Hodgkin Lymphoma-derived Cell Lines Research Articles

Mutations in CD58 and Other Immune System Related Genes in Hodgkin Lymphoma

Hodgkin Lymphoma (HL) is a B cell derived malignancy characterized by a minority of tumor cells, known as Hodgkin Reed-Sternberg (HRS) cells. The background is composed of a wide variety of inflammatory cells with T cells representing the largest population. Chemokines and cytokines produced by HRS cells and by the infiltrating cells shape the environment and provide proliferative and survival signals to the HRS cells. Despite this critical dependence on the microenvironment, HRS cells also need to apply mechanisms to escape from both antigen-dependent and innate immune responses. HRS cells have evolved multiple mechanisms to evade cytotoxic T cell (CTL) and natural killer (NK) cell mediated anti-tumor responses. These mechanisms include secretion of immune-suppressive factors (IL10, TGFβ and others), recruitment of regulatory and helper T cells, expression of PDL1 and CD95 and loss of HLA expression. Recent publications show that mutations in immune system related genes might represent a mechanism of HRS cells to evade detection by immune cells. The aim of this study was to validate whole exome sequencing results of seven HL cell lines focusing on immune system associated genes. We previously showed that B2M mutations affect the ATG start codon in L428 (heterozygous) and DEV (homozygous) cells. B2M mRNA levels were reduced in both cell lines as compared to L1236, whereas HLA-A, HLA-B and HLA-C mRNA levels were in the same range. Consistent with these findings we observed no membranous B2M and HLA class I expression by flow cytometry in the two cell lines with mutated B2M genes. In primary diagnostic HL tissue we showed lack of membranous B2M in 51% of the cases. We now studied two additional genes in more detail. CD58 gene mutations were observed in KMH2 and DEV cells. By manual inspection of the alignments using the Integrative Genomics Viewer (IGV), we also noticed a lack of reads of exons 1, 2 and 3 in SUPHD1. Heterozygous mutations and homozygous loss of exons 1-3 were confirmed for all three cell lines. CD58 mRNA levels were low or absent in SUPHD1 and KMH2 cells and normal in DEV. CD58 protein expression as determined by flow, western blot and IHC was low or absent in all 3 mutated HL cell lines in comparison to four cell lines with wild type CD58. Tumor cells of 36 primary HL cases with good treatment outcome showed a strong CD58 expression in all cases. As HL cell lines are derived from end stage HL patients, we next studied CD58 expression in relapsed HL patients. No or weak CD58 staining was observed in HRS cells in 6 out of 45 patients who experienced a relapse. Our results indicate that mutations in CD58 and loss of CD58 expression are common in HL derived cell lines and that loss of CD58 expression in tumor cells is restricted to relapsed HL patients. Heterozygous CSF2RB mutations in KMH2, SUPHD1, DEV and L1236 were validated by RNA-seq and Sanger-seq. As CSF2RB encodes the common β chain (CD131) shared by the interleukin-3 (IL-3), granulocytic macrophage colony-stimulating factor (GM-CSF) and IL-5 receptors, we also measured the expression of these 3 α chain receptors. We observed the same expression pattern between CD131 and CD116 (GM-CSF α receptor chain) in HL cell lines by flow cytometry suggesting that these mutations mainly affect the GM-CSF receptor. In conclusion, we show that mutations of immune system genes are common in HL. Deleterious mutations in B2M explain the lack of HLA class I expression, indicating that this genetic alteration is responsible for defective antigen presentation. Deleterious mutations or deletions of CD58 exons result in loss of CD58 protein expression. This will lead to loss of binding to CD2 expressed on T cells and will result in a defect in T cell adhesion and activation. Overall these results indicate that mutations are likely to contribute to the immune escape mechanisms applied by the HRS cells. DisclosuresNo relevant conflicts of interest to declare.

Bruton's tyrosine kinase (Btk) is a useful marker for Hodgkin and B cell non-Hodgkin lymphoma.

