ALBERT

Description: Abstract 3620 Introduction: Waldenstrom macroglobulinemia (WM) is a rare indolent non-Hodgkin lymphoma, characterized by bone marrow infiltration of clonal lymphoplasmacytic cells. Despite recent advances in understanding the pathogenesis of this disease, the molecular basis of WM etiology has not been clearly defined. We therefore performed genome-wide analysis of RNA polymerase II (pol II) binding sites and gene expression profiling in primary WM cells in order to comprehensively define the aberrant transcriptional regulation and related genes in WM. Methods: Primary CD19+ bone marrow derived WM cells and normal primary bone marrow were used. Genomic DNA was extracted using genome isolation kit (QIAGEN) after cross linking. All the DNA samples were sent for Chip assay and human promoter 1.0R array (Genepathway Inc.) which comprised of over 4.6 million probes tiled through over 25.500 human promoter regions. Each promoter region covers approximately 7.6kb upstream through 2.45kb downstream of the transcription start sites. For over 1,300 cancer associated genes, coverage of promoter regions was expanded to additional genomic content; for selected genes total coverage spans from 10kb upstream through 2.45kb downstream of transcription start sites. The published gene expression datasets (GDS2643) which included 10 CD19+ B cell from bone marrow of 10 WM patients and 8 normal controls was analyzed by d-chip software and normalized to normal control. The motif analysis was performed using Cistrome online tools from the Dana Farber Cancer Institute. The gene sets enrichment analysis (GSEA) was performed using GSEA online software from Broad institute. Results: A total of 13,546 high-confidence pol II sites were identified in WM samples and share a small percentage of overlap (11.5%) with the binding sites identified in normal controls. Combining the expression microarray data of WM patient samples and normal controls, we demonstrated a significant correlation between high levels of gene expression and enriched promoter binding of pol II. Notably, we also observed that the WM-unique pol II binding sites are localized in the promoters of 5,556 genes which are involved in important signaling pathways, such as Jak/STAT and MAPK pathways by applying gene set enrichment analysis (GSEA). Interestingly, we found that STAT, FOXO and IRF family binding sites motifs were enriched in the pol II-bound promoter region of IL-6 which plays a crucial role in cell proliferation and survival of WM cells. Moreover, the CpG island associated c-fos promoter was enriched for Pol II binding as compared to the normal control. Conclusion: The presence of increased Pol II binding and the identification of transcription factor motifs in the promoters of key oncogenes may lead to a better understanding of WM. Our findings suggest that altered transcriptional regulation may play an important role in the pathogenesis of WM. In addition, this study will provide novel insights into the molecular mechanism of WM etiology, and may lead to discovery of novel diagnostic molecular biomarkers and therapeutic targets for WM. Disclosures: Leleu: Celgene: Consultancy, Research Funding; Janssen Cilag: Consultancy, Research Funding; Leo Pharma: Consultancy; Amgen: Consultancy; Chugai: Research Funding; Roche: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Ghobrial:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

Description: Abstract 4914 Background. We and others have previously demonstrated that microRNA-155 (miR-155) is overexpressed in patients with Waldenstrom's Macroglobulinemia (WM) and Chronic lymphocytic leukemia (CLL) compared to healthy subjects; indicating that miR-155 may play a crucial role in regulating the pathogenesis of these diseases. We therefore evaluated the role of the novel locked nucleic acid (LNA) antimiR against miR-155 in WM and CLL-derived cell lines. Methods. WM and CLL-derived cell lines (BCWM.1, MEC1) have been treated with 20μM antisense LNA antimiR-155 or scramble probe (Santaris Pharma, Hørsholm, Denmark). Efficiency of delivered LNA oligos into cells was determined by using FAM-labeled LNA, followed by Flow cytometry (FACS). Survival and cell proliferation were assessed by MTT assay and thymidine uptake, respectively. Serum levels of miR-155 from peripheral blood of WM patients or healthy subjects and the level of miR-155-targeted genes were detected by quantitative RT-PCR (qPCR). Results. Efficiency of delivered LNA oligos into both WM and CLL-derived cell lines was higher than 90%. LNA antimiR-155 reduced proliferation of WM and CLL-derived cell lines, as compared to LNA scramble probe used as a control. In contrast, LNA antimiR-155 did not exert any effect on survival of them. We demonstrated increased expression of miR-155-targeted genes, such as CEBPβ, SOCS1, and TP53DINP1 in WM cells upon LNA antimiR-155 treatment. Moreover, we found miR-155 could be detected in peripheral blood serum of WM patients, but not the serum of healthy subjects. Summary. These data provide preclinical evidence for using the novel antisense LNA antimiR-155 in WM and CLL. Moreover, serum levels of miR-155 could potentially be used as a biomarker in patients with WM. