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Epigenetic modifiers frequently harbor loss-of-function mutations in lung cancer, but their tumor-suppressive roles are poorly characterized. Histone methyltransferase KMT2D (a COMPASS-like enzyme, also called MLL4) is among the most highly inactivated epigenetic modifiers in lung cancer. Here we show that lung-specific loss of Kmt2d promotes lung tumorigenesis in mice and upregulates pro-tumorigenic programs, including glycolysis. Pharmacological inhibition of glycolysis preferentially impedes growth of human lung cancer cells bearing KMT2D-inactivating mutations. Mechanistically, Kmt2d loss widely impairs epigenomic signals for super-enhancers/enhancers, including the super-enhancer for the circadian rhythm repressor Per2. Loss of Kmt2d decreases expression of PER2, which regulates multiple glycolytic genes. These findings indicate that KMT2D is a lung tumor suppressor and KMT2D deficiency confers a therapeutic vulnerability to glycolytic inhibitors. SIGNIFICANCE: Lung cancer is the leading cause of cancer deaths. The overall survival rate for lung cancer patients remains low despite recent therapeutic advances, and a majority of lung cancer patients lack a druggable target. Therefore, there is a great need for a better understanding of the molecular mechanisms driving lung cancer. Epigenetic modifiers are frequently lost in lung cancer, but how this provokes lung tumorigenesis remains unclear. We show that KMT2D, which is recurrently mutated in lung cancer, is a lung tumor suppressor. Our results uncover a tumor-promoting epigenetic mechanism by which KMT2D-inactivating mutations induce aberrant metabolic reprogramming via super-enhancer impairment in lung cancer. Our findings support a glycolysis-inhibitory approach as a therapeutic intervention strategy against KMT2D-mutant lung cancer.

Chromatin deregulation is an emerging hallmark of cancer. However, the extent of epigenetic aberrations during tumorigenesis and their relationship with genetic aberrations are poorly understood. Using ChIP-sequencing for enhancers (H3K27ac and H3K4me1), promoters (H3K4me3), active transcription (H3K79me2) and polycomb (H3K27me3) or heterochromatin (H3K9me3) repression we generated chromatin state profiles in metastatic melanoma using 46 tumor samples and cell lines. We identified a strong association of NRAS, but not BRAF mutations, with bivalent states harboring H3K4me3 and H3K27me3 marks. Importantly, the loss and gain of bivalent states occurred on important pro-metastasis regulators including master transcription factor drivers of mesenchymal phenotype including ZEB1, TWIST1, SNAI1 and CDH1. Unexpectedly, a subset of these and additional pro-metastatic drivers (e.g. POU3F2, SOX9 and PDGFRA) as well as melanocyte-specific master regulators (e.g. MITF, ZEB2, and TFAP2A) were regulated by exceptionally wide H3K4me3 domains that can span tens of thousands of kilobases suggesting roles of this new epigenetic element in melanoma metastasis. Overall, we find that BRAF, NRAS and WT melanomas may use bivalent states and broad H3K4me3 domains in a specific manner to regulate pro-metastatic drivers. We propose that specific epigenetic traits – such as bivalent and broad domains – get assimilated in the epigenome of pro-metastatic clones to drive evolution of cancer cells to metastasis.

SUMMARY The dynamic evolution of chromatin state patterns during metastasis, their relationship with bona fide genetic drivers, and their therapeutic vulnerabilities are not completely understood. Combinatorial chromatin state profiling of 46 melanoma samples reveals an association of NRAS mutants with bivalent histone H3 lysine 27 trimethylation (H3K27me3) and Polycomb repressive complex 2. Reprogramming of bivalent domains during metastasis occurs on master transcription factors of a mesenchymal phenotype, including ZEB1, TWIST1, and CDH1. Resolution of bivalency using pharmacological inhibition of EZH2 decreases invasive capacity of melanoma cells and markedly reduces tumor burden in vivo, specifically in NRAS mutants. Coincident with bivalent reprogramming, the increased expression of pro-metastatic and melanocyte-specific cell-identity genes is associated with exceptionally wide H3K4me3 domains, suggesting a role for this epigenetic element. Overall, we demonstrate that reprogramming of bivalent and broad domains represents key epigenetic alterations in metastatic melanoma and that EZH2 plus MEK inhibition may provide a promising therapeutic strategy for NRAS mutant melanoma patients., Graphical abstract, In brief Terranova et al. provide a comprehensive epigenome resource for melanoma encompassing 284 chromatin maps. They find key regulatory roles for bivalent and broad domains in expression of pro-metastatic genes and identify EZH2 plus MEK inhibition as a therapeutic strategy for NRAS mutant melanomas.

Epigenetic modifiers often harbor loss-of-function mutations in lung cancer, but their tumor-suppressive roles are poorly characterized. Here we show that lung-specific loss of the gene encoding the histone methyltransferase MLL4 (alias KMT2D; a COMPASS-like enzyme), which is ranked the most highly inactivated epigenetic modifier in lung cancer, strongly promotes lung adenocarcinoma in mice. Mll4 loss upregulated tumor-promoting programs, including glycolysis. The pharmacological inhibition of glycolysis preferentially impeded tumorigenic growth of human lung cancer cells bearing MLL4-inactivating mutations. Mll4 loss widely impaired epigenomic signals for super-enhancers and enhancers, including the super-enhancer for the circadian rhythm repressor gene Per2, and decreased Per2 expression. Per2 downregulated several glycolytic pathway genes. These findings uncover a distinct tumor-suppressive epigenetic mechanism in which MLL4 enhances Per2-mediated repression of pro-tumorigenic glycolytic genes via super-enhancer activation to suppress lung adenocarcinoma tumorigenesis and also implicate a glycolysis-targeting strategy as a therapeutic intervention for the treatment of MLL4- mutant lung cancer.

SUMMARY Histone methyltransferase KMT2D harbors frequent loss-of-function somatic point mutations in several tumor types, including melanoma. Here, we identify KMT2D as a potent tumor suppressor in melanoma through an in vivo epigenome-focused pooled RNAi screen and confirm the finding by using a genetically engineered mouse model (GEMM) based on conditional and melanocyte-specific deletion of KMT2D. KMT2D-deficient tumors show substantial reprogramming of key metabolic pathways, including glycolysis. KMT2D deficiency aberrantly upregulates glycolysis enzymes, intermediate metabolites, and glucose consumption rates. Mechanistically, KMT2D loss causes genome-wide reduction of H3K4me1-marked active enhancer chromatin states. Enhancer loss and subsequent repression of IGFBP5 activates IGF1R-AKT to increase glycolysis in KMT2D-deficient cells. Pharmacological inhibition of glycolysis and insulin growth factor (IGF) signaling reduce proliferation and tumorigenesis preferentially in KMT2D-deficient cells. We conclude that KMT2D loss promotes tumorigenesis by facilitating an increased use of the glycolysis pathway for enhanced biomass needs via enhancer reprogramming, thus presenting an opportunity for therapeutic intervention through glycolysis or IGF pathway inhibitors., In Brief Through an in vivo epigenome-focused pooled RNAi screen, Maitituoheti et al. identify KMT2D as a tumor suppressor in melanoma. KMT2D-deficient tumors show substantial reprogramming of key metabolic pathways by reduction of H3K4me1-marked active enhancers, conferring sensitivity to glycolysis and IGFR inhibitors in melanoma with KMT2D-inactivating mutations., Graphical Abstract

Over the last few years, a detailed map of genetic and epigenetic lesions that underlie multiple myeloma (MM) has been created. Regulation of microRNA (miR)-dependent gene expression and mRNA splicing play significant roles in MM pathogenesis; however, to date an interplay between these processes is not yet delineated. Here we investigated miR-mediated regulation of splicing networks at the transcriptome level. Our studies show that a significant number (78%) of miRs which are either up- or down-regulated in patient CD138+ MM cells, but not in healthy donors (HD) CD138+ plasma cells (PC), target genes involved in early stages of pre-mRNA splicing. We also identified deregulated miRs that target core splicing factors (SF) and modifiers (SM, enhancers/silencers) which cause altered splicing in MM. Our studies suggest that Let-7f, in combination other miRs which are frequently and significantly deregulated in patients with overt MM, targets genes that regulate intron excision. Importantly, deregulated expression of certain miRs in MM promote increased intron retention, a novel characteristic of the MM genome, by inducing deregulated expression of the genes that regulate the splicing network. Our studies, therefore, provide the rationale for therapeutically targeting deregulated miRs to reverse aberrant splicing and improve patient outcome in MM.