Bruton's tyrosine kinase (Btk) is a member of the Tec family of protein tyrosine kinases involved in B cell development and proliferation in neoplastic human lymphoid tissues. We used immunohistochemistry to evaluate a polyclonal anti-Btk antibody on formalin-fixed paraffin-embedded tissue blocks. The tested samples included normal lymphoid tissues, tissue samples of 395 different lymphomas and 14 malignant lymphoid cell lines. Btk was expressed more often in B cell lymphomas than in T cell lymphomas. This correlated well with the results obtained on B cell lymphoma cell lines, which strongly expressed Btk, in contrast to T cell lymphoma cell lines. More than 60% of myelomas expressed Btk. Among Hodgkin lymphomas, the nodular lymphocyte predominant variant was more often positive (14/16) than the classical variant (6/27). Only one out of three Hodgkin lymphoma-derived cell lines showed a few atypical large cells expressing Btk. Btk represents a useful marker to identify B cell non-Hodgkin lymphomas. Furthermore, Btk might help to distinguish the nodular lymphocyte predominant variant of Hodgkin lymphomas from the classical form. Finally, in view of the recently discovered therapeutic potential of Btk inhibitors in lymphoma, we report the pattern of expression of Btk in a large collection of different types of lymphoma.

Glycoproteomic Profiling of Hodgkin Lymphoma Reveals Novel Proteins that Contribute to the Microenvironment and Have Diagnostic Utility

Background: Proteins with N-linked glycosylation are frequently expressed on cell membranes and/or secreted, and represent idea biomarkers with diagnostic and therapeutic utility. In this study, we carried out unbiased identification of N-linked glycoproteins of Hodgkin lymphoma-derived cell lines.

Recurrent somatic mutations of PTPN1 in primary mediastinal B cell lymphoma and Hodgkin lymphoma

Classical Hodgkin lymphoma and primary mediastinal B cell lymphoma (PMBCL) are related lymphomas sharing pathological, molecular and clinical characteristics. Here we discovered by whole-genome and whole-transcriptome sequencing recurrent somatic coding-sequence mutations in the PTPN1 gene. Mutations were found in 6 of 30 (20%) Hodgkin lymphoma cases, in 6 of 9 (67%) Hodgkin lymphoma-derived cell lines, in 17 of 77 (22%) PMBCL cases and in 1 of 3 (33%) PMBCL-derived cell lines, consisting of nonsense, missense and frameshift mutations. We demonstrate that PTPN1 mutations lead to reduced phosphatase activity and increased phosphorylation of JAK-STAT pathway members. Moreover, silencing of PTPN1 by RNA interference in Hodgkin lymphoma cell line KM-H2 resulted in hyperphosphorylation and overexpression of downstream oncogenic targets. Our data establish PTPN1 mutations as new drivers in lymphomagenesis.

The EBV oncogene LMP1 protects lymphoma cells from cell death through the collagen-mediated activation of DDR1

AbstractThe malignant Hodgkin and Reed-Sternberg (HRS) cells of Hodgkin lymphoma are surrounded by a tumor microenvironment that is composed of a variety of cell types, as well as noncellular components such as collagen. Although HRS cells harbor oncogenic Epstein-Barr virus (EBV) in approximately 50% of cases, it is not known if the tumor microenvironment contributes to EBV-driven lymphomagenesis. We show that expression of the EBV-encoded latent membrane protein-1 (LMP1) in primary human germinal center B cells, the presumed progenitors of HRS cells, upregulates discoidin domain receptor 1 (DDR1), a receptor tyrosine kinase activated by collagen. We also show that HRS cells intimately associated with collagen frequently overexpress DDR1 and that short-term exposure to collagen is sufficient to activate DDR1 in Hodgkin lymphoma-derived cell lines. The ectopic expression of DDR1 significantly increased the survival of collagen-treated DG75 Burkitt lymphoma cells, following etoposide treatment. Conversely, knockdown of DDR1 significantly decreased the survival of collagen-treated L428 Hodgkin lymphoma cells in the absence of specific apoptotic stimulus, suggesting that DDR1 also influences baseline survival. Our results identify a hitherto unknown function for collagen in protecting Hodgkin lymphoma cells from apoptosis and suggest an important contribution of the tumor microenvironment in promoting the oncogenic effects of EBV.