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 453 Introduction: The interaction of multiple myeloma (MM) cells with the bone marrow (BM) microenvironment, which includes stromal (BMSCs) and endothelial cells (ECs), plays a crucial role in MM pathogenesis and drug resistance. We have previously shown that the chemokine stromal cell-derived factor-1 (SDF-1), its receptor-CXCR4, and GTPases in the downstream signaling of the receptor regulate this interaction. Selectins are adhesion molecules which are involved in the primary interaction of lymphocytes with the endothelium. In this study, we have tested the expression of selectins and their ligands in MM, and explored their role in the interaction with the BM milieu and its potential therapeutic applications. Methods AND RESULTS: Flow cytometry and immunohistochemical (IHC) staining of tissue microarrays revealed that P-selectin glycoprotein ligand-1 (PSGL-1, CD162) was over expressed in MM cells from patients (n=20) and cell lines (MM1s, H929, RPMI, OPM1 and OPM2) compared to normal plasma cells (n=3). Gene expression profiling (GEP) analysis showed that the expression of PSGL-1 was directly correlated with MM stage of progression (normal plasma cells, n=15 〈 MGUS, n=20 〈 smoldering MM, n=23 〈 MM, n=68 p

Description: Abstract 3622 Introduction: Epigenetic factors such as DNA methylation have been shown to play a crucial role in the pathogenesis and progression of multiple myeloma (MM), yet studies of DNA methylation in MM are still limited. Therefore, in order to better understand the role of DNA methylation and identify specific genes that may be affected by differential methylation in MM patients, we conducted genome-wide DNA methylation profiling in cd138+ plasma cells purified from bone marrow of the patients with MM and normal donors. Methods: Genomic DNA of CD138+ Plasma cell selected from both MM patients and normal primary bone marrow was extracted using QIAGEN genome isolation kit. Following extraction, methylated DNA was isolated by Chip and hybridized to Affymetrix Human 2.0 tiling arrays. Chip assay and array hybridization was performed by Genepathway Inc. CEL files were processed and normalized using the MAT program, and methylation peaks were called from the resulting MAT scores using a custom segmentation method. Peak annotation and characterization of different genomic regions was done with custom tools and using genome annotation files from the UCSC genome database. All peaks were visualized by IGB online software. Medip-PCR was done in human MM cell lines to validate the methylation status. Methylated gene expression was determined by both Semi-quantitative PCR and real-time PCR. 5′aza was used for demethylation in human MM cell lines. Methylated gene expression with or without 5′aza treatment was determined by both Semi-quantitative PCR and real-time PCR. Results: Genomic DNA from CD138+ plasma cells from bone marrow of MM patients showed a significant increase in methylation levels compared to normal controls. We demonstrated that the hypermethylated sites were distributed across the genome in the following proportions: 3.2% in the promoter region; 45.6% in the intragenic region; 5.4 % in the 3′ end region; and 46.8 % in the intergenic region. Furthermore, around 9 % promoter CpG islands (CGIs); 11% intragenic CGIs; 15 % CGIs in 3′end region; and 14.3 % intergenic CGIs of patients genomic DNA were methylated. Moreover 2.1% promoter CGIs; 2.3 % intragenic CGIs; 2.5% CGIs in 3′end region; and 4.7% intergenic CGIs were methylated for the normal control. Medip-PCR showed that the identified methylation pattern in MM patients showed similar results in MM cell lines. Expectedly, we also observed that suppressor of cytokine signaling 1 (SOCS1) was hypermethylated at the promoter region (MAT score=19.986) as has been reported in human cell lines. Importantly, another member of SOCS family SOCS3 showed much stronger signal in the promoter region with CpG island (MAT score=31.707) in MM patients compared to normal control. Notably, the expression of two members of TNFR superfamily TNFRSF18 and TNFRSF4 which play an important role in development and programmed cell death of lymphocyte significantly have increased 283 and 141-fold after treatment with 5′aza in MM cell lines. Conclusion: These findings enhance our understanding of the role of DNA methylation in MM, as one of the epigenetic changes that may contribute to the pathogenesis of this disease. The identification and functional characterization of