Discovery of cancer drivers has traditionally focused on the identification of protein-coding genes. Here we present a comprehensive analysis of putative cancer driver mutations in both protein-coding and non-coding genomic regions across >2,500 whole cancer genomes from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium. We developed a statistically rigorous strategy for combining significance levels from multiple driver discovery methods and demonstrate that the integrated results overcome limitations of individual methods. We combined this strategy with careful filtering and applied it to protein-coding genes, promoters, untranslated regions (UTRs), distal enhancers and non-coding RNAs. These analyses redefine the landscape of non-coding driver mutations in cancer genomes, confirming a few previously reported elements and raising doubts about others, while identifying novel candidate elements across 27 cancer types. Novel recurrent events were found in the promoters or 5’UTRs ofTP53, RFTN1, RNF34,andMTG2,in the 3’UTRs ofNFKBIZandTOB1,and in the non-coding RNARMRP.We provide evidence that the previously reported non-coding RNAsNEAT1andMALAT1may be subject to a localized mutational process. Perhaps the most striking finding is the relative paucity of point mutations driving cancer in non-coding genes and regulatory elements. Though we have limited power to discover infrequent non-coding drivers in individual cohorts, combined analysis of promoters of known cancer genes show little excess of mutations beyondTERT.

miRs play a critical role in tumor pathogenesis as either oncogenes or tumor-suppressor genes. However, the role of miRs and their regulation in response to proteasome inhibitors in multiple myeloma (MM) is unclear. In the current study, miR profiling in proteasome inhibitor MLN2238-treated MM.1S MM cells shows up-regulation of miR33b. Mechanistic studies indicate that the induction of miR33b is predominantly via transcriptional regulation. Examination of miR33b in patient MM cells showed a constitutively low expression. Overexpression of miR33b decreased MM cell viability, migration, colony formation, and increased apoptosis and sensitivity of MM cells to MLN2238 treatment. In addition, overexpression of miR33b or MLN2238 exposure negatively regulated oncogene PIM-1 and blocked PIM-1 wild-type, but not PIM-1 mutant, luciferase activity. Moreover, PIM-1 overexpression led to significant abrogation of miR33b- or MLN2238-induced cell death. SGI-1776, a biochemical inhibitor of PIM-1, triggered apoptosis in MM. Finally, overexpression of miR33b inhibited tumor growth and prolonged survival in both subcutaneous and disseminated human MM xenograft models. Our results show that miR33b is a tumor suppressor that plays a role during MLN2238-induced apoptotic signaling in MM cells, and these data provide the basis for novel therapeutic strategies targeting miR33b in MM.

Purpose: The transcription factor specificity protein 1 (Sp1) controls number of cellular processes by regulating the expression of critical cell cycle, differentiation, and apoptosis-related genes containing proximal GC/GT-rich promoter elements. We here provide experimental and clinical evidence that Sp1 plays an important regulatory role in multiple myeloma (MM) cell growth and survival. Experimental Design: We have investigated the functional Sp1 activity in MM cells using a plasmid with Firefly luciferase reporter gene driven by Sp1-responsive promoter. We have also used both siRNA- and short hairpin RNA–mediated Sp1 knockdown to investigate the growth and survival effects of Sp1 on MM cells and further investigated the anti-MM activity of terameprocol (TMP), a small molecule that specifically competes with Sp1-DNA binding in vitro and in vivo. Results: We have confirmed high Sp1 activity in MM cells that is further induced by adhesion to bone marrow stromal cells (BMSC). Sp1 knockdown decreases MM cell proliferation and induces apoptosis. Sp1-DNA binding inhibition by TMP inhibits MM cell growth both in vitro and in vivo, inducing caspase-9–dependent apoptosis and overcoming the protective effects of BMSCs. Conclusions: Our results show Sp1 as an important transcription factor in myeloma that can be therapeutically targeted for clinical application by TMP. Clin Cancer Res; 17(20); 6500–9. ©2011 AACR.

The transformation from monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma (MM) is thought to be associated with changes in immune processes. We have therefore used serologic analysis of recombinant cDNA expression library to screen the sera of MGUS patients to identify tumor-associated antigens. A total of 10 antigens were identified, with specific antibody responses in MGUS. Responses appeared to be directed against intracellular proteins involved in cellular functions, such as apoptosis (SON, IFT57/HIPPI), DNA and RNA binding (ZNF292, GPATCH4), signal transduction regulators (AKAP11), transcriptional corepressor (IRF2BP2), developmental proteins (OFD1), and proteins of the ubiquitin-proteasome pathway (PSMC1). Importantly, the gene responsible for the oral-facial-digital type I syndrome (OFD1) had response in 6 of 29 (20.6%) MGUS patients but 0 of 11 newly diagnosed MM patients. Interestingly, 3 of 11 (27.2%) MM patients after autologous stem cell transplantations showed responses to OFD1. We have confirmed T-cell responses against OFD1 in MGUS and observed down-regulation of GLI1/PTCH1 and p-β-catenin after OFD1 knock-down with specific siRNA, suggesting its functional role in the regulation of Hh and Wnt pathways. These findings demonstrate OFD1 as an important immune target and highlight its possible role in signal transduction and tumorigenesis in MGUS and MM.

PREX2 (phosphatidylinositol-3,4,5-triphosphate-dependent Rac-exchange factor 2) is a PTEN (phosphatase and tensin homolog deleted on chromosome 10) binding protein that is significantly mutated in cutaneous melanoma and pancreatic ductal adenocarcinoma. Here, genetic and biochemical analyses were conducted to elucidate the nature and mechanistic basis of PREX2 mutation in melanoma development. By generating an inducible transgenic mouse model we showed an oncogenic role for a truncating PREX2 mutation (PREX2(E824)*) in vivo in the context of mutant NRAS. Using integrative cross-species gene expression analysis, we identified deregulated cell cycle and cytoskeleton organization as significantly perturbed biological pathways in PREX2 mutant tumors. Mechanistically, truncation of PREX2 activated its Rac1 guanine nucleotide exchange factor activity, abolished binding to PTEN and activated the PI3K (phosphatidyl inositol 3 kinase)/Akt signaling pathway. We further showed that PREX2 truncating mutations or PTEN deletion induces down-regulation of the tumor suppressor and cell cycle regulator CDKN1C (also known as p57(KIP2)). This down-regulation occurs, at least partially, through DNA hypomethylation of a differentially methylated region in chromosome 11 that is a known regulatory region for expression of the CDKN1C gene. Together, these findings identify PREX2 as a mediator of NRAS-mutant melanoma development that acts through the PI3K/PTEN/Akt pathway to regulate gene expression of a cell cycle regulator.

Multiple myeloma is a hematological cancer of plasma B cells and remains incurable. Two major subtypes of myeloma, hyperdiploid MM (HMM) and non-hyperdiploid MM (NHMM), have distinct chromosomal alterations and different survival outcomes. Transcription factors (TrFs) have been implicated in myeloma oncogenesis, but their dysregulation in myeloma subtypes are less studied. Here, we developed a TrF-pathway coexpression analysis to identify altered coexpression between two sample types. We apply the method to the two myeloma subtypes and the cell cycle arrest pathway, which is significantly differentially expressed between the two subtypes. We find that TrFs MYC, nuclear factor-κB and HOXA9 have significantly lower coexpression with cell cycle arrest in HMM, co-occurring with their overactivation in HMM. In contrast, TrFs ESR1 (estrogen receptor 1), SP1 and E2F1 have significantly lower coexpression with cell cycle arrest in NHMM. SP1 chromatin immunoprecipitation targets are enriched by cell cycle arrest genes. These results motivate a cooperation model of ESR1 and SP1 in regulating cell cycle arrest, and a hypothesis that their overactivation in NHMM disrupts proper regulation of cell cycle arrest. Cotargeting ESR1 and SP1 shows a synergistic effect on inhibiting myeloma proliferation in NHMM cell lines. Therefore, studying TrF-pathway coexpression dysregulation in human cancers facilitates forming novel hypotheses toward clinical utility.

