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LMO2 regulates gene expression by facilitating the formation of multipartite DNA-binding complexes. In B cells, LMO2 is specifically up-regulated in the germinal center (GC) and is expressed in GC-derived non-Hodgkin lymphomas. LMO2 is one of the most powerful prognostic indicators in diffuse large B-cell (DLBCL) patients. However, its function in GC B cells and DLBCL is currently unknown. In this study, we characterized the LMO2 transcriptome and transcriptional complex in DLBCL cells. LMO2 regulates genes implicated in kinetochore function, chromosome assembly, and mitosis. Overexpression of LMO2 in DLBCL cell lines results in centrosome amplification. In DLBCL, the LMO2 complex contains some of the traditional partners, such as LDB1, E2A, HEB, Lyl1, ETO2, and SP1, but not TAL1 or GATA proteins. Furthermore, we identified novel LMO2 interacting partners: ELK1, nuclear factor of activated T-cells (NFATc1), and lymphoid enhancer-binding factor1 (LEF1) proteins. Reporter assays revealed that LMO2 increases transcriptional activity of NFATc1 and decreases transcriptional activity of LEF1 proteins. Overall, our studies identified a novel LMO2 transcriptome and interactome in DLBCL and provides a platform for future elucidation of LMO2 function in GC B cells and DLBCL pathogenesis. © 2012 by The American Society of Hematology., E.C. is partially supported by the Fundacion Alfonso Martin Escudero. I.S.L. is supported by National Institutes of Health (NIH) grants CA109335, CA122105, and U56 CA112973, and the Dwoskin Family and Recio Family foundations. I.S.G. is supported by MICINN (SAF2009-08 803), and by MEC OncoBIO Consolider- Ingenio 2010 (Ref. CSD2007-0017). All Spanish funding is cosponsored by the European Union FEDER program.

Abstract 438 The LMO2 is a cysteine-rich protein containing two zinc binding LIM domains indirectly regulating gene expression by mediating protein-protein interactions with other transcriptional factors (TFs), facilitating the formation of multipartite DNA-binding complexes. It is mainly expressed in endothelial and hematopoietic cells forming cell type specific complexes containing LDB1, TAL1, E2A and GATA2 proteins in endothelial cells and hematopoietic stem cells and LDB1, TAL-1, GATA1 and E proteins in the erythroid lineage. In these cells LMO2 is involved in angiogenesis and erythroid hematopoiesis. In T cells where LMO2 is only expressed in immature CD4/CD8 double-negative thymocytes, the LMO2 complex consist of LDB1, TAL1 and E2A, but may also bind to GATA3. Aberrant expression of LMO2 in T cells induces leukemogenesis. In the B cells, LMO2 is specifically up regulated in Germinal Center (GC) B cells. LMO2 is also expressed in GC-derived non-Hodgkin's lymphomas and is one of the most powerful survival predictors in DLBCL patients. However, its function in GC B cells and DLBCL is currently unknown. In the present study we aimed to characterize the LMO2 transcriptome and interactome in DLBCL cells. Gene expression arrays were performed in Rck8 cells expressing low levels of endogenous LMO2, in which LMO2 was stably overexpressed to levels observed in GCB-like DLBCL. A total of 311 differentially expressed genes (DEGs) between control cell lines transfected with mock vector and LMO2 stably transfected samples were identified at FDR of 0.05. Sixty-four genes were down-regulated by LMO2 transfection, while 247 were up-regulated. Prominent amongst these were 27 genes encoding proteins of the histone cluster 1, colocalized on chromosome 6 and consistently higher expressed in LMO2 expressing cell lines. At the same time, multiple cell-cycle-related genes were also up-regulated by LMO2 transfection, including centromere proteins (CENPE, CENPI, and CENPN), the kinetochore associated protein NDC80 and the mitotic protein CDC25C. Expression of several randomly selected genes (SPIC, LAX1, DLEU2, DOCK3, CHND2 and TNFRSF9) was validated by real time PCR and confirmed the observed gene expression changes upon LMO2 overexpression. To obtain a higher-level view of expression changes, we compared the list of DEGs to Gene Ontology (GO) categories. This revealed significant induction of genes involved in chromosome, nucleosome, and chromatin and protein-DNA complex assembly. We screened our microarray data against the Broad Institute Molecular Signatures Database, to identify TFs whose target genes were significantly up- or down-regulated by stable LMO2 transfection. This candidate list was filtered to include only those TFs whose target genes significantly overlapped with genes which had previously identified binding motifs for LMO2 complexes in their promoter sequences. Our analysis identified Sp1, NFAT, Elk1, and LEF1 as potential novel LMO2 co-factors, but not classical TAL1 and GATA partner proteins. We examined the expression of previously reported and new potential interaction partners of LMO2 in DLBCL cell lines and GC lymphocytes by Western blotting and analyzed the interaction of the expressed proteins with LMO2 by co-immunprecipitation. We identified that in DLBCL the LMO2 complex contains some of the traditional partners such as LDB1, E2A, HEB, lyl1 and ETO2, but not TAL1 or GATA proteins. Furthermore, we demonstrated LMO2 interaction also with SP1, ELK1, NFATc1 and LEF1 proteins. All the identified LMO2 interacting partners are also expressed in GC B lymphocytes, suggesting the presence of a similar protein complex in both normal and malignant B cells. LMO2 interaction with LEF protein affected its transcriptional activity, as measured by reporter assays. A Chromatin immunoprecipitation assay using promoter region of the DLEU2 gene, whose expression was up-regulated by LMO2 expression, confirmed the direct and specific LMO2 binding to a region harboring E2A and a SP1 binding sites. Overall, our studies identified an LMO2 transcriptome in DLBCL as well as its interactome, which contains novel previously unknown interacting partners. These findings suggest that LMO2 may affect unique and novel cellular functions in GC lymphocytes which may potentially contribute to DLBCL pathogenesis and are currently being investigated in our laboratory. Disclosures: No relevant conflicts of interest to declare.

