by IL-17 from BM stromal cells alone and in the presence of MM cells. Production of IL-6 induced by IL-17 was abrogated (40–50%) by transduction of siRNA targeting IRF-4 into co-culture components (stromal and myeloma cells). The inhibition of IL-17-induced IL-6 production in stromal cells was also observed with JAK-2 (70%) and MEK (76%) inhibitors suggesting role of multiple signalling pathways in induction of IL-6. Finally, we have also observed negative effects of TH17- related cytokines, especially IL-17 and IL-22, on development of IFN-gamma producing TH1 cells suggesting immunosuppressive effect. Taken together these results suggest significant role of IL-17 and related cytokines in myeloma cell growth, as well as, immune dysfunction observed in these patients. This data provides the preclinical rationale to target IL-17 in myeloma to suppress growth as well as improve immune responsiveness.

Stathmin(Op18) is a ubiquitous cytosolic 18Kda protein regulating microtubule (MT) dynamics via binding and stabilizing activities. The activity of Op18 is down regulated during cell cycle by phosphorylation at four Ser residues. The phospho-Op18 is unable to bind to tubulin allowing the progression of the cell cycle. Two additional Op18-binding proteins, KIS prevents Op18 dephosphorylation and activation while iASPP (inhibitor of apoptosis stimulatory protein phosphatase) binds to WT-p53 and inhibits p53-related apoptosis. We have identidfied Op18 as a differentially expressed gene by suppression subtractive hybridization in myeloma. We have also confirmed differential expression of KIS in myeloma cells compared to normal plasma cells. Overexpression of Op18 was confirmed at both RNA and at protein level by RT –PCR and Western blotting in human myeloma cell lines as well as primary samples compared to normal plasma cells and normal human fibroblasts. In contrast, none of the tonsillar CD138+ plasma cells and normal bone marrow mononuclear cells showed Op18 overexpression. To establish the role of Op18 overexpression in MM cell transformation, the human MM cell lines were treated with antisense Op18 oligodeoxynucleotides (ODN). The growth rate of the ODN treated human myeloma cells was significantly reduced compared to the control cells along with cell cycle arrest in G2/M phase and increase in apoptosis as measured by immunohistochemical staining. Additionally, we have silenced Op18 by transfecting Op18-specific siRNA in MM cells and observed that KIS is translocated to cytoplasm, from nucleus as well as MM cells are arrested in G2/M. Importantly, Op18 silencing increased sensitivity of MM cells to microtubule drugs suggesting possible combination approach for therapeutic application. Its role in networking cellular signal transduction pathways in myeloma is under investigation. Recent publication identifying Op18 as one of the 15 most relevant genes determining outcome in myeloma (Avet-loiseau et al, JCO 2008) adds to the validity of Op18 as a molecule playing important role in myeloma cell growth and survival and warrant investigation as a novel therapeutic target.