Background Genome-wide expression signatures are emerging as potential marker for overall survival and disease recurrence risk as evidenced by recent commercialization of gene expression based biomarkers in breast cancer. Similar predictions have recently been carried out using genome-wide copy number alterations and microRNAs. Existing software packages for microarray data analysis provide functions to define expression-based survival gene signatures. However, there is no software that can perform survival analysis using SNP array data or draw survival curves interactively for expression-based sample clusters. Results We have developed the survival analysis module in the dChip software that performs survival analysis across the genome for gene expression and copy number microarray data. Built on the current dChip software's microarray analysis functions such as chromosome display and clustering, the new survival functions include interactive exploring of Kaplan-Meier (K-M) plots using expression or copy number data, computing survival p-values from the log-rank test and Cox models, and using permutation to identify significant chromosome regions associated with survival. Conclusions The dChip survival module provides user-friendly way to perform survival analysis and visualize the results in the context of genes and cytobands. It requires no coding expertise and only minimal learning curve for thousands of existing dChip users. The implementation in Visual C++ also enables fast computation. The software and demonstration data are freely available at http://dchip-surv.chenglilab.org.

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.

Alternative splicing is a crucial mechanism for gene regulation, which enhances the diversity of transcriptome and proteome. Misregulation of alternative splicing has been implicated in number of disease processes including cancer. Our data utilizing exon array profile from 170 uniformly treated newly diagnosed patients with MM confirms clinical relevance of splicing as demonstrated by impact of level and extent of alternate splicing on both progression free and overall survival. Fox2, a RNA splicing factor, is one of the most important genes predicting clinical outcome in these patients. We confirmed Fox2 expression in 10 MM cell lines at both RNA and protein levels. Immunohistochemistry staining showed a predominant nuclear localization of Fox2. Importantly, we also observed that MM cell - bone marrow stromal cells (BMSC) interaction led to significant inhibition of Fox2 expression in MM cells. Similar response was also observed using BMSC supernatants. While IL6 treatment significantly downregulated the expression of Fox2 in MM1S and RPMI8226 cells in a dose-dependent manner, IGF-1 treatment had no significant impact on Fox2 expression in MM cell lines. Since Fox2 has been described to plays a role in the maintenance of cell cytoskeleton, we therefore evaluated whether Fox2 might influence the migration and adhesion in MM cells. Transwell migration assay showed enhanced migration rate of Fox2-knocking down- MM1S and RPMI8226 cells versus controls. We also observed the increased cell adhesion to fibronetin in both cell lines upon Fox2 knockdown. Actin polymerization evaluated by Alexa488-conjugated phalloidin staining and confocal microscope analysis showed Fox2 knocking down cells with increased actin polymerization in both MM1S and RPMI8226 cell lines. Interstingly, we observed that Fox2 knockdown in MM cell lines did not affect the cell proliferation and survival. As Fox-2 is a splicing factor, we further evaluated the molecular impact of Fox2 expression in multiple myeloma by RNA-seq analysis. Our data revealed that Fox2 functions in regulating both protein-coding and non-coding RNA alternative splicing. Knockdown of Fox2 resulted in significant isoform up-regulation (60 in MM1S and 151 in RPMI8226) and down-regulation (70 in MM1S and 69 in RPMI8226). Gene enrichment analysis showed these genes are clustered in cell cytoskeleton regulation, microtubule-based movement, ATP binding, amongst others. Our study then focused on Fox2 knockdown-induced significant isoform switch in MM cell lines. We designed the primers testing the spliced exons and confirm the isoform switch in MM cells by PCR analysis (e.g. Pyk2 and PFDN6). Importantly, our RNA seq data showed that Fox2 regulates the expression of a series of microRNAs and long-noncoding RNAs (e.g. MALAT1 RPMI8226 CNT (58) vs Fox2 knockdown (108)), which provides us a new insight into impact of Fox2 on non-protein coding RNAs. We have also validated the RNA-seq data by Q-PCR analysis. In summary, our results identify Fox2 as a biologically important RNA binding protein that is regulated by bone marrow microenvironment interaction and with essential function and potential clinical implications in multiple myeloma. Disclosures No relevant conflicts of interest to declare.

Alternate splicing is an important post translational change that alters specificity of gene function. Misregulation of alternative splicing has been implicated in number of disease processes including cancer. We have analyzed alternate splicing in myeloma using high throughput GeneChip Human Exon 1.0 ST Arrays in 170 uniformly treated patients and identified pattern of splicing as well as their impact on both overall and event free survival in myeloma. We have now further analyzed this data and identified Fox2, a RNA alternative splicing regulator, as one of the most important genes predicting clinical outcome in these patients. We observe that the expression level of Fox2 correlates with the frequency of RNA splicing and disease prognosis in MM patients. We have now further investigated the molecular role of Fox2 in myeloma. Fox2 expression was detected in all 10 MM cell lines tested at both RNA and protein levels. Immunohistochemistry staining showed a predominant nuclear localization of Fox2. We next evaluated impact of IL-6 on Fox2 expression in MM1S and RPMI8226 MM cell lines and observed dose-dependent reduction in Fox2 expression. Importantly, MM cell - bone marrow stromal cells (BMSC) interaction also led to significant inhibition of Fox2 expression in MM1S and RPMI8226 cells. Similar response was also observed using BMSC supernatants. On the other hand, IGF-1 stimulation showed slight upregulation of Fox2 in MM cell lines. We have also evaluated impact of IL-6 on Fox2 and splicing using genomewide RNA-seq and confirmed the results. Fox2 was downregulated 33% in MM1S and 37% in RPMI8226 at gene expression level. To study its role in MM, we knocked down the expression of Fox2 in MM1S and RPMI8226 cell lines by using Fox2-directed siRNA. Compared to control cell lines, Fox2 knockdown in MM cell lines did not affect the cell proliferation and survival, as measured by cell titer glo luminescent cell viability assay and annexin V and PI staining respectively. Since Fox2 has been described to plays a role in the maintenance of cell cytoskeleton, we therefore evaluated whether Fox2 might influence the migration and adhesion in MM cells. Transwell migration assay showed enhanced migration rate of Fox2-knocking down- MM1S and RPMI8226 cells versus controls. We also observed the increased cell adhesion to fibronetin in both cell lines upon Fox2 knockdown. Actin polymerization evaluated by Alexa488-conjugated phalloidin staining and confocal microscope analysis showed Fox2 knocking down cells with increased actin polymerization in both MM1S and RPMI8226 cell lines. Currently, RNA seq data following Fox2 knock down in MM cell lines is being evaluated to define the molecular mechanisms of bone marrow microenvironment-mediated Fox2-regualted alternative splicing events in MM. In summary, our results identify Fox2 as a biologically important splicing factor with essential function and potential clinical implications in multiple myeloma. Disclosures: No relevant conflicts of interest to declare.