Abstract 584 The Human Germinal center Associated Lymphoma (HGAL) gene is exclusively expressed in germinal center (GC) B-lymphocytes and GC-derived lymphomas. In patients with diffuse large B-cell lymphomas (DLBCL), HGAL expression identifies a subgroup of patients with biologically distinct tumors associated with improved survival. Our previous in vitro studies demonstrated that HGAL decreases spontaneous and chemoattractant-induced cell motility by activating the RhoA signaling pathway and by directly interacting and augmenting F-actin and myosin II binding. However, the major function of HGAL in GC lymphocytes remains largely unknown. Based on our previous observation of tyrosine phosphorylation of a modified ITAM motif in the HGAL by Lyn, we hypothesized that HGAL may be involved in B-cell receptor (BCR) signaling. Indeed, following BCR stimulation of two GCB-like lymphoma cell lines (Raji and VAL), we observed marked reduction of Syk, Btk and PLCγ phosphorylation upon knockdown of endogenous HGAL by specific but not control siRNAs. Concordantly, HGAL knockdown in BCR-stimulated Raji cells reduced Ca2+ mobilization and decreased NFAT transcriptional activity as analyzed by a luciferase reporter assay. HGAL expression in the BCR-stimulated HBL1 lymphoma cell line (lacking endogenous HGAL protein) resulted in increased Syk, Btk and PLCγ phosphorylation. Syk plays a major role in coupling BCR activation to downstream effectors. Endogenous HGAL was detected in immunoprecipitates of endogenous Syk and vice versa. Nanoscope microscopy studies confirmed co-localization of HGAL and Syk proteins in cell membranes, which was enhanced following BCR stimulation. In BCR-stimulated cells, Syk kinase activity was markedly increased following addition of HGAL protein as measured by an in vitro Syk kinase activity assay. To comprehensively examine HGAL effects on immune system and BCR signaling, we generated a transgenic mouse model in which HGAL is expressed under the control of the mouse Ly-6E.1 promoter in Sca1+ hematopoietic stem cells and progenitors of C57BL/6 × CBA mice. The Sca1-HGAL transgenic mice showed normal embryonic and post natal development, and at 8 weeks of age demonstrated normal lymphoid development without any significant changes in the major hematopoietic compartments (bone marrow (BM), spleen, thymus and peripheral lymph nodes) and in peripheral blood. They also exhibited normal GC development in response to a T-cell dependent antigen immunization. In contrast, at 12 months of age the Sca1-HGAL mice developed a decrease in BM immature B-cells at the expense of recirculating B-cells (B220+IgDhi) compared to the age-matched normal littermates, suggesting a defect in B-cell lymphopoiesis. All the Sca1-HGAL transgenic mice became ill from approximately 12 months of age and all died between 12 to 22 months of age with statistically shorter survival as compared to the wild type controls. Analysis of these animals showed massive splenomegaly with marked white pulp hyperplasia and presence of multiple, frequently contiguous nodules predominantly composed of polyclonal follicular (B220+CD21intCD23hi) B lymphocytes. Extra-lymphatic infiltration by similar B lymphocytes was observed in the liver, lungs and kidneys of Sca1-HGAL mice with advanced disease. IgG isotype titers in these animals tended to be higher than in the wild-type controls, reaching a statistically significant difference for the IgG1 isotype. Follicular hyperplasia in the Sca1-HGAL transgenic mice is likely attributable to increased RhoA activation and enhanced BCR signalling manifested by increased Syk phosphorylation, Ca2+ mobilization and in vitro B cell proliferation following BCR stimulation, in agreement with similar data observed in human DLBCL cell lines expressing HGAL. Gene expression profiling of lymphoid tissues confirmed significantly enhanced BCR signalling and RhoA pathway activation in Sca1-HGAL transgenic mice, corresponding to similar pathway activation in human lymphoma cell lines over-expressing HGAL. Overall, our findings demonstrate that HGAL, specifically expressed in GC B cells, enhances responsiveness to antigens by stimulating Syk kinase activity that without appropriate regulation may lead to lymphoproliferation. Further studies are needed to examine the role of HGAL in the pathogenesis of GC-derived lymphomas. Disclosures: No relevant conflicts of interest to declare.