Human Germinal Center-associated Lymphoma Protein Research Articles

Role of an adaptor protein human germinal center-associated lymphoma (HGAL) in cell signaling and lymphomagenesis

Human germinal center (GC)-associated lymphoma (HGAL) is a multi-domain adaptor protein expressed in GC B lymphocytes, T follicular helper (Tfh) cells and lymphomas derived from these cells. HGAL expression is an independent predictor of longer survival of diffuse large B-cell lymphoma (DLBCL) and classical Hodgkin’s lymphoma (HL) patients. HGAL regulates B cell receptor (BCR) signaling and immunological synapse formation by binding to either the downstream effectors [e.g., spleen tyrosine kinase (Syk)] or other signaling regulators [e.g., growth factor receptor-bound protein 2 (Grb2)]. HGAL regulates the cytoskeleton that reshapes B cell morphology during BCR signaling and cell motility by at least two molecular mechanisms: enhanced Ras homolog gene family member A (RhoA) signaling and inhibition of myosin-actin translocation. These effects on the cytoskeleton decrease lymphoma dissemination in animal models and contribute to decreased lymphoma dissemination in patients. The latter may contribute to the association of HGAL protein expression with longer survival of patients with DLBCL and HL tumors. The ability to regulate multiple and distinct functions simultaneously in B cells implies that the HGAL protein level is tightly regulated. It was demonstrated that HGAL can be regulated by PR/SET domain 1 (PRDM1)/B lymphocyte-induced maturation protein-1 (BLIMP1) and interleukin-4 (IL-4) at the transcription level, by microRNA-155 (miR-155) at the post-transcriptional level, and by F-box protein 10 (FBXO10) at the post-translational level. Constitutive enforced expression of HGAL at physiological levels leads to lymphoid hyperplasia and DLBCL in mice. Future studies need to focus on identifying HGAL interactome, dissecting its interaction network, and understanding HGAL spatiotemporal signaling in live cells in physiological conditions. Further, the recent demonstration of HGAL expression in Tfh cells requires the determination of its function in these cells. These studies will contribute to new insights into the biology of these cellular subsets and how immune dysregulation contributes to lymphomagenesis.