novel key molecules affected by DNA methylation will provide deeper insight into the molecular basis of MM disease. Disclosures: Leleu: Celgene: Consultancy, Research Funding; Janssen Cilag: Consultancy, Research Funding; Leo Pharma: Consultancy; Amgen: Consultancy; Chugai: Research Funding; Roche: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Description: Abstract 2958 PURPOSE: Recent advances in understanding of the molecular alterations that occur at the genetic and epigenetic levels in Multiple Myeloma (MM) have led to major leaps in identifying molecular pathways that regulate progression and resistance to therapeutic agents. However, despite great scientific advances at the genomic level, studies to identify signaling pathways deregulated at the functional proteomic level in MM are limited. Using an antibody-based protein microarray technique, we identified Citron Rho Interacting Kinase (CRIK) as a protein that was highly expressed in primary multiple myeloma (MM) cells compared to normal plasma cell. We therefore sought to investigate the functional role of CRIK in MM cells. Methods: Primary CD138+ sorted MM cells were obtained from the bone marrow of patients after informed consent. We determined the protein expression level of 512 polypeptides in 12 samples of newly diagnosed patients with MM and 4 healthy control using high-throughput proteomic analysis with antibody-based protein microarray (Clontech, CA). MM.1S, OPM2, RPMI8226, and INA6 were used to perform functional validation. Protein expression has been studied by immunoblotting. Gene expression analysis has been assessed using the Affymetrix U133A platform and qPCR. Lentivirus was used to knockdown CRIK in MM cell lines (MM.1S, RPMI8226, OPM2, and INA6). DNA synthesis, cell survival, cell cycle profiling and apoptosis were assessed by BrdU, MTT, PI and Apo2.7 staining, and flow cytometry analysis, respectively. Results: We first showed that CRIK was overexpressed in 12 patients with MM compared to normal CD138+ cells obtained from healthy controls using high-throughput protein microarray. We further confirmed CRIK expression using immunohistochemistry in the same samples of MM patients. We next correlated CRIK gene expression level (CIT) with prognosis using previously published gene expression datasets and found that CRIK correlated with poor prognosis. Knockdown of CRIK in MM cell lines led to an anti-proliferative and pro-apoptotic effect in all MM cell lines tested. Indeed, CRIK-knockdown MM cells were characterized by a reduction of 60–80% in the proliferation rate, supported by reduction of DNA synthesis and G2/M phase cell cycle arrest. Moreover, induction of cytotoxicity of caspase-3, caspase-8, caspase-9, and parp cleavage was also demonstrated in CRIK knockdown cells compared to scramble probe transfected cells. We also showed that CRIK knockdown led to cytokinesis in MM cell lines, indicating a possible mechanism of inhibition of proliferation of these cells. Conclusion: In this study, we show that MM cells express a high level of a novel protein CRIK, and that inhibition of this protein leads to significant inhibition of proliferation and survival of MM cells. CRIK could be a critical therapeutic target in MM. Disclosures: Anderson: Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Ghobrial:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

Description: Abstract 1943 Introduction: Plerixafor (Mozobil®), a potent CXCR4 inhibitor, is approved in combination with G-CSF to mobilize hematopoietic stem cells (HSCs) for autologous transplantation in multiple myeloma (MM) and non Hodgkin's lymphoma (NHL). Another area of investigation consists of exploring whether disruption of the CXCR4 pathway by plerixafor could potentiate the effect of chemotherapy in active disease. This study aimed to establish the maximum tolerated dose (MTD) of plerixafor in combination with bortezomib in patients who have active relapse/refractory MM. This was informed by preclinical studies showing that plerixafor induces de-adhesion of MM cells with sensitization to combination therapy with bortezomib in pre-clinical animal models. Methods: Eligibility criteria include: 1) patients with relapsed or relapsed/refractory MM with any prior lines of therapy including bortezomib, 2) measurable disease, 3) not receiving chemotherapy〉 3weeks, or biological/novel therapy for MM 〉 2 weeks. Patients with active disease received plerixafor at the recommended dose sc on days 1–6 of every cycle. Dose levels include 0.16, 0.24, 0.32, 0.40, and 0.48 mg/kg. Bortezomib was given at the recommended dose twice a week on days 3, 6, 10, and 13 every 21 days. Dose levels include 1.0 and 1.3 mg/m2. Bortezomib was given 60–90 minutes after plerixafor. Patients were assessed after every cycle. Patients who had response or stable disease went on to receive a total of 8 cycles without planned maintenance therapy. 