2386 Multiple myeloma (MM) is a clonal plasma cell malignancy with a heterogeneous genetic background. Extensive gene expression profile analysis have provided interesting insight into the disease biology and its correlation with clinical outcome; however, we have begun to realize the significant limitations of expression profile data alone. Therefore there is a growing understanding that additional genomic correlates need to be incorporated to develop an integrated oncogenomic analysis. We have here developed and defined multi-gene transcriptional and post-transcriptional feed-forward loop (FFL) These conceptual FFLs consist of a master TF which regulates a miR and together with it controls a set of specific common gene/s. These recurrent and important network motifs form functional nodes in the larger regulatory network, and are considered linchpins of disease causing genomic alterations in cancer and MM in particular. We have developed a comprehensive novel integrative analysis method, dChip-GemiNI (Gene and miRNA Network-based Integration), which combines gene and miR expression profiles, and also incorporates regulatory network structure in the form of computationally identified TF–miRNA FFLs. The dChip-GemiNI method statistically ranks computationally predicted FFLs by their explanatory power to account for differential gene and miRNA expression. We have next applied dChip-GemiNi to a training dataset of 60 MM patients and 5 normal plasma cells (NPCs) with both gene expression (GE) and miR profiles (dataset [GSE16558][1]) in order to identify FFLs containing TF-miR-gene networks with loss of negative feedback regulation in MM, supporting the uncontrolled growth, anti-apoptosis and/or other oncogenic effects. We have identified 20 FFLs significantly aberrant between NPC and MM cells. Prominent FFLs involve known MM dysregulated TFs such as MYC, TP53 and Sp1. In addition, we have utilized 3 available myeloma datasets with both miR and GE profiles ([GSE16558][1], [GSE17306][2], [GSE17498][3]), and classified MM samples into hyperdiploid MM (HMM) and non-hyperdiploid MM (NHMM) subtype groups by GE profiles at an accuracy >85%. These two groups have different survival outcomes (p-value < 0.01). We have identified 55 FFLs altered between these two MM subtypes. In particular we have observed that the FFL involving CREB1- miR-20a and target genes RRAGD, PIP4K2A, RHOC and CCND2 is altered between HMM and NHMM and is common between the 3 datasets. We have now begun to statistically ranks computationally predicted FFLs and develop a motif score to develop an integrated risk stratification model. In conclusion, FFLs form critical regulatory loops driving the functional behavior of MM cells. Analyzing the molecular impact of FFLs as a unit combining the aggregate impact of TF-miR and the target gene/s in MM will be instrumental in understanding the biology of the disease, developing clinically relevant integrated risk models, and translating basic research into targeted therapy. The ultimate goal is to develop strategies to regulate homeostatic control of these loops and overcome their oncogenic effects that drive the malignant phenotype. Disclosures: Munshi: Celgene: Consultancy; Millenium: Consultancy; Merck: Consultancy; Onyx: Consultancy. [1]: /lookup/external-ref?link_type=NCBIGEO&access_num=GSE16558&atom=%2Fbloodjournal%2F120%2F21%2F2386.atom [2]: /lookup/external-ref?link_type=NCBIGEO&access_num=GSE17306&atom=%2Fbloodjournal%2F120%2F21%2F2386.atom [3]: /lookup/external-ref?link_type=NCBIGEO&access_num=GSE17498&atom=%2Fbloodjournal%2F120%2F21%2F2386.atom

We describe here a novel method for integrating gene and miRNA expression profiles in cancer using feed-forward loops (FFLs) consisting of transcription factors (TFs), miRNAs and their common target genes. The dChip-GemiNI (Gene and miRNA Network-based Integration) method statistically ranks computationally predicted FFLs by their explanatory power to account for differential gene and miRNA expression between two biological conditions such as normal and cancer. GemiNI integrates not only gene and miRNA expression data but also computationally derived information about TF–target gene and miRNA–mRNA interactions. Literature validation shows that the integrated modeling of expression data and FFLs better identifies cancer-related TFs and miRNAs compared to existing approaches. We have utilized GemiNI for analyzing six data sets of solid cancers (liver, kidney, prostate, lung and germ cell) and found that top-ranked FFLs account for ∼20% of transcriptome changes between normal and cancer. We have identified common FFL regulators across multiple cancer types, such as known FFLs consisting of MYC and miR-15/miR-17 families, and novel FFLs consisting of ARNT, CREB1 and their miRNA partners. The results and analysis web server are available at http://www.canevolve.org/dChip-GemiNi.

Abstract 1929 Considerable efforts have been spent evaluating impact of global gene expression profile on clinical outcome. Although significant correlation has been described between outcome and expressed gene signature, the overall predictability of such models have reached a plateau. Biologically this is expected as gene function is modulated at multiple levels. Besides the change in level of expression, post-transcriptional changes such as alternate splicing alter specificity of gene function and may affect the eventual outcome. Although various genes have normal alternate spliced form, dysregulation of alternative splicing that alters protein function has been implicated in number of disease processes including cancer. We have observed significant level of dysregulated splicing events in multiple myeloma (MM). We hypothesize that a combined model that includes dysregulated splicing events, besides level of expressed genes, may provide superior survival model in MM. To develop a combined model we have hybridized RNA isolated from CD138+ purified MM cells collected at the time of diagnosis from 170 newly-diagnosed patients treated homogeneously in tandem transplantation IFM trials, 23 MM cell lines and 6 Healthy donors on Affymetrix Exon 1.0 ST GeneChip arrays. Exon array not only provides an accurate measure of expression levels for genes, but also allows simultaneous identification of alternative splicing events. Pre-processing and normalization methods in aroma, affymetrix and robust multichip analysis model in FIRMA, followed by t-tests with Benjamini-Hochberg multiple hypothesis corrections were used respectively to identify differential expression and alternative splicing. We identified 1454 differentially expressed genes and 759 differential splicing events between healthy donors and MM patients, and 5476 differentially expressed genes and 4012 differential splicing events between healthy donors and MM cell lines. There are 1071 differentially expressed genes and 286 alternative spliced exons shared between MM samples and MM cell lines. Univariate survival analysis using FIRMA scores of exons identified a total of 89 genes with more than 10 alternative splicing events between healthy donors and MM patients associated with survival with Cox proportional hazard model and log-rank tests. We have now built 3 different survival models considering: 1) gene expression only, 2) alternative splicing only, and 3) a combined model integrating gene expression and alternative splicing events. We utilized refined regularized variable selection methods to handle these high-dimensional feature space. Our analysis suggests that composite model using gene expression and alternative splicing information performs significantly better than the gene expression only model in identifying high-risk patients, when the data were divided in median or quartiles. Specifically, the difference in overall survival is 32.6 months to 38.5 months using the median survival, and 18 months vs 23 months for median event free survival. We are currently in the process of validating the combined model. Our data suggests the need for inclusion of modifiers of transcriptome to develop a comprehensive model that will have higher predicative power for risk stratification as well as for selection of therapeutic intervention. Disclosures: Anderson: Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Munshi:Millennium Pharmaceuticals: Honoraria, Speakers Bureau.

Abstract 306 Current therapy for multiple myeloma (MM) remains empiric. With advancement in the understanding of its molecular basis, newer therapies are emerging faster than ever with increasing the difficulty in the selection of treatment regime to maximize response and minimize the rising cost of therapy. In recent years, treatment response prediction using gene expression profiling is being evaluated to identify expression signature that can classify patients likely to benefit from chemotherapy, e.g., there are several multi-gene expression assays available to predict treatment responses. However, expression signatures and predictive power vary significantly among these assays. Here, we have assessed the ability of gene expression profile to predict complete response (CR) in patients with MM. We evaluated 128 newly-diagnosed patients with MM enrolled on IFM protocol and treated uniformly with high-dose melphalan followed by autologous stem cell transplant. Seventy one of 128 patients (56%) had achieved CR while the rest 57 (44%) had partial response (PR) or less to this therapeutic intervention. CD138+ MM cells collected at the time of diagnosis were profiled for gene expression and processed using the dChip and aroma.affymetrix module in R software. We have used all common machine learning packages in R/Bioconductor and BRB-array Tools software to build response signature models; the packages used but not limited to were: Decision tree, Support Vector Machines (SVM), Prediction Analysis of Microarray (PAM), K-Nearest Neighbors, Bayesian Additive Regression Trees (BART), Lasso, Ridge regression, amongst others. For accurate assessment of model prediction ability, the dataset was split into training and test sets. Classifier gene models were built, trained and evaluated using K-fold cross-validation followed by model selection based on minimum prediction error. We built several models using different classification methods and experimented with gene inclusion criteria in our datasets according to those features most differentially expressed between CR and non-CR patients. Final model from each of these methods was applied to test dataset to predict CR vs non-CR, and prediction results were evaluated using area under the ROC curve (AUC) as a predictive measure. The maximum AUC among all the training-testing splits was 0.63. The true positive rate (Sensitivity) to correctly predict CR case reached maximum 70% or more at the cost of higher false negative, which is to misclassify a patient as non-CR who might have responded to the treatment. Among the number of methods employed, our best predictive capability provided 66% sensitivity, 60% specificity, 67% positive predictive value and 59% negative predictive value. Importantly, comparing real CR proportion (71/128 = 56%) with that of predicted by the best model (66%), no statistically significant difference was observed (Chi-square; p-value: 0.09). We observe similar results using two independent datasets available in public data repository. Based on our analysis, we recognize and in fact foresee that the expression profile alone has limited ability to predict treatment response especially when response rate is high. This lack of predictability using current approach of response prediction with gene expression alone may be related to several limitations, like alternate splicing, miRNA-based gene regulation, post-translational modifications, binary distribution of response status, inherent variability of new samples, and developing unified signature without consideration of myeloma subtypes. A comprehensive model needs to be developed using global genomic changes to have meaningful output for clinical application. Disclosures: Munshi: Millennium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees.