FBXO10 Targets HGAL for Degradation

Expression of the Human Germinal center Associated Lymphoma (HGAL) gene is restricted to germinal center (GC) B-lymphocytes and GC-derived lymphomas. HGAL expression identifies lymphomas characterized by a better prognosis. We previously showed that HGAL is a unique adaptor protein that regulates both cell motility and B-cell receptor (BCR) signaling, processes that are central for successful completion of the GC reaction. In our previous studies we demonstrated that upon BCR activation HGAL binds to and increases Syk kinase activity, resulting in enhanced BCR signaling. Syk induces HGAL phosphorylation, leading to its redistribution from lipid rafts to the bulk membrane and cytoplasm, followed by its rapid degradation. The mechanism of HGAL degradation is currently unknown.To elucidate the mechanism of HGAL degradation, we hypothesized that HGAL is degraded by a SKP1-CUL1-F-box protein (SCF) ubiquitin ligase complex. Consequently, we examined the ability of HGAL to be recruited to the SCF by screening a panel of F-box proteins transfected into HEK293T cells by reciprocal coimmunoprecipitations (Co-IPs). These studies revealed that HGAL specifically bound only FBXO10. Concordantly, HGAL Co-IPed with endogenous SKP1 and CUL1 in the presence of FBXO10. Furthermore, a dose dependent reduction of HGAL levels was observed in Raji cells transfected with increasing quantities of FBXO10 expressing plasmid. In contrast, none of the other FBXO11 tested F-box proteins affected HGAL levels. The reduction in HGAL protein level by FBXO10 was due to enhanced proteolysis, as demonstrated by the decrease in HGAL protein half-life and the rescue of its levels by either addition of proteasome inhibitor MG132 or expression of a dominant-negative CUL1 mutant (CUL1 (1-242)). Furthermore, depletion of FBXO10 by two short hairpin RNA (shRNA) constructs in Raji cells increased HGAL stability and half-life.SCF complexes mediates protein degradation by polyubiquitination. We therefore examined if HGAL protein can be ubiquinated. To this end we cotransfected 293T cells with HGAL-V5, FBXO10-FLAG, HA-ubiquitin with or without CUL1 (1-242) and blotted immunoprecipitated HGAL for ubiquitin. FBXO10 expression markedly increased HGAL ubiquitination and this was significantly decreased in cells coexpressing CUL1 (1-242). Similarly, HGAL ubiquitination was markedly decreased in cells expressing FBXO10 (FBXO10- ΔF-box), in which the F-box motif, necessary for linking the target protein to the SCF ubiquitin ligase moieties, is deleted. These results support the function of FBXO10 in binding HGAL and facilitating its ubiquitination by the SCF complex prior to its degradation.We next analyzed the functional significance of HGAL degradation by FBXO10 on BCR activation, as measured by intracellular and transmembrane Ca2+ mobilization. Overexpression of FBXO10 in Raji cells expressing endogenous HGAL led to decreased levels of HGAL and in Ca2+ mobilization. In contrast, FBXO10 did not affect BCR-induced Ca2+ mobilization in Raji cells in which HGAL was deleted by crispr/Cas9 targeting. These results suggest that FBXO10 affects BCR signaling via regulating HGAL levels.While FBXO10 mediates HGAL degradation post BCR-induced HGAL phosphorylation, FBXO10 also bound HGAL in the absence of BCR activation. The binding of FBXO10 to HGAL was unaffected in Raji cells incubated with anti-IgM antibodies. Treatment of cellular lysates with l-phosphatase did not affect FBXO10 binding to HGAL. These findings indicate that FBXO10-dependent degradation of HGAL is phosphorylation independent. In contrast, HGAL degradation by FBXO10 was dependent on HGAL lipid raft localization, since HGAL myristoylation and palmitoylation mutants (G2A, C43A/C45A, and G2A/C43A/C45A) exhibited decreased Co-IP with the FBXO10 protein. To further map FBXO10 binding motifs of HGAL, we generated HGAL deletion and mutant constructs and examined their ability to Co-IP with FBXO10. These studies revealed that HGAL amino acids 91-95(HRVLC) mediate HGAL binding to FBXO10.In summary, our results demonstrate that SCFFBXO10 is the ubiquitin ligase for HGAL and regulates BCR signaling via controlling HGAL expression. Some diffuse large cell lymphomas expressing HGAL harbor inactivating mutations or express low levels of FBXO10, thus predisposing to enhanced BCR signaling- a phenomenon implicated in lymphomagenesis. DisclosuresNo relevant conflicts of interest to declare.

Grb2 Directly Interacts with HGAL and Ameliorates Its Effects on B-Cell Receptor Signaling