4 dose levels were initially planned at a maximum of 0.24 mg/kg plerixafor. The protocol was then modified to include 3 higher doses of plerixafor, to further evaluate the hypothesis that higher doses may induce better chemosensitization. To examine the in vivo effect of plerixafor and bortezomib on de-adhesion of MM cells and other accessory cells of bone marrow, blood samples were obtained from patients at 0, 2, 4 and 24 hours post-plerixafor injection on days 1 and 3, and time points 0, 2, and 4 hours on days 6, 10 and 13 of cycle 1 and examined for the presence of plasma cells or CD34+ cells using flow cytometry. Results: Thirteen patients have been treated to date, three in each cohort with cohort 5 currently enrolling. The median age is 60, the median lines of prior therapy is 2. All of the patients received prior bortezomib. Three patients were assessed by light chain, two patients had extramedullary disease. The median number of cycles on therapy was 5 (1-8). None of the patients came off study due to toxicity. To date, there have been no dose-limiting toxicities. Overall, the combination is very well tolerated. Grade 3 possibly related toxicities include lymphopenia (30%), hypophosphatemia (15%), anemia (8%), and hyponatremia (8%). No grade 2 or higher neuropathy has been noted in these patients. Twelve patients are evaluable for response, including 1 (8%) complete remission (CR) and 1 (8%) minimal response (MR), with an overall response rate including MR of 2 (16%) in this relapsed/refractory population. In addition, 8 (66%) patients had stable disease (SD), and 2 (18%) had progressive disease (PD). We also examined the number of plasma cells, CD34+ HSCs, and other accessory bone marrow cells (including endothelial progenitor cells and plasmacytoid dendritic cells) in the peripheral blood. Analysis of these samples is ongoing, but preliminary data indicate de-adhesion of plasma cells. Conclusions: : The combination of plerixafor and bortezomib is very well tolerated with minimal neuropathy or other toxicities. The responses observed are encouraging in this relapsed/refractory population. The ability to demonstrate transient de-adhesion of MM cells and accessory cells in vivo indicates that these cells can be separated from their protective stromal environment which may make them more sensitive to chemotherapy. This study was supported by R01CA133799-01, and by Genzyme. Disclosures: Ghobrial: Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Munshi:Millennium Pharmaceuticals: Honoraria, Speakers Bureau. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees.

Description: Abstract 142 Introduction: Receptor tyrosine kinases (RTKs) are key regulators of the development and progression of many types of cancer. The Eph receptors represent the largest family of RTKs, which are used extensively throughout embryogenesis and development but detected in adult normal tissues. Elevated levels of expression of Eph receptors was found in a wide range of cancers and correlated with aggressive disease and metastasis. This is, triggered by a wide array of cellular responses, including cell boundary formation, motility, adhesion, and repulsion. Waldenstrom macroglobulinemia (WM) is characterized by widespread involvement of the bone marrow, indicating homing and adhesion of the malignant cells to specific niches in the bone marrow, which provides a protective environment for the survival and proliferation of these cells. Direct adhesion to endothelial cells and several growth factors and cytokines secreted by endothelial cells in the BM were shown to regulate tumor proliferation. In this study we aimed to characterize the role of RTKs, specifically Eph-B2 receptor, in the interaction of WM cells with the BM microenvironment including cell adhesion, proliferation, and cell-cycle. Methods and Results: We first examined the expression and activation (phosphorylation) of different RTKs in primary WM samples and cell lines using an antibody-based RTK-array. Eph-B2 receptor was activated in all patient samples compared to control, with a 5-fold increase in CD19+ WM cells, and we further confirmed that Eph-B2 was phosphorylated in WM cell lines, BCWM.1, and in IgM secreting cell lines RL, MEC-1. We next examined the activity of Ephrin-B2/Eph-B2 on adhesion and migration of WM cells. Treating starved WM cells with recombinant ephrin-B2 (the ligand of Eph-B2) activated cell-adhesion signaling, including focal adhesion kinase (FAK), Src, p130, paxilin and cofilin, but had no effect on WM cell chemotaxis. Moreover, we found that ephrin-B2 was highly expressed on endothelial cells isolated from the BM of WM patients and on HUVEC cells. Inhibition of ephrin-B2 on the endothelial cells or Eph-B2 on the WM cells with blocking antibodies inhibited the adhesion of the WM cells to endothelial cells, as well as the