Abstract 134 Alteration in expression and function of transcription factors has been frequently associated with neoplastic transformation. We here provide both experimental and clinical evidence that Sp1, a transcription factor that controls number of cellular processes, plays an important regulatory role in MM cell growth and survival. Although Sp1 is ubiquitously expressed, its nuclear localization observed in MM is functionally both relevant and important. We have confirmed high Sp1 activity in MM cells both by demonstrating increased DNA binding as well as increased Sp1-responsive promoter activity measured by luciferase reporter assay. MM cell-BMSC interaction led to Sp1 activation which was completely abrogated by the ERK pathway inhibitor U0126 but not by the AKT inhibitor LY29004. Furthermore, using both SiRNA and ShRNA mediated Sp1 knock-down, we have confirmed the growth and survival effects of Sp1 on MM cell growth. Using gene expression profile of MM cells from 172 uniformly treated patients, we have further confirmed these in vitro results by observing that overexpression of Sp1-related genes, including HIF-1a, HDAC1 and MAPK genes, correlate with poor prognosis in MM. This clinical correlation further suggests the role of Sp1 in MM biology, providing the rationale to preclinically target Sp1 in MM. We have investigated TMP, a derivative of the plant lignan nordihydroguaiaretic acid (NDGA) which disrupts the interaction between Sp1 and GC-rich motifs inhibiting Sp1 activity. We have previously confirmed specific inhibition of both Sp1 binding and transcriptional activity in MM cells by TMP, including in the context of MM-BMSC interaction. Along with inhibition of Sp1 activity, we observed both in vitro and in vivo in murine models of human myeloma, anti-myeloma effect of TMP. Importantly, there was no significant synergistic effect when MM cells transfected with Sp1 siRNA were treated with TMP, confirming specificity of TMP's mechanism of action. We have further observed activation of the mitochondrial apoptotic pathway by TMP via activation of caspase-3, -9 and -7 and PARP cleavage while caspase-8 was not activated suggesting possible synergism with activators of alternate apoptotic pathways. We have also observed that lenalidomide and dexamethasone upregulate Sp1 activity, suggesting that Sp1 may be a common resistance mechanism. We have confirmed that the increased Sp1 activity by lenalidomide or dexamethasone is abrogated by TMP and the combination provides synergistic cytotoxicity, in MM cell lines as well as primary MM cells. In conclusion, we report significant role of Sp1 in myeloma cell growth, survival and drug resistance with its influence on clinical outcome in MM. Our results suggest that specific inhibition of Sp1 activity may be an important therapeutic target in MM. Disclosures: Avet-Loiseau: Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Munshi:Millennium Pharmaceuticals: Honoraria, Speakers Bureau.

Abstract 1926 Gene regulatory networks with regulatory circuits at different domains are the fundamental mechanism in phenotypic expression of the underlying genome. Regulation by transcription factors (TF) and microRNA (miRNA) are important such domains beside other epigenetic and post-translational gene regulation. With many genomic data sets in recent years, improved understanding of such regulatory network motifs would be of substantial value to find causal genomic alternations in the cancer. A particular example of one such TF-miRNA-gene feed-forward loop (FFL) is, TF (e.g. MYC) regulates the transcription of one or a set of genes/TFs (e.g. E2F1), and the TF (MYC) also regulates the transcription of one or a set of miRNAs (e.g. mir-17-5p and mir-20a), which in turn regulate the same gene or set of genes (E2F1). We hypothesize that an integrative analysis of network motifs and gene expression data can discover experimentally testable TF-miRNA regulation relationships. We have developed computational algorithms based on network motifs and expression data for identifying TF-miRNA pairs important in biological processes and disease states. We construct candidate regulatory networks and rank them using network structure and expression data. We based our prediction on a score-based algorithm, and analyzed publicly available paired gene expression-miRNA profile data from 60 multiple myeloma (MM) patients and 5 samples from healthy donors. We have identified 65 FFLs containing TF-miRNA-gene networks with loss of negative feedback regulation in MM, which supports the uncontrolled growth, anti-apoptosis or other oncogenic effects in MM. Two of the prominent examples involve known proto-oncogenes, c-Myc and FOXA2. We also observed FFL involving c-Myc - mir-629 and target gene PRDM16. Here, concurrently up-regulated c-Myc (TF) and down-regulated mir-629 favors overexpression of target gene, PRDM16, with reported role in myelodysplastic syndrome and acute myeloid leukemia. Similarly, we observe FFL involving HOXA2 – mir-15a and mir-214 and target genes RUNX1T1, HOXC13, NKD1 and PRDM16. Using independent gene expression and miRNA public datasets, we have validated the loss of the negative regulation of these FFLs in MM. We are in the process of functionally validating these identified loops and the associated changes. We will present the predicted FFLs and their biological and functional significance in MM. Such integrative analysis of network motifs and gene expression data can discover experimentally testable TF-miRNA-gene regulatory network and will be instrumental in understanding the biology of the disease, developing clinically relevant integrated models, and translating basic research into targeted therapy. Disclosures: Anderson: Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Munshi:Millennium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees.

Abstract 120 The transcription factor Sp1 transactivates expression of genes containing proximal GC/GT-rich promoter elements controlling cell differentiation, cell cycle and apoptosis affecting growth and survival of tumor cells. Based on previous observation that key multiple myeloma (MM) cell growth and survival genes such as NF-kB p65, IGF-IR, VEGF, and IL-6 are controlled by Sp proteins, we have previously investigated and observed high Sp1 expression and activity in MM cells and confirmed its role in MM by Sp1 knock down using both siRNA and lentiviral shRNA constructs specific for Sp1. We further evaluated the role of Sp-1 in WM and observed high nuclear Sp1 protein expression along with increased Sp1 activity in WM cells compared to normal peripheral blood mononuclear cells (PBMC). Moreover, adhesion of WM cells to bone marrow stromal cells (BMSC) further induces Sp1 activity in WM cells. Based on these data, we have investigated the anti-WM activity of Terameprocol (TMP), a small molecule suitable for clinical application,which specifically competes with Sp1-specific DNA binding domains within gene promoter regions. It disrupts the interaction between Sp1 and GC-rich motifs inhibiting Sp1 activity without direct effect on its expression. We have confirmed inhibition of both basal and BMSC-induced binding and transcriptional activity of Sp1 in WM cells using an ELISA assay specific for measuring Sp1 binding activity and using Sp1 sensitive luciferase reporter plasmid. TMP treatment did not affect Sp1 protein levels. Importantly, TMP significantly inhibited WM cell growth in a dose-dependent fashion (IC50 between 5–20 μ M at 24 hours) and was able to overcome the protective effects of BMSCs. TMP activates the mitochondrial apoptotic pathway via induction of caspase-3, -9 and -7 and PARP cleavage but without caspase-8 activation. TMP treatment also led to downregulation of expression of survivin, a known anti-apoptotic gene transcriptionally regulated by Sp1. We have also confirmed in vivo activity of TMP in a murine xenograft model of MM. Finally based on the data suggesting that both dexamethasone and revlimid increase Sp1 activity, we have combined TMP with these agents and observed synergistic activity on cell growth and survival. In conclusion, our results demonstrate Sp1 as an important transcription factor in WM and provides preclinical rationale for clinical development of TMP as a specific Sp1 inhibitor alone and in combination with conventional and novel agents in WM. Disclosures: Anderson: Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. 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, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees.