The EBV Oncogene LMP1 Protects Lymphoma Cells From Cell Death Through The Collagen-Mediated Activation Of Discoidin Domain Receptor 1

The malignant Hodgkin/Reed-Sternberg (HRS) cells of Hodgkin lymphoma are surrounded by a tumour microenvironment which is composed of a variety of cell types, as well as non-cellular components such as collagen. Although HRS cells harbour oncogenic Epstein-Barr virus (EBV) in approximately 50% of cases, it is not known if the tumour microenvironment contributes to EBV-driven lymphomagenesis. We show that expression of the EBV-encoded latent membrane protein-1 (LMP1) in primary human germinal centre B cells, the presumed progenitors of HRS cells, up-regulates discoidin domain receptor 1 (DDR1), a receptor tyrosine kinase activated by collagen. We show that HRS cells intimately associated with collagen frequently over-express DDR1 and that short-term exposure to collagen is sufficient to activate DDR1 in Hodgkin lymphoma-derived cell lines. We also show that ectopic expression of DDR1 significantly increased the survival of collagen-treated DG75, Burkitt lymphoma cells, following etoposide treatment. Conversely, inhibition of DDR1, by RNA interference, significantly decreased the survival of collagen-treated L428 Hodgkin lymphoma cells in the absence of specific apoptotic stimulus suggesting that DDR1 also influences baseline survival. Our results identify a hitherto unknown function for collagen in protecting Hodgkin lymphoma from apoptosis and suggest an important contribution of the tumour microenvironment in promoting the oncogenic effects of EBV. Disclosures:No relevant conflicts of interest to declare.

The pan-deacetylase inhibitor panobinostat induces cell death and synergizes with everolimus in Hodgkin lymphoma cell lines

AbstractThe pan-deacetylase inhibitor panobinostat (LBH589) recently has been shown to have significant clinical activity in patients with relapsed Hodgkin lymphoma, but its mechanism of action in Hodgkin lymphoma remains unknown. In this study, we demonstrate that panobinostat has potent antiproliferative activity against Hodgkin lymphoma–derived cell lines. At the molecular level, panobinostat activated the caspase pathway, inhibited STAT5 and STAT6 phosphorylation, and down-regulated hypoxia-inducible factor 1 α and its downstream targets, glucose transporter 1 (GLUT1) and vascular endothelial growth factor. Paradoxically, panobinostat inhibited LKB1 and AMP-activated protein kinase, leading to activation of mammalian target of rapamycin (mTOR) that promotes survival. Combining panobinostat with the mTOR inhibitor everolimus (RAD001) inhibited panobinostat-induced mTOR activation and enhanced panobinostat antiproliferative effects. Collectively, our data demonstrate that panobinostat is a potent deacetylase inhibitor against Hodgkin lymphoma–derived cell lines, and provide a mechanistic rationale for combining panobinostat with mTOR inhibitors for treating Hodgkin lymphoma patients. Furthermore, the effect of panobinostat on GLUT1 expression suggests that panobinostat may modulate the results of clinical diagnostic imaging tests that depend of functional GLUT1, such as fluorodeoxyglucose positron emission tomography.

