Runnels, Judith

e14640 Background: MicroRNAs (miRNAs) are non-coding RNAs that act as negative regulators of gene expression. They have been described to play roles in solid tumors and hematologic malignancies. However the role of miRNAs in multiple myeloma (MM) has not been yet fully described. Methods: We performed miRNA-profiling of primary bone marrow-derived CD138+ MM cells, compared to their normal cellular counterparts and validated data by qRT-PCR. In vitro and in vivo functional studies were performed on miRNA-15a- and -16–1-precursors-transfected MM cells. Effect of miRNA-15a and -16–1 on signaling cascades have been evaluated by western blot and immunofluorescence. NF-kB activity has been studied using the Active Motif TransAM kit. In vivo MM cell growth has been evaluated by either using an in vivo imaging model or bioluminescence. Angiogenesis has been studied both in vitro and in vivo using the chorioallantoic membrane model. Results: We identified a MM-specific miRNA signature characterized by down-expression of miRNA-15a, -16–1 and over-expression of miRNA-222/-221/-382/-181a/-181b (P<0.01). Data were confirmed by qRT-PCR on matched samples. Predicted targets for the decreased miRNAs in MM patients included pro-angiogenic cytokines, oncogenes, cell cycle regulators, NFkB activators. Conversely, predicted targets for the increased miRNAs included cell cycle inhibitors, suppressors of cytokine signaling, and pro-apoptotic factors. We demonstrated that miRNA-15a and - 16–1 regulate proliferation and growth of MM cells. Indeed, transfected cells showed decreased DNA synthesis; decreased cyclinD1/cyclinD3/CDC25a/pRb protein expression; phase G1 cell cycle arrest. Moreover, transfected cells showed inhibition of NFkB pathway as shown by reduced p65-/p50-/p52-NFkB activities; downregulation of p-p65/p50/p52 nuclear protein level; upregulation of cttosolic phospho-IkB; and inhibited translocation of p-p65 from the cytolplasm to the nucleus. Similarly, inhibition of MM cell growth was confirmed in vivo; and anti-angiogenic properties of miRNA-15a and -16–1 were demonstrated both in vitro and in vivo. Conclusions: These data indicate that miRNAs play a pivotal role in the biology of MM; providing the basis for the development of new miRNA-based targeted therapies in this disease. No significant financial relationships to disclose.

Abstract 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. In this study, we used a rapid and reproducible antibody-based protein microarray technique to screen the functional differences between malignant plasma cells in samples obtained from patients with MM compared to normal plasma cells (NPC) from the bone marrow of healthy volunteers. METHODS: We determined the protein expression level of 512 polypeptides in 12 samples of newly diagnosed patients with MM using high-throughput proteomic analysis with antibody-based protein microarray. Primary CD138+ sorted MM cells were obtained from the bone marrow of patients after informed consent. MM1.S was used in this study. Using immunohistochemistry and immunoblotting were confirmed. Lentivirus was used to knockdown CRIK. Gene expression datasets from the Mayo Clinic (accession number GSE 6477) and the UAMS (accession number GSE 2658) were obtained from the Gene Expression Omnibus for analysis. The Mayo dataset was generated using Affymetrix U133A platform whereas the UAMS dataset was generated using Affymetrix U133plus 2.0 platform. RESULTS: We identified four subgroups of MM using unsupervised clustering analysis. We confirmed overexpression of some of these proteins including CRIK and CDK4 using immunohistochemistry and immunoblotting. Many of these proteins are known to be deregulated in MM, indicating that this technique can accurately identify proteins that are over or under-expressed in MM in a high-throughput fashion. In addition, we identified novel proteins that are not previously known to be differentially expressed in MM, including the small GTPase member of the Rho family, CRIK protein. We then showed using knockdown of CRIK that this novel protein specifically regulates migration and adhesion in MM. There was no effect on survival of MM cells using the CRIK knockdown. Analyzing the GEP data of the 15 NPC, 46 monoclonal gammopathy of undetermined significance (MGUS) or smoldering myeloma (SMM) and 101 MM samples from the Mayo Clinic, there was a significant increase in expression of CIT from NPC to MM. Among the 351 patients entered into TTII trials from UAMS, CIT expression was similar across the different TC class. Using a cut-off normalized expression level of 1.25 (a level above expression in NPC), MM with a high CIT expression (n=81) had a significantly shorter survival than the other patients. CONCLUSION: In this study, we show that MM cells express a high level of CRIK, and that inhibition of this protein leads to significant inhibition of adhesion and migration of MM cells. In addition, CRIK protein expression correlated with CIT gene expression, with high expression in MM samples compared to NPC. In addition, high CIT expression correlated with poor survival in patients with MM.