Abstract 4042 Plasmacytomas are rare clonal proliferations of plasma cells that though cytologically identical to plasma cell myeloma, present with osseous or extraosseous growth pattern. Understanding their molecular characteristics can provide crucial insights into their pathogenesis and risk of progression to multiple myeloma (MM). To investigate the differences between extramedullary (EMP) and medullary plasmacytomas (MP) and MM without plasmacytomas, we sought to molecularly profile these tumors by tissue microarrays, gene expression, microRNA, and FISH. We identified 85 patients from our data base with a pathological diagnosis of plasmacytoma. Of the 85 patients, 13 patients presented with EMP, and 72 had MP. Among the patients with EMP (n=13), 2 patients presented with multiple lesions. Three of 13 (23%) patients progressed to develop MM at a median of 12 months. 72 patients presented with MP, of which 21 had solitary lesions and 27 (37%) progressed to MM at a median of 20.5months. There was a male preponderance (67% vs 33%) and the median age at diagnosis was 60.5 years (range 27.7–87.6). The mean overall survival for patients with EMP was 121 months (95% confidence interval[CI] 97–144 months) and for patients with MP was 102 months (95% CI 93–128 months) {p=0.025}. MicroRNA (miRNAs) profiling was performed on MP (n=19), EMP (n=7) and MM samples (n=66). Data was normalized using U6 endogenous control. Gene expression profiling was performed and correlated with the miRNA data to identify genes and transcripts of interest. miRNA 127, which regulates SET D8, was upregulated four fold in both MP and EMP compared to MM. miRNA 493, which regulates cadherin 11 and PTCH 1, both of which have been associated with metastatic potential in solid tumors, was similarly downregulated four fold in both MP and EMP compared to MM. A tissue microarray was created on 52 patients (8: EMP, 44: MP,) in whom paraffin-embedded tissue was available. Additional evaluation using SET 8, cadherin 11 antibodies and validation of additional functional targets is ongoing and will be reported. Differential expression patterns of factors involved in proliferation, survival, adhesion, and stroma-tumor cell interactions may help explain plasmacytoma biology and identify factors responsible for progression to MM. These insights may help identify new therapeutic approaches and targets in the treatment of these plasma cell disorders. Disclosures: No relevant conflicts of interest to declare.

Abstract 1924 Homologous recombination (HR), by inducing DNA rearrangements, plays an important role in cancer cell proliferation and progression via induction of oncogene activation, and/or anti-oncogene inactivation (or loss of heterozygosity at tumor suppressor loci), as well as telomere-expansion. We have observed that recombinase (RAD51) expression and homologous recombination (HR) activity are constitutively elevated in multiple myeloma (MM) as well as other cancers, and the suppression of recombinase (hsRAD51) reduces HR and significantly inhibits genomic instability and development of drug resistance in MM. We have further extended these observations to investigate whether elevated HR in myeloma is also implicated in karyotypic instability and if prominent HR genes can be used to predict clinical outcome in MM. Myeloma cells, transduced with control and RAD51 shRNAs, were cultured for 30 days and evaluated for karyotypic changes using “day 0” cells as reference. G-band karyotype of baseline control cells showed number of chromosomal abnormalities including three copies of chromosome 7; and three copies of chromosome 14 each with different aberrations: add(14)(q32), add(14)(q24.3), and der(14)t(3;14)(p13;p11.2)add(14)(q24.3). We observed acquisition of new karyotypic change in the control shRNA transduced cells after 30 days. Besides retaining the same stemline karyotype as baseline, it showed a sideline (3 of 20 cells) with additional aberrations in chromosomes 7 and 14. In the sideline, one chromosome 7 showed additional material on the distal short arm, add(7)(p22), while the other abnormal chromosome 7 showed a deletion of the short arm, del(7)(p12). Only a single change was identified in the chromosomes 14 involving the loss of the 3p material from the short arm of the der(14)t(3;14). Interestingly, the recombinase-shRNA transduced cells showed the same stemline aberrations as the baseline reference cells, without acquisition of new change. We have confirmed similar results with spectral karyotyping as well as by analyzing copy number change using high-density SNP array. To further analyze the impact of HR on outcome in MM, we have analyzed, gene expression data from 170 uniformly treated patients using expression levels of 155 HR-related genes. We observed that several important HR-related genes including recombinase (HsRAD51), XRCC2, ERCC1, and endonucleases are significantly upregulated in MM patients (P Disclosures: Munshi: Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees.

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 1809 Poster Board I-835 Bone marrow infiltration by myeloma cells and osteolytic bone lesions are the major features of Multiple Myeloma. Magnetic Resonance Imaging (MRI) has been used in MM not only to image bone marrow (BM) and to identify lytic bone disease but to also evaluate therapeutic response and prognosis. Gadolinium (Gd)-based contrast agents are frequently used to enhance MRI resolution. We evaluated effect of the most common Gd-containing agent, Omniscan, on myeloma cells. We observed that Omniscan induced both time and dose dependent MM cell growth in vitro (8-20 fold increase relative to control). Importantly, the presence of BMSC enhanced the effect of Omniscan on growth of both MM cell lines and primary MM cells. However, Omniscan was not able to overcome cytotoxic effects of conventional and novel agents in MM. This growth promoting effects were not observed on normal BM stromal cells. Evaluating the molecular mechanism of action of Omniscan on MM cells, we observed time dependent ERK1/2 phosphorylation as well as reversal of growth promoting effects of Omniscan by specific inhibition of ERK signaling; however, Omniscan had no effect on STAT3 and AKT signaling pathways. Next, we investigated in vivo effect of Omniscan in a murine xenograft model of MM. Following detection of tumor, mice were treated with either iv Omniscan or PBS. Treatment with Omniscan significantly induced MM tumor growth compared to control mice (1042 ±243 mm3 vs 502 ±137 mm3 respectively; p=0.0001). Finally in autopsies in 8 MM patients with repeated exposure to Omniscan, we quantified gadolinium in various tissues using Inductively-coupled mass spectrometry. We observed massive quantities of gadolinium accumulation in tissues of these MM patients regardless of their renal function. These results, confirming both in vitro and in vivo growth promoting effects of Gd-containing contrast agent on MM, suggest the need for further analysis of the mechanism of its action on myeloma cells and careful analysis of its clinical impact in MM patients undergoing MRI evaluation. Disclosures No relevant conflicts of interest to declare.

Abstract 605 Despite therapeutic advances and high response rates, most if not all patients with multiple myeloma (MM) develop drug resistance and relapse and curative outcomes remain elusive. A prominent feature of MM is striking genomic instability that evolves with the progression of disease. This genomic instability is considered responsible for development of aggressive phenotype associated with relapsed disease and for development of drug resistance. The molecular basis for the generation of this genetic diversity in cancer cells thus has important implication in understanding cancer progression and therapy. However the genomic evolution in MM patient samples has not been documented. Here, we have utilized single nucleotide polymorphism (SNP) arrays to monitor genome-wide changes in heterozygosity and copy number, in two CD138+ MM cell samples collected 6 months apart from 14 MM patients. Genomic changes acquired in the late tumor samples were identified using early samples as baseline. We defined an event as detectable change in heterozygosity/copy number in three or more consecutive SNPs. All 14 MM patients acquired new genomic change in the later sample at a frequency ranging from 0.021 - 2.674 % (i.e. per 100 informative loci investigated). Although the rate of mutation varied, 12 out of 14 patients had acquired >100 mutational events. Chromosomes 1, 13, and × showed large areas of copy number change in several patients. We also evaluated if genomic changes correlate with changes in expression of corresponding genes. Selecting larger areas of genome, we observed that copy number changes correlate well with the change in expression of genes in these areas. As expected, we also observed a correlation among changes in copy number, heterozygosity, and gene expression at several chromosomal loci. In a number of instances frequently recurrent changes were observed. For example, recurrent copy number changes in areas spanning 1q42.13-1q44 and 1p12-1p12 of chromosome 1 were seen in majority (12 out of 14 and 13 out of 14) patients, whereas copy number changes in the p arm of chromosome × were present in all patients. Similarly the region of chromosome × spanning xq42.13-xq44 showed change in heterozygosity in majority of patients. We also observed that some of the newly acquired changes in late samples correlated with genomic markers of poor clinical outcome. We evaluated prognostic significance of these changes in 192 uniformly treated patients with MM with genomic gains and losses data from SNP array and survival information. Changes in chromosomal regions 1p12 and xp22.1-xp22.33 frequently observed in late samples were significantly (p = 0.017 and 0.037) associated with poor survival in these patients. These data suggest that MM cells acquire changes associated with aggressive phenotype and shorter survival. In conclusion we observe that MM patients acquire genomic changes at a very high rate; and certain chromosomal regions are more vulnerable predicting poor clinical outcome. These data also suggest a need to target mechanisms mediating genomic instability for therapeutic application. Disclosures: Richardson: Keryx Biopharmaceuticals: Honoraria. Anderson:Millenium: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding.