Human Germinal center Associated Lymphoma (HGAL) is specifically expressed in germinal center (GC) B-cells and GC-derived lymphomas. High expression of HGAL is an independent predictor of prolonged survival of Diffuse Large B-Cell (DLBCL) and classical Hodgkin (cHL) lymphoma patients. HGAL is a unique adaptor protein that regulates both cell motility and B-cell receptor (BCR) signaling, processes that are central for the successful completion of the GC reaction. HGAL increases BCR signaling by binding to and enhancing Syk kinase activity. However, our previous studies also suggested that other proteins may be involved in HGAL-mediated regulation of BCR signaling.In vitro kinase assays demonstrated that both Syk and Lyn can phosphorylate HGAL. Mass spectrometry (μ LC/MS/MS) demonstrated that these kinases can phosphorylate HGAL's tyrosines Y80, Y86, Y106Y107, Y128 and Y148. The HGAL Y106Y107 comprise a YYENV motif (aa 106-110) similar to the phosphopeptide motif pYXNX frequently used as a binding site to the SH2 domain of Growth Factor Receptor bound protein 2 (Grb2). Grb2 signaling in B cells controls lymphoid follicle organization and the GC reaction. Specifically, Grb2 is an integral component of the BCR signalosome and decreases BCR-induced Ca2+influx. The presence of the phosphorylated YYENV motif in HGAL raised the hypothesis that HGAL-Grb2 interactions may play a role in HGAL -mediated regulation of BCR signaling. To address this possibility, we performed reciprocal coimmunoprecipitations (Co-IPs) of endogenous HGAL and Grb2 in Raji and VAL lymphoma cell lines. These studies demonstrated that HGAL Co-IPs with Grb2. The interaction between these two proteins is dependent on the presence and phosphorylation of tyrosines in the YYENV motif, since an HGAL mutant in which these tyrosines were mutated to phenylalanine (FFENV) failed to Co-IP with Grb2. Isothermal titration calorimetry confirmed that phosphorylated (pYEN) but not unphosphorylated (YEN) HGAL-derived 12-mer peptides bind to the SH2 domain of Grb2 with an affinity of 5µM. GST-Grb2 pull down assays with recombinant Trx-HGAL(FFENV) and Trx-HGAL proteins confirmed that the HGAL-Grb2 interaction is direct and occurs only if the HGAL tyrosines are phosphorylated. Concordantly, addition of phosphatase to cellular lysates decreased the HGAL-Grb2 interaction. Furthermore, CO-IP studies demonstrated that HGAL's interaction with Grb2 increases following BCR stimulation-induced HGAL phosphorylation. Concordantly, confocal microscopy studies demonstrated HGAL-Grb2 colocalization in the cell membrane following BCR signaling activation.We next examined the functional significance of the HGAL-Grb2 interaction on BCR activation as measured by intracellular and transmembrane Ca2+ mobilization and phosphorylation of proximal BCR effectors (Syk (Y352), BLNK (Y84), BTK (Y551) and PLCγ2 (Y753) in several lymphoma cell lines (U2942, TMD8 and Mino) stablly transfected to express HGAL protein. HGAL expression markedly increased Ca2+ influx and phosphorylation of these proteins, while Grb2 knockdown only slightly increased transmembrane Ca2+ mobilization. Of note, concomitant HGAL expression and Grb2 knockdown further increased intracellular and transmembrane Ca2+ influx and phosphorylation of BCR effectors in comparison to HGAL expression alone. Expression of the HGAL (FFENV) mutant also enhanced Ca2+ influx and phosphorylation of BCR effectors in comparison to wild type HGAL. Concordantly, expression of the dominant negative Grb2 (W193K) mutant also enhanced HGAL's effects on BCR signaling. These observations suggest that Grb2's interaction with HGAL ameliorates HGAL's effects on BCR signaling.We previously showed that HGAL interacts with Syk and enhances Syk kinase activity. We now demonstrate that Grb2 Co-IPs with both Syk and HGAL and thus may potentially interfere with HGAL-Syk interaction. Indeed, knockdown of Grb2 increased HGAL Co-IP with the Syk kinase and this was associated with increased BCR signaling. These findings indicate that Grb2 ameliorates HGAL-mediated enhancement of BCR signaling by decreasing HGAL binding to Syk.In summary, out data demonstrates that Grb2 directly interacts with HGAL and ameliorates HGAL-enhanced BCR signaling. These interactions may play an important function in regulating the magnitude of BCR signaling during the GC reaction. DisclosuresNo relevant conflicts of interest to declare.