We have previously demonstrated that a consistent feature of malignant plasma cells of multiple myeloma (MM) is the aberrant expression of genes important in patterning and development, such as members of Hedghehog (Hh) pathway (FE Davies et al, Blood 2003). These findings suggest that overexpression of genes of this pathway, already involved in many solid tumors and recently implicated in maintaining a proposed MM stem cell compartment (CD Peacock et al, PNAS 2007), might be one of the mechanism through which Hh-signaling contributes to tumorigenesis in MM. Therefore, several small molecule modulators of Hh-pathway, which work as agonists and antagonists, are currently under development. We evaluated, by microarray analysis, the expression of Hh pathway genes in MM cell lines and primary MM cells vs. plasma cells from healthy donors. We found that primary MM cells overexpress Sonic (Shh), Smoothened (Smo), Patched (Ptc), Gli-1 and Gli-3 (relative expression ratios ranging from +1.8 to +5.0). Overexpression of Patched was also observed in most of the MM cell lines analyzed (+5.0 ratio in 5 of 6 MM cell lines). Additionally, we confirmed the expression of Shh and of Gli-1, by flow cytometry and western blotting respectively, in a large panel of MM cell lines. These data suggest an activation of the Hh-pathway in MM that, in some cell lines, is Shh-dependent. Therefore, we investigated the therapeutic potential of Hh-inhibitors in MM. We assayed the cell viability and proliferation, by MTT and Thymidine uptake respectively, in 8 MM cell lines after 72 hours of treatment with the small molecule Smo-inhibitor CUR-0199691 (Genentech). We observed a reduction in MM cell viability, with IC50 values ranging between 4.5–9.5 μM in these 8 cell lines and an inhibition of MM cell proliferation with IC50 values ranging between 0.5 and 2.5μM in the same cell lines. MM cell sensitivity to this compound appears to be related to the level of expression of Gli-1, since the cell lines with lower level of expression of Gli-1 were more sensitive. The treatment of these MM cell lines with Cyclopamine, another Hh-inhibitor, showed an IC50 between 7.5μM and 10μM after at least 96 hours of treatment in 4 of the MM cell lines tested. CUR-0199691 is also active in primary MM cells, triggering inhibition of proliferation by 50% at 5μM after only 24h of treatment, while cyclopamine reduces MM cell proliferation (normalized to the effect of tomatidine, its inactive analog) by 30% at 20μM after a 48 hour treatment. Annexin V-PI staining of Hh inhibitor-treated KMS11 cells, which are one of the most sensitive MM cell lines, showed induction of apoptosis, evidenced by detection of 12 and 15% of MM cells being Annexin V+/PI- after 48h and 72h respectively with 5μM of CUR-0199691. These results, taken together, show that the Hh-pathway is fuctionally active in MM and that the novel Hh pathway inhibitor CUR-0199691 is 4–5 times more effective than cyclopamine in both MM cell lines and primary MM cells. These studies provide the framework for further preclinical evaluation of CUR-0199691 in MM models towards possible future clinical trials.

We have identified a panel of 11 novel antigens with induced antibody response in patients with Monoclonal Gammopathy of Undetermined Significance (MGUS) by screening a Multiple Myeloma (MM) cDNA library with patient serum using Serological Analysis of Recombinant Expression cloning (SEREX). Among these antigens, a specific antibody response against OFD1 protein was observed in 6/29 (20.6%) MGUS patients, while 0/12 newly diagnosed, 1/11 (8.3%) relapsed and 3/11 (27.2%) post-transplant MM patients in complete or partial remission showed an antibody response against this protein. Interestingly, in 1 of these patients the antibody response appeared only after autologous transplantation. No responses were observed in 25 healthy donors. OFD1 is widely expressed in embryonic stage with important role in organogenesis; however its expression decreases significantly in adult tissues. Moreover, OFD1 that is involved in the functional regulation of the Hedgehog-pathway, is dysregulated in solid tumors, suggesting a potential role in the tumorigenesis. We have observed the OFD1 expression at high levels in all 11 MM cell lines and 4 primary MM patient cells tested as compared with low or no expression in PBMC and normal bone marrow stromal cells. Specific sequences, derived from three clones corresponding to three different parts of OFD1 protein, were used to identify 15 peptides with potential binding to HLA-A*0201 and selected two peptides (3 and 8) on the basis of their high binding affinity. We observed presence of OFD1 specific CD8+ T cells, as measured by HLA-A2+ pentamers, on all 6 HLA2-A2+ MGUS patients tested. Presence of these cells was also observed in 5 MM patients. However, T cell proliferation following incubation with OFD1 peptides for 7days was observed only in MGUS (=6) while T cell proliferation was not observed in MM (n=5) patients indicating intact OFD1-specific responses in MGUS as compared with MM. These results highlight a possible role of OFD1 in the immune regulation in MGUS and provide the framework for further evaluation of OFD1 as a new potential target for immunotherapeutic approaches in both MGUS and MM.