Abstract Abstract 1825 Poster Board I-851 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. We have previously demonstrated that Citron Rho Interacting Kinase (CRIK) is overexpressed in primary multiple myeloma (MM) cells, as compared to the normal plasma cell counterpart, using an antibody-based protein microarray technique. We therefore sought to investigate the functional role of CRIK in MM cells. Methods We determined the protein expression level of 512 polypeptides in 12 samples of newly diagnosed patients with MM using high-throughput proteomic analysis with antibody-based protein microarray. Primary CD138+ sorted MM cells were obtained from the bone marrow of patients after informed consent. MM.1S, RPMI8226, and INA6 MM cell lines were used in this study. Protein expression has been studied by immunoblotting. Gene expression analysis has been assessed using the Affymetrix U133A platform. Lentivirus was used to knockdown CRIK in MM cell lines (MM.1S, RPMI8226, INA6). DNA synthesis, cell survival, cell cycle profiling and apoptosis were assessed by thymidine uptake, MTT, PI and Annexin/PI staining and flow cytometric analysis, respectively. Results Overexpression of CRIK has been confirmed in primary CD138+ tumor cells isolated from bone marrow of 12 patients with MM, as compared to normal plasma cells obtained from healthy donors. We found that CRIK-knockdown exerted an anti-proliferative and pro-apoptotic effect only in IL-6-dependent MM cell line INA6; in contrast, no effect on proliferation and survival was observed in MM1.S and RPMI8226. Indeed, INA6 CRIK-knockdown cells were characterized by a reduction in the proliferation rate, associated with decreased S-phase and G2/M phase cell cycle arrest. Moreover, induction of cytotoxicity was also demonstrated in CRIK knockdown cells compared to scramble probe transfected or non-transfected cells. We also showed that CRIK knockdown led to cytokinesis in INA6, indicating a possible mechanism for inhibition of proliferation of these cells. We next correlated CRIK gene expression level (CIT) with prognosis using previously published gene expression datasets and found that CRIK correlated with poor prognosis. Conclusion In this study, we show that MM cells express a high level of CRIK, and that inhibition of this protein leads to significant inhibition of proliferation and survival of IL-6 dependent MM cells. Moreover, CRIK protein expression correlated with poor survival in patients with MM. Disclosures Anderson: Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Ghobrial:Millennium: Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.

Abstract Detailed genomic studies have shown that cytogenetic abnormalities contribute to multiple myeloma (MM) pathogenesis and disease progression. Nevertheless, little is known about the characteristics of MM at the epigenetic level and specifically how microRNAs regulate MM progression in the context of the bone marrow milieu. Therefore, we performed microRNA expression profiling of bone marrow derived CD138+ MM cells versus their normal cellular counterparts and validated data by qRT-PCR. We identified a MM-specific microRNA signature characterized by down-expression of microRNA-15a/-16 and overexpression of microRNA-222/-221/-382/-181a/-181b (P < .01). We investigated the functional role of microRNA-15a and -16 and showed that they regulate proliferation and growth of MM cells in vitro and in vivo by inhibiting AKT serine/threonine-protein-kinase (AKT3), ribosomal-protein-S6, MAP-kinases, and NF-κB-activator MAP3KIP3. Moreover, miRNA-15a and -16 exerted their anti-MM activity even in the context of the bone marrow milieu in vitro and in vivo. These data indicate that microRNAs play a pivotal role in the biology of MM and represent important targets for novel therapies in MM.

Abstract The nuclear factor-κB (NF-κB) path-way has been implicated in tumor B-cell survival, growth, and resistance to therapy. Because tumor cells overcome single-agent antitumor activity, we hypothesized that combination of agents that target differentially NF-κB pathway will induce significant cytotoxicity. Therapeutic agents that target proteasome and Akt pathways should induce significant activity in B-cell malignancies as both pathways impact NF-κB activity. We demonstrated that perifosine and bortezomib both targeted NF-κB through its recruitment to the promoter of its target gene IκB using chromatin immunoprecipitation assay. This combination led to synergistic cytotoxicity in Waldenstrom macroglobulinemia (WM) cells that was mediated through a combined reduction of the PI3K/Akt and ERK signaling pathways, found to be critical for survival of WM cells. Moreover, a combination of these drugs with the CD20 monoclonal antibody rituximab further increased their cytotoxic activity. Thus, effective WM therapy may require combination regimens targeting the NF-κB pathway.

Abstract The mechanisms by which multiple myeloma (MM) cells migrate and home to the bone marrow are not well understood. In this study, we sought to determine the effect of the chemokine SDF-1 (CXCL12) and its receptor CXCR4 on the migration and homing of MM cells. We demonstrated that CXCR4 is differentially expressed at high levels in the peripheral blood and is down-regulated in the bone marrow in response to high levels of SDF-1. SDF-1 induced motility, internalization, and cytoskeletal rearrangement in MM cells evidenced by confocal microscopy. The specific CXCR4 inhibitor AMD3100 and the anti-CXCR4 antibody MAB171 inhibited the migration of MM cells in vitro. CXCR4 knockdown experiments demonstrated that SDF-1–dependent migration was regulated by the PI3K and ERK/MAPK pathways but not by p38 MAPK. In addition, we demonstrated that AMD3100 inhibited the homing of MM cells to the bone marrow niches using in vivo flow cytometry, in vivo confocal microscopy, and whole body bioluminescence imaging. This study, therefore, demonstrates that SDF-1/CXCR4 is a critical regulator of MM homing and that it provides the framework for inhibitors of this pathway to be used in future clinical trials to abrogate MM trafficking.