Interaction of HGAL with Grb2 Enhances RhoA and B-Cell Receptor Signaling

AbstractAbstract 681Expression of the Human Germinal center Associated Lymphoma (HGAL) gene is restricted to germinal center (GC) B-lymphocytes and GC-derived lymphomas. HGAL expression identifies lymphomas characterized by improved survival. Previously we have demonstrated that HGAL decreases spontaneous and chemoattractant-induced cell motility by 1) interaction with F-actin and myosin II, inhibiting the ability of myosin to translocate actin; 2) activating RhoA signaling by direct interactions with RhoA-specific guanine nucleotide exchange factors. We recently demonstrated that HGAL enhances B-cell receptor (BCR)-mediated Syk activation by directly binding to and enhancing Syk kinase activity. However, the precise molecular mechanisms of the HGAL effects on these signaling pathways and requirement for cofactors have yet to be described. Examination of the HGAL protein sequence revealed the presence of a putative growth factor receptor-bound protein 2 (Grb2) binding motif (YYENV; aa106-110). Grb2 is an abundant adaptor protein with important functions in multiple intracellular pathways, including BCR and RhoA signaling. We examined whether Grb2 may directly interact with the HGAL protein and be involved in regulation of HGAL-mediated signaling. Confocal microscopy studies demonstrated colocalization of HGAL and Grb2 proteins in Raji and HeLa cell lines, the latter transiently expressing HGAL protein. Concordantly, coimmunoprecipitation experiments in VAL and Raji lymphoma cell lines detected endogenous Grb2 in immunoprecipitates of endogenous HGAL and vice versa. GST pull down assay using recombinant GST-Grb2 failed to pull down non-phosphorylated TRX-HGAL protein. In contrast, TRX-HGAL protein in vitro phosphorylated by Lyn kinase was detected in the GST-Grb2 pull down, confirming a direct interaction between these proteins and demonstrating requirement for HGAL phosphorylation, as would be predicted from the YYENV binding motif. We further elucidated the relevance of this interaction by evaluating the effects of siRNA mediated knockdown of Grb2, HGAL or both proteins together on BCR and RhoA signaling in lymphoma cell lines. In accordance with our previous data, HGAL knockdown led to a significant decrease in phosphorylated Syk (Y352) following BCR stimulation. A similar decrease in Syk(P352) phosphorylation was observed following knockdown of Grb2 alone or simultaneously with HGAL. Concordantly, BCR-induced Ca2+ mobilization was decreased significantly and to a similar extent following HGAL or Grb2 knockdown. Concomitant knockdown of HGAL and Grb2 did not lead to additive or synergistic decrease in BCR-induced Ca2+ mobilization. Similarly, knockdown of HGAL or Grb2 decreased fibronectin(FN)-induced RhoA-GTP levels. To further elucidate the importance of the HGAL-Grb2 interaction, we have generated a plasmid encoding a HGAL(Y106FY107F) protein in which the putative Grb2 binding site was mutated. The HGAL(Y106FY107F) protein is not interacting with the Grb2 protein, as demonstrated by coimmunoprecipitation studies. Expression of the wild type HGAL protein in HeLa cells markedly increased both FN-induced RhoA-GTP levels and RhoA-mediated serum response factor (SRF)-induced transcriptional activity from the serum responsive element (SRE). In contrast, the HGAL(Y106FY107F) mutant did not affect either FN-induced RhoA-GTP levels or SRF-induced transcriptional activity from the SRE. These observations suggest that the HGAL-Grb2 interaction is important for mediating HGAL's effect on RhoA signaling pathway. HGAL was previously shown to inhibit lymphoma cell motility by activating the RhoA signaling pathway. Indeed, siRNA mediated HGAL knockdown significantly increased lymphoma cell chemotaxis in response to IL-6 and SDF1. Knockdown of Grb2 similarly increased IL-6-induced lymphoma chemotaxis, with no further increases in IL-6 induced chemotaxis observed flowing concomitant HGAL and Grb2 knockdown. In contrast, compared to the HGAL knockdown, knockdown of Grb2 significantly increased lymphoma cell chemotaxis in response to SDF-1. Concomitant knockdown of both Grb2 and HGAL resulted in an ever greater increase in lymphoma cell motility in response to SDF-1. In summary, our findings suggest that interaction of HGAL with Grb2 protein enhances both RhoA and B-cell receptor signaling and may be necessary for mediating at least some of the HGAL effects. Disclosures:No relevant conflicts of interest to declare.

HGAL-a Germinal Center Specific Protein, Enhances B-Cell Receptor Signaling by Activation of Syk, Leading to Follicular Lymphoproliferation

Abstract 584The 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.

HGAL Protein Expression Persists in Disorders of Germinal Center Dissolution

Human Germinal Center-associated Lymphoma (HGAL) is a germinal center (GC) B-cell marker associated with a favorable outcome in diffuse large B-cell and classic Hodgkin lymphomas (CHL). To test its potential role in GC function, 75 cases involving GC disruption including 23 progressive transformation of germinal centers (PTGC), 25 follicle lysis and 27 Castleman disease (CD) were studied. HGAL protein expression uniformly correlated with GC B-cells in all except a subset of hyaline-vascular CD that showed severe regression of GCs. HGAL staining highlighted dismantled GCs in PTGC, in contrast to weak or absent CD10 and BCL6 staining. In follicle lysis, HGAL staining was comparable to that of CD10, BCL6, and CD21 in highlighting lysed follicles. Our findings show that HGAL protein expression effectively discriminates clusters of GC B-cells in disrupted follicles. Its persistence in disrupted GCs, suggests that it may be necessary for GC maintenance and supports its proposed role of confining B-cells to the GC microenvironment.