The Pan-Deacetylase Inhibitor Panobinostat Downregulates HIF-1α and VEGF and, Synergizes with Everolimus In Hodgkin Lymphoma Cell Lines

AbstractAbstract 2851Deacetylases (DAC) inhibitors are promising new class of anticancer agents. Panobinostat (LBH589) is a pan-DAC inhibitor with evidence of clinical activity in patients with relapsed classical Hodgkin Lymphoma (HL). However the mechanisms of action of this new drug are not completely understood. The aim of this study was to investigate the mechanisms of antiproliferative effect induced by LBH589 in HL-derived cell lines. Experiments were performed in HL derived-cell lines (HDLM-2, KM-H2 and L-428). Cells were treated with 0.01–1μM of LBH589 alone or in combination with 0.01–1μM of everolimus (RAD001), an inhibitor of the mammalian target of rapamycin (mTOR), for 24–72 hours. Growth inhibition and apoptosis were analyzed in response to treatment using MTS assay, Western blotting and flow cytometric analyses. Effect of panobinostat on a panel of 30 cytokines and chemokines was assayed on cells after incubation of 24 hours using a multiplex assay. Panobinostat demonstrated antiproliferative activity in HL cell lines in a dose-and time-dependant manner with an IC50 ranging between 20 and 40nM at 72 hours. The antiproliferative activity was associated with downregulation of the X-linked inhibitor of apoptosis protein (XIAP), activation of caspase 9 and caspase 3, cleavage of Poly ADP Ribosome polymerase (PARP) and induction of apoptosis. In addition, panobinostat downregulated the level of the transcription factor hypoxia-inducible factor 1 alpha (HIF-1α) a major regulator of tumor cell adaptation to hypoxic stress. Furthermore, panobinostat decreased the level of the vascular endothelial growth factor (VEGF), an HIF-1α target gene, in the cell culture supernatants.The combination of panobinostat and everolimus had synergistic antiproliferative activity in the cells lines. This synergy was due to reciprocal inhibition of negative feedback loops induced by mTOR inhibitor and panobinostat therapy. Collectively, our data demonstrate that panobinostat induces apoptosis in HL cell lines by modulating several survival pathways. Furthermore, the observed synergy between panobinostat and everolimus provides rationale for combining these two active agents in a phase I/II clinical trial in patients with relapsed HL. Disclosures:No relevant conflicts of interest to declare.

HDAC inhibitors induce cell cycle arrest, activate the apoptotic extrinsic pathway and synergize with a novel PIM inhibitor in Hodgkin lymphoma-derived cell lines

HDAC inhibitors induce cell cycle arrest, activate the apoptotic extrinsic pathway and synergize with a novel PIM inhibitor in Hodgkin lymphoma-derived cell lines

Depsipeptide induces cell death in Hodgkin lymphoma-derived cell lines

A variety of genetic and epigenetic abnormalities were characterized over the last years in Hodgkin and Reed-Sternberg (H-RS) cells of classic Hodgkin Lymphoma (cHL). It was speculated that simultaneous inhibition of multiple signalling pathways might be a promising strategy to target this tumor entity. In the present study we tested the effect of histone deacetylase (HDAC) inhibition using depsipeptide (also known as romidepsin, FK228, FR901228 or NSC-630176) in cHL cell lines in vitro. Molecular mechanisms of toxicity were analyzed using RNA expression analysis and functional assays. It is shown that depsipeptide is effective at submicromolar concentrations and acts mainly by apoptosis induction, upregulation of p21 and cell cycle inhibition in G2/M. Of special note, HDAC mediated toxicity in H-RS cells does not require RelA/p65 downregulation, which was previously shown to drive the malignant phenotype of H-RS cells. In summary, depsipeptide induced protein acetylation results in transcriptional changes of a large number of pathogenetically relevant genes and increased RelA/p65 binding activity in cHL cell lines. Our preclinical data suggest that HDAC inhibition using depsipeptide might be a promising approach for the treatment of cHL patients.

Novel Findings Support a Patophysiological Role of the Arachidonic Cascade in Hodgkin Lymphoma.