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 833 A growing body of evidence suggests that the genome of a many organisms, particularly mammals is controlled not only by transcription factors but also by post-transcriptional programs that are modulated by the family of small RNA molecules including microRNAs (miRs). miRs can block mRNA translation and affect mRNA stability. We have evaluated profiles of 384 human miRs in CD138+ cells from 79 patients with multiple myeloma (MM), 11 MM cell lines and 9 healthy donors (HD) using qRT-PCR based microRNA array. This analysis has identified a MM specific miRNA signature that significantly correlates with OS (p=0.05) and EFS (p=0.017) of patients. Based on this signature one group of patients clustered with HD suggesting indolent disease while other with cell lines indicating aggressive disease. We identified significant modulation of expression of 61 microRNAs in MM cells compared to normal plasma cells. Specific miRs with established oncogenic and tumor suppressor functions such as miR-155, miR-585 and Let7-f were significantly dysregulated in MM (p Disclosures: No relevant conflicts of interest to declare.

Abstract 2846 Poster Board II-822 Background: A prominent feature of most cancers is striking genetic instability and ongoing accrual of mutational changes associated with tumor progression, including acquisition of invasiveness, drug resistance, and metastasis. Methods: We first utilized single nucleotide polymorphism (SNP) arrays (Affymetrix) to evaluate genome-wide gains and losses in copy number and heterozygosity in CD138+ multiple myeloma (MM) cells collected from 14 patients at two time points at least 6 months apart. To estimate the extent of genomic instability in each patient, the number of events leading to copy number or heterozygosity changes throughout the genome were calculated. An event was defined as detectable change in copy number or heterozygosity in three or more consecutive SNPs. Two cases were also investigated for genome-wide rearrangements utilizing a paired-end approach on next generation sequencing. Results: In a period of six months, all MM patients analyzed acquired multiple new mutational events including changes in copy number and heterozygosity, ranging from 0.021 - 2.674 %, indicating a wide range of genetic instability. Although the rate of mutation varied, the majority (71%) of MM patients had acquired > 100 mutational events within the six months period, thus indicating a striking genetic instability. Chromosomes 1, 13, and X were unique with respect to copy number changes and showed large areas of change, spanning the entire length of a chromosome in several patient samples analyzed. Chromosomes 1 and 13 also showed large areas of loss or gain of heterozygosity in several patients, indicating areas of recurrent changes. We were also able to correlate genomic changes with changes in expression of corresponding genes. In two cases, we investigated genome-wide rearrangements utilizing a massively parallel sequencing approach. Short insert (400bp) libraries from two samples collected 6 months apart were constructed and subjected to paired-end sequencing utilizing 37bp readlengths on the Illumina GAII instrument. Approximately 80 million reads were generated for each of the 4 samples. Read pairs were mapped back to the reference genome, and those mapping aberrantly (incorrect orientation, different chromosomes, incorrect genomic distance) were further analyzed. Bespoke PCR assays defining each breakpoint were designed and used to verify the somatic nature of the mapped rearrangement. Further, PCR fragments spanning somatic genomic rearrangements were sequenced to generate base-pair resolution of breakpoints. To date, 29 somatic rearrangements have been sequenced, including three that were present only in the second sample. One of these was on chromosome 13. Breakpoint sequencing revealed a 64.9Kb homozygous (no wild-type readpairs found) deletion removing the first two exons of the RB1 gene. No reads spanning this breakpoint were found in the matching sample taken six months earlier. Conclusions: This is the first study utilizing massively parallel sequencing to investigate the MM genome and provides important insight into the pathogenesis of disease progression .as well as confirms the potential of whole genome sequencing to inform biology of the disease that may affect the therapeutic approach in future. Disclosures: 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. Richardson:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millennium Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Anderson:Celgene: Consultancy, Honoraria, Research Funding; Millennium: 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.

Alternate splicing is an important post translational change that alters specificity of gene function. Misregulation of alternative splicing has been implicated in number of disease processes including cancer. We have here analyzed alternate splicing in myeloma using high throughput exon array analysis. The GeneChip Human Exon 1.0 ST Arrays used in this investigation not only provides information on expression levels for genes, but as the probe sets are spread evenly over each exon, the array data also provides information on presence of each exon and identify recurrent alternately spliced genes. We conducted a study in series of 170 newly-diagnosed patients with multiple myeloma treated homogeneously in tandem transplantation IFM trials. RNA isolated from CD138 purified MM cells collected at the time of diagnosis were analyzed using the GeneChip Human Exon 1.0 ST Arrays. The dChip software was modified to analyze exon array data. We first normalized gene-level expression values across samples, and then identified differentially expressed exons between two sample groups. These exons are candidates for alternatively splicing events. We observed over 100 genes with candidate alternate splicing events, which have up or down-regulation of an exon relative to the baseline group in more than 20% patients. Dividing the group based on response, 52 alternately spliced exons were identified as influencing response to therapy. The genes and their exons with highest frequency of alternate splicing were CD79A exon 5 and 6, EDNRB exon 2, RASSF1 exon 8, CD1d exon9, TGFBetaRII exon 1, Calmin exon2, CEACAM1 exon 7, MADH1 exon 7, TBX5 exon 2, Amyloid beta exon 14, Nit protein 2 exon 10, Thymidylate synthetase exon 7. Amongst these genes, 32 genes had the most influence on response with over 50% differential frequency in patients achieving CR. Similarly we have identified 85 alternately spliced genes as influencing overall survival between groups divided by less than or more than 48 month survival. The genes with highest frequency of alternate splicing were NOTCH2 exon 18, CXCL3 exon 8, CD9 exon 3 Calmin exon 2, TIMELESS exon 12, SPOUTY homolog 1 exon 4, Amyloid beta exon 14, Nit protein 2 exon 10, Cyclin A2 exon7, lymphocyte antigen 6 exon 7. Forty-nine genes within this group had the most influence on overall survival. Number of spliced variants were shared between both response and survival groups suggesting their biological and functional significance. Further validation of these alternate splicing is under investigations. This study highlights significant frequency of alternate splicing and points to the need for evaluation of not only the expression level of genes but also post translational modifications. The genes identified here are important target for therapy as well as possible immune modulation.