Germinal center-specific protein human germinal center associated lymphoma directly interacts with both myosin and actin and increases the binding of myosin to actin

Human germinal center associated lymphoma (HGAL) is a germinal center-specific gene whose expression correlates with a favorable prognosis in patients with diffuse large B-cell and classic Hodgkin lymphomas. HGAL is involved in negative regulation of lymphocyte motility. The movement of lymphocytes is directly driven by actin polymerization and actin-myosin interactions. We demonstrate that HGAL interacts directly and independently with both actin and myosin and delineate the HGAL and myosin domains responsible for the interaction. Furthermore, we show that HGAL increases the binding of myosin to F-actin and inhibits the ability of myosin to translocate actin by reducing the maximal velocity of myosin head/actin movement. No effects of HGAL on actomyosin ATPase activity and the rate of actin polymerization from G-actin to F-actin were observed. These findings reveal a new mechanism underlying the inhibitory effects of germinal center-specific HGAL protein on lymphocyte and lymphoma cell motility.

Human Germinal Center-Associated Lymphoma (HGAL) Protein Expression Is Associated with Improved Failure-Free Survival in Brazilian Patients with Classical Hodgkin Lymphoma.

Abstract 4632 BACKGROUNDThe HGAL gene has prognostic value in diffuse large B-cell lymphoma and expression of its cognate protein is germinal center–specific. A previous study had suggested that HGAL protein expression might also be related to outcome in patients with Hodgkin lymphoma. PATIENTS AND METHODSThe aim of the current study was to confirm the prognostic impact of HGAL protein expression in an independent, well-characterized cohort of 232 classic Hodgkin lymphoma patients treated uniformly with the ABVD regimen from 1997 to 2004 in Brazil. The median follow-up was 6.2 years. RESULTSTissue microarray analysis showed HGAL staining in 188 specimens (81%). The overall survival was better in patients with young age, early-stage, absence of B symptoms, low-risk international prognostic score (IPS) and good performance status. Failure-free survival was superior in patients with early-stage disease, low-risk IPS and HGAL-positive patients. The estimated 5-year failure-free survival for HGAL-positive and HGAL-negative patients was 82% and 67%, respectively (P=0.03). When stage, B symptoms and performance status were included along with HGAL in a multivariate analysis, advanced stage and absence of HGAL staining were independent predictors of a worse failure-free survival. CONCLUSIONThis study confirms and validates recent findings of a correlation between HGAL expression and outcome in classical Hodgkin lymphoma. Disclosures:No relevant conflicts of interest to declare.

Human germinal center-associated lymphoma protein expression is associated with improved failure-free survival in Brazilian patients with classical Hodgkin lymphoma

The human germinal center-associated lymphoma (HGAL) gene has prognostic value in diffuse large B-cell lymphoma, and expression of its cognate protein is germinal center-specific. A previous study had suggested that HGAL protein expression might also be related to the outcome in patients with Hodgkin lymphoma (HL). The aim of this study was to confirm the prognostic impact of HGAL protein expression in an independent, well-characterized cohort of 232 patients with classic HL treated uniformly with doxorubicin, bleomycin, vinblastine and dacarbazine (ABVD). Tissue microarray analysis showed HGAL staining in 188 specimens (81%). Failure-free survival (FFS) was superior in patients with early-stage disease, low-risk IPS, and HGAL-positive patients. The estimated 5-year FFS for HGAL-positive and HGAL-negative patients was 82% and 67%, respectively (p = 0.03). In the multivariate analysis, advanced stage and absence of HGAL staining were independent predictors of a worse FFS. This study confirms and validates recent findings of a correlation between HGAL expression and outcome in classical HL.

Human germinal center-associated lymphoma protein expression is associated with improved failure-free survival in Brazilian patients with classical Hodgkin lymphoma

Human germinal center-associated lymphoma protein expression is associated with improved failure-free survival in Brazilian patients with classical Hodgkin lymphoma

HGAL - A Germinal Center-Like Diffuse Large B-Cell Lymphoma Prognostic Biomarker Interacts with the Cytoskeleton and Influences Cell Migration.