Lipoxygenases (LO) are a family of structurally related enzymes which catalyze the conversion of the fatty acid arachidonic acid to biologically active metabolites. The key enzyme in leukotriene synthesis, 5- LO, catalyzes the first step in the metabolism of arachidonic acid to leukotriene (LT) C4. This compound and its metabolites LTD4 and LTE4, collectively called cysteinyl leukotrienes (cysLT), can bind to two high-affinity receptors, named cysLT1 and cysLT2. A sister enzyme to 5-LO is 15-LO-1 which also can catalyze the formation of bioactive metabolites. Arachidonic acid metabolites have traditionally been linked to inflammation and asthma but several studies also indicate a role of these metabolites in carcinogenesis In our studies of arachidonic acid metabolism in human lymphomas, we have identified the expression of 15-LO-1 and receptors for cysLT in primary as well as cultured Hodgkin-Reed Sternberg (H-RS) cells. In tumor tissue, H-RS cells positive for 15-LO-1 and the cysLT1 receptor were detected immunohistochemically in 13/15 and 12/16 cases, respectively. The presence of mRNA for 15-LO-1 and the cysLT1 and 2 receptors was confirmed by microarray analysis of laser dissected H-RS cells. Studies of 15-LO-1 gene transcription revealed that STAT6 activation and chromatin remodeling by DNA demethylation and histone acetylation are crucial for transcriptional activation of this gene in cultured H-RS cells. Incubation of the Hodgkin lymphoma-derived cell lines KMH2 and L1236 with LTD4 led to a robust calcium response that was completely abolished in presence of the cysLT1 receptor inhibitor zafirlukast. In L1236 cells, LTD4 induced increased DNA synthesis, interleukin-13 transcription and release of TNF-alpha, interleukin-6 and interleukin-8 in a dose-dependent manner, all of which were inhibited by zafirlukast. Thus, as the H-RS cells lack the expression of 5-LO, we hypothesize that that tumor-associated cysLT-producing inflammatory cells, such as eosinophils and mast cells, contribute to the pathogenesis of Hodgkin lymphoma through induction of tumor cell proliferation and cytokine release. Furthermore, immunohistochemical studies of 20 non-Hodgkin lymphomas revealed the expression of cysLT1 receptors also in primary mediastinal B cell lymphomas (PMBCL), and the functionality of these receptors was confirmed in the PMBCL-derived cell line MedB1. Taken together, these novel findings support a role for arachidonic acid metabolites in the pathogenesis of lymphoma.

Comprehensive identification of proteins in Hodgkin lymphoma-derived Reed–Sternberg cells by LC-MS/MS

Mass spectrometry-based proteomics in conjunction with liquid chromatography and bioinformatics analysis provides a highly sensitive and high-throughput approach for the identification of proteins. Hodgkin lymphoma is a form of malignant lymphoma characterized by the proliferation of Reed–Sternberg cells and background reactive lymphocytes. Comprehensive analysis of proteins expressed and released by Reed–Sternberg cells would assist in the discovery of potential biomarkers and improve our understanding of its pathogenesis. The subcellular proteome of the three cellular compartments from L428 and KMH2 Hodgkin lymphoma-derived cell lines were fractionated, and analyzed by reverse-phase liquid chromatography coupled with electrospray ionization tandem mass spectrometry. Additionally, proteins released by Hodgkin lymphoma-derived L428 cells were extracted from serum-free culture media and analyzed. Peptide spectra were analyzed using TurboSEQUEST® against the UniProt protein database (5.26.05; 188 712 entries). A subset of the identified proteins was validated by Western blot analysis, immunofluorescence microscopy and immunohistochemistry. A total of 1945 proteins were identified with 785 from the cytosolic fraction, 305 from the membrane fraction, 441 from the nuclear fraction and 414 released proteins using a minimum of two peptide identifications per protein and an error rate of <5.0%. Identification of proteins from diverse functional groups reflected the functional complexity of the Reed–Sternberg proteome. Proteins with previously reported oncogenic function in other cancers and from signaling pathways implicated in Hodgkin lymphoma were identified. Selected proteins without previously demonstrated expression in Hodgkin lymphoma were validated by Western blot analysis (B-RAF, Erb-B3), immunofluorescence microscopy (Axin1, Tenascin-X, Mucin-2) and immunohistochemistry using a tissue microarray (BRAF, PIM1). This study represents the first comprehensive inventory of proteins expressed by Reed–Sternberg cells of Hodgkin lymphoma and demonstrates the utility of combining cellular subfractionation, protein precipitation, tandem mass spectrometry and bioinformatics analysis for comprehensive identification of proteins that may represent potential biomarkers of the disease.