Multiple myeloma (MM) is associated with significant immune dysfunction. Although various mechanisms mediating immune dysregulation in MM have been studied, its molecular and cellular basis is ill defined. IL-6, TGF-β and IL-1β have been implicated in this process, but their mechanism of effects on immune function have not been studied in MM. Together, IL-6 and TGF-β enhance the generation of TH17 cells, important in the development of immunity and auto-immunity. Additionally, TH17 cells are differentiated by number of inflammatory cytokines including, IL-21, IL-22, IL-23, and IL-27. Therefore, we evaluated the immune dysfunction and the role of TH17 cells and associated pro-inflammatory cytokines in myeloma. We have previously characterized that the production of TH1 mediated cytokines including IFN-γ following anti-CD3-mediated activation is significantly lower in myeloma PBMC compared to normal PBMC. We hypothesize that this may be regulated via skewing the immune system towards TH17 pathway. We observed that TH17 cells, measured by intra-cellular flow cytometry, are significantly increased in number in myeloma (16.9%) and MGUS (6.2%) compared to normal (3.3%). Furthermore, we analysed supporting pro-inflammatory cytokine network for the generation of TH17 cells in myeloma, which may be responsible for the observed TH17 skewing of T cell subsets. Sera from MGUS (n=12) and myeloma (n=17) patients were evaluated for the presence of these pro-inflammatory cytokines compared with normal sera (n=6) using ELISA. We observed significant increase in serum IL-21, IL-22 and IL-23 in MGUS (373 pg/ml, 14 pg/ml and 147 pg/ml respectively; p

The pursuit of rationale, targeted therapies relies on a detailed understanding of the mechanisms that subvert normal growth control and lead to development of Multiple Myeloma (MM). To further define the mechanistic steps that contribute to MM pathogenesis, we examined mRNA expression profiles of CD138+ plasma cells obtained from normal, Monoclonal Gammopathy of Unknown Significance (MGUS), and MM patient samples. Using genomic results in combination with molecular and cellular-based assays, we demonstrate a critical role for UBC9 and the sumoylation pathway in myeloma cell growth and survival. Notably, Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) demonstrated a ten-fold induction in UBC9; a gene that encodes the sole Sumo-conjugating enzyme in human cells. We demonstrated an elevation of UBC9 in both MM primary patient cells and in a number of patient-derived MM cell lines. In addition, a number of other sumoylation pathway components were induced in primary MM cells at the gene and protein level relative to normal plasma cells. We believe that induction of UBC9 is an early genetic event in the pathogenesis of MM since the induction was observed at the gene and protein level in plasma cells from patients with MGUS. Importantly, UBC9 induction was functionally significant since a different pattern of sumoylation was observed in total cell lysate from MM patient plasma cells relative to that of normal plasma cells. Furthermore, overexpresion of a mutant form of UBC9 deficient in Sumo-conjugating activity increased the sensitivity of plasma cells to apoptosis by chemotherapeutic agents and these cells were impaired in other essential functions that included cellular proliferation, DNA synthesis, resistance to apoptosis and adhesion to bone marrow stroma. Immunoblotting of MM patient cell lysates also demonstrated a similar induction of the UBC9 gene product (Ubc9) as well as induction of the Sumo ligases Nse2 and PIAS1. These studies identify UBC9 as a target upregulated early in the pathogenesis of MM and indicate a critical role for sumoylation in disrupting the controls that govern normal plasma cell growth. To further develop prognostically relevant molecular signatures and classifications of MM subtypes, we analyzed the survival outcome of patients that expressed induced levels of UBC9, as well as other sumoylation components, and demonstrate significantly reduced survival of such patients following current treatment modalities. The results provide evidence for critical role of UBC9 and sumoylation in MM pathogenesis. Furthermore, sumoylation pattern may serve as a therapeutic target in MM, help stratify clinical management and provide a framework for the identification of sumoylation pathway targets that govern MM cell growth and progression.