Gene expression profiling studies of lymphoid malignancies have led to the discovery of previously unrecognized lymphoma subtypes and associated novel genes. We recently cloned a germinal center (GC) specific gene - human germinal center-associated lymphoma (HGAL), whose expression correlates with survival in patients with diffuse large B-cell lymphoma (DLBCL) and classical Hodgkin lymphoma (HL). HGAL protein contains an ITAM motif and harbors 6 tyrosines that may be potentially phosphorylated. Mice deficient in the HGAL homologue M17 form normal GCs with the exception of reduced-sized Peyer's patches, undergo efficient class-switch recombination and somatic hypermutation, and mount T-cell-dependent antibody responses similar to wild-type controls (Schenten et al., Blood 2006). The biological functions of HGAL in normal GC lymphocytes and lymphoma are unknown. Herein we demonstrate that HGAL plays a role in regulation of cell migration. Ex-vivo exposure of lymphoma or HGAL-transfected HeLa cell-lines to pervanadate resulted in tyrosine phosphorylation of HGAL protein. Stimulation of lymphoma and HGAL-transfected HeLa cell-lines with interleukin (IL)-6 led to transient HGAL tyrosine phosphorylation, peaking at 5–15 minutes. The phosphorylated HGAL had a markedly shortened life time compared to the unphosphorylated protein. No HGAL tyrosine phosphorylation was observed upon stimulation with anti IgM, interferon-g or IL-4. Deletion of the two C-terminal ITAM tyrosines in a truncated HGAL mutant (amino-acids 1-118) totally abrogated HGAL tyrosine phosphorylation upon IL-6 stimulation or exposure to pervanadate. Immunofluorescence and confocal miscroscopic studies of the SUDHL6 lymphoma and transfected HeLa cell-lines demonstrated HGAL redistribution from mainly perinuclear cytoplasmic localization to podosomes and spike-like filopodia. The time-frame of the HGAL relocalization was similar to that of HGAL tyrosine phosphorylation, being most prominent at 5 and 10 minutes in the SUDHL6 and transfected HeLa cells, respectively. Immunofluorescence studies demonstrated HGAL colocalization with actin, myosin and WASP proteins. HGAL immunoprecipitation studies followed by MAS spectroscopy or Western blotting confirmed HGAL interaction with actin and myosin type 2, but not with WASP protein. The N terminal portion of HGAL protein (aa 1-118) was sufficient for these interactions, however HGAL phosphorylation at its C-terminal tyrosines increased and extended the duration of its interaction with the myosin type 2.We further assessed the effects of HGAL on cell migration. Upon IL-6 stimulation for 24 hours, the migration of the Neo-HeLa cells in wound assay was markedly increased. However this effect of IL-6 was significantly ameliorated in the HGAL-HeLa cells. Moreover, siRNA knockdown of the HGAL in the HGAL-HeLa cells markedly reversed the HGAL-induced inhibition of IL-6 stimulated cell migration. This IL-6 effect on HGAL-HeLa and control Neo-HeLa cells was not attributable to differences in cell proliferation. Collectively, our results suggest that HGAL is involved in negative regulation of lymphocyte migration, thus constraining lymphocytes to the GC. Furthermore, inhibition of lymphocyte migration might contribute to the less aggressive clinical behavior of HGAL expressing DLBCL and HL tumors.

Expression of the Human Germinal Center Associated Lymphoma (HGAL) Protein Identifies a Subset of Classical Hodgkin Lymphoma of Germinal Center Derivation and Improved Outcome.

Classical Hodgkin lymphoma (CHL) exhibit crippled somatic mutations in rearranged Ig genes and an immunophenotype unlike any other hematolymphoid malignancy. Expression profiles of CHL cell lines show that CHL is most closely related to EBV-transformed peripheral blood B-cells with marked loss of B lineage-specific gene expression. The Human Germinal center-Associated Lymphoma (HGAL) gene was identified as a candidate molecule that predicted improved outcome in diffuse large B-cell lymphoma. Previously, we showed that HGAL mRNA and protein are expressed specifically in germinal centers (GC), GC-derived lymphomas and a substantial proportion of CHL. Here, we sought to investigate whether HGAL protein expression could serve to distinguish biologically distinct subgroups of CHL. Ninety informative cases of CHL on a tissue microarray of 1.0 mm representative cores of paraffin embedded tissue were used. Staining was performed and the percent of stained Hodgkin cells was scored on a 3-tiered scale as negative (0%), weak (<30%), and strong (>30%), as previously described (Natkunam et al. Blood 2005, 105:3979–86). These cutoffs were chosen before results were correlated with clinical endpoints. Twenty-five patients (28%) showed no staining for HGAL and 25 patients (28%) had weak staining; HGAL was strongly expressed in 40 patients (44%). Thirteen of the 90 patients had died and 19 had failed (i.e. relapsed or died). The overall 5-year survival (OS) and failure-free survival (FFS) rates were 87% + 4% and 79% + 4%, respectively. HGAL expression was associated with improved OS in univariate analysis (p=0.04, no vs weak/strong staining), as were age < 45 years (p<0.001), stage I or II (p=0.02), and possibly histology (nodular sclerosing vs other, p=0.09). However, in multivariate analysis with age, stage and histology, HGAL expression no longer had an impact (p=0.93). HGAL did not impact FFS in either univariate (p=0.13) or multivariate analysis (p=0.87). The expression of the GC-specific marker HGAL in a subset of CHL suggests that a substantial proportion of CHL retain characteristics of lymphomas of GC derivation. The association with improved OS in univariate but not multivariate analysis suggests HGAL may be a biologic marker related to known clinical parameters of improved OS and FFS. Comparative studies with well-characterized GC markers, BCL6 and CD10, and with BCL2, MUM1 and Blimp-1 are underway to further define biologically distinct subsets of CHL. Analysis of additional cases of CHL is also on-going to substantiate these findings in multivariate analysis.