Sp1 transcription factor has an important role in regulating expression of cell differentiation, cell cycle and apoptosis related genes. Increased Sp1 activity has been shown to augment growth and metastatic potential of tumor cells. In multiple myeloma (MM), modulation of autocrine IL-6 secretion by Sp1 has been described. Using genome expression profiling analysis we observed high expression of Sp1 in primary myeloma cells obtained from bone marrow aspirates of MM patients. This observation was confirmed by western blot analysis. Therefore, we evaluated Sp1 as a novel therapeutic target in MM, analyzing both in vitro and in vivo anti-MM activity of a Sp1 inhibitor (Terameprocol, EM-1421). Terameprocol significantly (≥80% inhibition, P≤0.001) inhibited DNA synthesis in 12 MM cells lines tested in a dose-dependent fashion at 24 hour with an IC50 of 1–10 uM. Longer exposure of MM cells to Terameprocol resulted also in cytotoxity, as assessed by MTT assay, in all cell lines tested (IC50 of 20–40 uM at 72 hours). Terameprocol induced apoptosis in a time- and dose-dependent manner in all MM cell lines examined, as assessed by Annexin V+/PI staining, and triggered caspase-9/3 and PARP cleavage. Since Sp1 modulates transcription of survivin and survivin is an anti-apoptotic protein overexpressed in most human cancers that inhibits caspases-3 and -9 activity, we next examined effect of Terameprocol on survivin protein expression. Consistent with induction of caspase-3 and apoptosis, Terameprocol significantly reduced survivin protein in a time-dependent manner. To identify additional potential downstream targets of Sp1 inhibition in MM, we evaluated change in expression profiling in MM cells following Terameprocol treatment (10 uM for 12 hours). We observed alteration (>2-fold changes) in 68 genes. In particular, we recognized significant down-regulation of c-Jun and up-regulation of p53 and p21 following exposure to Terameprocol. Changes in protein expression of these genes were also confirmed by western blot analysis. Since bone marrow stromal cells (BMSCs) trigger growth, survival, and drug resistance of MM cells we next confirmed the ability of Terameprocol to overcome protective effects of BMSCs on MM cell growth and survival. Finally, we have confirmed in vivo activity of Terameprocol on MM cell growth in a xenograft murine model of human MM. In conclusion, our results demonstrate that Sp1 may be an attractive target for MM therapy and provide the rationale for clinical evaluation of Terameprocol in MM.

Current therapy for multiple myeloma (MM) remains empiric. Only 30–40% of patients respond to any one agent. Moreover, with each new therapy attempted, patients often experience numerous complications and toxicities requiring specialized intervention. In last 4 years four new agents have been approved for use in MM making selection of effective agents more difficult as well as economically taxing. With the improved understanding of oncogenomics in MM and with an ultimate goal of selecting therapies based on their best chance of success, we have used expression profile of myeloma cells to identify expression signature associated with response or resistance to therapeutic agents. We evaluated 34 newly-diagnosed patients with MM enrolled on IFM protocol 2005-01 and treated with bortezomib and dexamethasone as an induction regimen. 18 patients had achieved CR/nCR while 16 patients had no response or progressive disease on therapy. MM cells were purified from bone marrow samples collected prior to initiation of therapy and expression profile was obtained using Affymetrix Human Exon 1.0 ST arrays and analyzed using the dChip software. We next used the “Sample Classification” module in the dChip software to explore predicting patient response using expression data. When we Use two-sample comparison or ANOVA methods to compare the response and to obtain a gene list, and then use a Linear Discriminant Analysis (LDA) to use these genes as features to train a classifier and predict samples, a prediction accuracy of 97% (33/34) was obtained. For unbiased prediction, we modified dChip to perform a leave-one-out cross-validation. Specifically, for each round, one of the 34 samples is left out and the remaining 33 samples are used to select genes and train the classifier. Then the classifier is used to predict the response of the left-out sample and compare it to the real response of this sample. With this analysis we observed 80% positive predictive value which compares favorably with the present CR ratio in the cohort from which these 34 samples come from. In addition, we also used the same cross-validation method to classify the 8 nCR (immunofixation (IFE) positive CR) versus the 10 CR (IFE negative CR) samples. Although the best achievable overall accuracy is 83%, the accuracy is much more variable than the CR versus NR classification when we vary the gene selection stringency. We also realize and in fact foresee that the expression profile will not be able to predict all patients and due to various factors some samples will not provide clear signature of resistance or sensitivity. A two-group comparison of the response Yes and No samples identified response-related genes. The genes down-regulated in the response group are significantly enriched by genes in Gene Ontology categories “biopolymer glycosylation”, “integral to Golgi membrane”, “transferase activity and gene on chromosome 11q.13. The genes up-regulated in the response group are significantly enriched by genes in Gene Ontology categories “cytoskeleton” and genes on chromosome 12p11 and 4p14. These genes provide a basis for investigating how gene expression and pathways change could affect response to the combination treatment with the two drugs. In conclusion, our preliminary pharmacogenomic studies have confirmed our ability to perform large-scale micro-array profiling from patient bone marrow samples and we have identified gene expression signature associated with responsiveness (CR) versus resistance (NR) to combination of Velcade and dexamethasone. The task ahead is to now prospectively validate our ability to predict whether the combination of bortezomib and dexamethasone will be effective in a given patient.

Waldenström’s macroglobulinemia (WM) is an incurable B cell malignancy characterized by the accumulation of IgM secreting clonally related bone marrow lymphoplasmacytic cells (LPC) including CD19+ B-cells and CD138+ plasma cells. Despite intense research efforts, the pathogenetic basis for this disease remains to be clarified. MicroRNAs are small noncoding RNAs that regulate the expression of protein-coding genes by inducing translational inhibition and through cleavage of targeted transcripts by partial or complete base pairing. We therefore evaluated the expression of 384 microRNAs in CD19+ and CD138+ sorted bone marrow lymphoplasmacytic from 13 untreated WM patients, and compared their expression profiling to analogous lymphoplasmacytic cells taken from the bone marrows of 13 healthy donors. Data obtained from microRNA arrays was analyzed using SDS, RQ manager, R and dChip softwares. Relative expression was calculated using the comparative Ct method through RQ manager and dChip softwares. Of the 384 microRNAs evaluated in CD19+ patient cells, miR-192, -125b, -21, -155 demonstrated significant upregulation, whereas miR-181c, -572, and -650 were significantly down regulated compared to healthy donor CD19+ bone marrow cells (p

Abstract 1806 Poster Board I-832 Plasmacytomas are rare clonal proliferations of plasma cells that though cytologically identical to plasma cell myeloma, present with osseous or extraosseous growth pattern. Understanding their molecular characteristics can provide crucial insights into their pathogenesis and risk of progression to multiple myeloma (MM). To investigate the differences between extramedullary (EMP) and medullary plasmacytomas (MP) and MM without plasmacytomas, we sought to molecularly profile these tumors by tissue microarrays, gene expression, microRNA, and FISH. We identified 85 patients from our data base with a pathological diagnosis of plasmacytoma. Of the 85 patients, 13 patients presented with EMP, and 72 had MP. Among the patients with EMP (n=13), 2 patients presented with multiple lesions. Three of 13 (23%) patients progressed to develop MM at a median of 12 months. 72 patients presented with MP, of which 21 had solitary lesions and 27 (37%) progressed to MM at a median of 20.5months. There was a male preponderance (67% vs 33%) and the median age at diagnosis was 60.5 years (range 27.7-87.6). The mean overall survival for patients with EMP was 121 months (95% confidence interval[CI] 97-144 months) and for patients with MP was 102 months (95% CI 93-128 months) { p=0.025} MicroRNA (miRNAs) profiling was performed on MP (n=19) and MM samples (n=66). Data was normalized using U6 endogenous control. Three hundred and one miRNAs out of a total 665 were significantly differentially expressed between MP vs MM samples. Gene expression profiling performed on MP will be correlated with the miRNA data to identify genes and transcripts of interest which will be functionally validated. Tissue microarrays were performed on 52 patients (8: EMP, 44: MP,) in whom paraffin-embedded tissue was available. Of samples analyzed, CD56 positivity was observed in 55% MP and 71% EMP samples (p=0.67). Additional staining for cyclin D1, Bcl 2 and FISH analysis will be reported. Differential expression patterns of factors involved in proliferation, survival, adhesion, and stroma-tumor cell interactions may help explain plasmacytoma biology and identify factors responsible for progression to MM. These insights may help identify new therapeutic approaches and targets in the treatment of these plasma cell disorders. Disclosures Hochberg: Enzon: Consultancy, Speakers Bureau; Biogen-Idec: Speakers Bureau; Genentech: Speakers Bureau; Amgen: Speakers Bureau. Anderson:Millennium: Research Funding. Raje:Celgene, Norvartis, Astrazeneca: Research Funding.