cytoskeletal signaling in WM cells induced by co-culture with endothelial cells. Consequently, inhibition of the ephrin-B2/Eph-B2 interaction reversed the proliferative and cell cycle effect in WM cell which was induced by co-culture with endothelial cells. This was confirmed using BrdU proliferation assay, flow cytometry for cell cycle, and by immuno-blotting to confirm the effect on proliferative signaling pathways including PI3K kinase and cell cycle related proteins including Cyclin-D, Cyclin-E and p-Rb. Conclusion: This study examines the interaction of Eph-B2 receptor in WM and shows that ephrin- B2/Eph-B2 axis regulates adhesion, activation of downstream signaling of integrin-related molecules, survival and cell cycle progression through the interaction of tumor cells with endothelial cells. This provides the basis for further studies to explore Eph-B2 as a novel therapeutic target in WM. Disclosures: Ghobrial: Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

Description: Abstract 4035 INTRODUCTION: The interaction of multiple myeloma (MM) cells with the bone marrow (BM) microenvironment plays a crucial role in MM pathogenesis, implying that progression of MM occurs through continuous interaction between the BM and MM cells, which controls the ability of MM cells to egress out of the BM and home into new BM niches. We have previously shown that the CXCR4/SDF1 axis as well as Rho GTPases downstream of the receptor was important for chemotaxis, adhesion, homing and egress of MM cells. However, the driving force for MM cells to leave the BM and metastasize to other BM sites is not well understood. Regions of severe oxygen deprivation (hypoxia) arise in tumors due to rapid cell division and are associated with poor patient prognosis, cell motility, associated angiogenesis and metastasis. In this study, we tested the role of hypoxia in the dissemination of MM cells in vivo, as well as regulation of the retention/egress of MM cells in and out of the BM. METHODS: To test the effect of hypoxia on induction of MM egress, MM1s-GFP+/Luc+ cells were injected into 12 SCID mice, and then mice with different stages of tumor development (based on the tumor size detected by bioluminescence) were treated with the hypoxia marker pimonidazole. Blood was drawn and BM was obtained from the femur. Mononuclear cells were then fixed, permeabilized, and stained with antibodies against pimonidazole, followed with an APC- secondary antibody, PE-mouse-anti-human CXCR4, and anti-cadherin antibody followed by an Alexa-Fluor-594 secondary antibody. MM cells in BM and peripheral blood were identified by gating on cells with high GFP signal. To confirm the effects of severe hypoxia found in vivo compared to physiologic mild hypoxia found in the BM, we tested the effect of mild hypoxic conditions (6% O2) and severe hypoxic conditions (0.5% O2) on MM expression of cadherins and CXCR4, as well on functional adhesion of MM cells to stromal cells and chemotaxis. RESULTS: Twelve mice with different stages of MM tumor progression were used. A bi-phasic correlation between tumor progression and the percent of hypoxic cells in BM was found, showing that severe hypoxic conditions in the BM correlated with tumor burden. The correlation between the tumor burden and the number of circulating cells was not linear; however, a direct linear correlation was observed between the number of circulating MM cells and hypoxia in the BM. Moreover, hypoxia in BM correlated directly with the expression of CXCR4 and negatively correlated with the expression of cadherins in MM cells isolated from the BM. To test the effect of the severe hypoxic conditions induced by tumor progression compared to mild hypoxic conditions found physiologically in the BM, we tested the effect of 0.5% O2 (severe hypoxia) and 6% O2 (mild hypoxia) compared to normoxia (21%) on MM cell adhesion to BMSCs, as well as on chemotaxis in response to SDF1, as well as expression of CXCR4 and cadherins. We found that severe hypoxic conditions decreased MM expression of cadherins and adhesion to BMSCs, as well as increased expression of CXCR4 and chemotaxis to SDF1 compared to cells in normoxia. In contrast, mild hypoxic conditions did not alter the expression of CXCR4 and cadherins, adhesion of MM cells to BMSCs, or chemotaxis of MM to SDF1 compared to normoxic cells. CONCLUSION: Hypoxia in the BM directly correlates with the number of circulating MM cells, and with changes in expression of cadherins and CXCR4 in vivo. Severe hypoxic conditions, but not mild hypoxic conditions, induce hypoxic responses in MM cells. Based on these findings, further studies to manipulate hypoxia in order to regulate tumor dissemination as a therapeutic strategy in MM are warranted. Disclosures: Anderson: Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Ghobrial:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.