Expression of the human germinal center–associated lymphoma (HGAL) protein, a new marker of germinal center B-cell derivation

AbstractWe identified the human germinal center-associated lymphoma (HGAL) in gene-expression profiling studies of diffuse large B-cell lymphoma (DLBCL). The expression of HGAL correlated with survival in patients with DLBCL. The HGAL gene is the human homolog of M17, a mouse gene expressed specifically in normal germinal center (GC) B cells. We generated a monoclonal antibody against the HGAL protein and show that HGAL is expressed in the cytoplasm of GC lymphocytes and in lymphomas of GC derivation. Among 727 lymphomas tested by immunohistochemistry on tissue microarrays, HGAL staining was found in follicular lymphomas (103 of 107), Burkitt lymphomas (40 of 40), mediastinal large B lymphomas (7 of 8), and in DLBCLs (103 of 151). Most marginal zone lymphomas lacked HGAL staining. Lymphocyte-predominant Hodgkin lymphomas (12 of 17) and, surprisingly, classical Hodgkin lymphomas (78 of 107) were found to be positive. Hierarchical clustering of comparative immunohistologic results in DLBCLs demonstrates that the expression of HGAL is similar to 2 other GC-associated proteins, BCL6 and CD10, but different from 2 markers associated with a non-GC phenotype, MUM1/IRF4 and BCL2. The restricted expression and GC specificity of HGAL protein suggest that it may have an important role in the diagnosis of specific lymphomas, and, potentially in the identification of subtypes associated with different prognoses.

Expression of the Human Germinal Center-Associated Lymphoma (HGAL) Protein, a New Marker of Germinal Center B Cell Derivation.

We identified the Human Germinal center-Associated Lymphoma (HGAL) gene in gene expression profiling studies of diffuse large B-cell lymphoma (DLBCL). The expression of HGAL was correlated with survival of patients with this diagnosis. The HGAL gene is the human homologue of M17, a mouse gene that had been previously identified to be expressed specifically in normal germinal center B cells. Here we generated a monoclonal antibody against the HGAL protein and show that HGAL is expressed in the cytoplasm of GC-lymphocytes and in lymphomas considered to be derived from the GC. Among 718 lymphomas tested by immunohistochemistry on tissue microarrays, staining for HGAL protein was present in 97% (103/107) of follicular lymphoma, 100% (40/40) of Burkitt lymphoma, 87% (7/8) of mediastinal large B-cell lymphoma and 70% (103/151) of DLBCL. Nodal and extranodal marginal zone lymphomas lacked HGAL expression. Lymphocyte predominant Hodgkin (70%, 12/17) and, surprisingly, a majority of cases of classical Hodgkin (73%, 78/107) lymphomas were found to be positive.Double-immunohistologic labeling experiments show that HGAL protein expression overlaps with the expression of BCL6 and CD10 proteins within GCs. Hierarchical clustering of comparative immunohistologic results in 151 cases of DLBCL demonstrates that the expression of HGAL is similar to two other GC-associated proteins BCL6 and CD10, but different from two markers associated with a non-GC phenotype, MUM1/IRF4 and BCL2. The restricted expression and GC-specificity of HGAL protein suggest that it may have an important role in the diagnosis of specific lymphomas, and, potentially in the identification of subtypes associated with different prognoses.