Autocrine NGFβ/TRKA signalling is an important survival factor for Hodgkin lymphoma derived cell lines

Hodgkin-Reed/Sternberg cells, the tumor cells of Hodgkin lymphoma, aberrantly express several receptor tyrosine kinases, among them TRKA whose stimulation supports B cell survival. We show here high expression of TRKA in Hodgkin-Reed/Sternberg cell lines as compared to normal B cells and other B cell lymphomas, without major increases in TRKA gene dosage. A fraction of TRKA is constitutively activated, likely due to the coexpression of NGFβ, the TRKA high affinity ligand. The TRK inhibitor K-252a decreased survival of Hodgkin-Reed/Sternberg cell lines accompanied by decreased AKT activation. Inclusion of TRK inhibitors in therapeutic regimens may thus be an interesting possibility.

Pharmacologic activation of p53-dependent and p53-independent apoptotic pathways in Hodgkin/Reed-Sternberg cells

The status of the p53 pathway in classical Hodgkin lymphoma (cHL) remains unclear, and a lack of proven TP53 mutations contrasts with often high expression levels of p53 protein. In this study, we demonstrate that pharmacologic activation of the p53 pathway with the murine double minute 2 (MDM2) antagonist nutlin-3 in Hodgkin lymphoma-derived cell lines leads to effective apoptosis induction and sensitizes the cells to other anticancer drugs. Cells with mutant p53 are resistant to nutlin-3, but sensitive to geldanamycin, a pharmacologic inhibitor of heat shock 90 kDa protein (HSP90), indicating that HSP90 inhibition can induce apoptosis in a p53-independent manner. Conversely, cells with defects in the HSP90/nuclear factor-kappa B pathway expressing wild-type p53 are more resistant to geldanamycin, but still sensitive to nutlin-3. Our results suggest that selective activation of p53 by MDM2 antagonists as a single agent or in combination with conventional chemotherapeutics and/or inhibitors of p53-independent survival pathways may offer effective treatment options for patients with cHL. Importantly, because nutlins and HSP90 inhibitors are non-genotoxic agents, their use might offer a means to reduce the genotoxic burden of current chemotherapeutic regimens.

Heat Shock Protein 90 (HSP90) Is Overexpressed in Primary and Cultured Hodgkin Lymphoma (HL) Cells: Role of Therapeutic Targeting Using the Small Molecule Inhibitor 17-AAG.

Conventional chemotherapy is the golden standard for therapy of Hodgkin Lymphoma (HL). Nevertheless, considerable toxicity and secondary malignancies indicate the need for targeted therapy that preferentially kills the malignant cells. The molecular chaperone heat shock protein 90 (HSP90) is expressed in all mammalian cells, but it is overexpressed in malignancy. 17-AAG, a small molecule inhibitor of HSP90, has been shown to induce apoptosis and cell cycle arrest in a variety of tumor types. In the present study we show that HSP90 is overexpressed in the primary Hodgkin and Reed-Sternberg (HRS) cells, as well as in HL derived cells lines. Inhibition of HSP90 17-AAG showed antiproliferative effect in HL derived cell lines in a dose dependent manner. Cell death was due to apoptosis, as determined by Annexin-V staining and FACS analysis. Apoptosis was mediated by the activation of the caspase pathway, especially by caspase 8, 9, and 3. Inhibition of caspase activity by the pancaspase inhibitor Z-VAD-FMK partially reversed the 17-AAG lethal effect. 17-AAG had no significant on the level of the antiapoptotic Bcl-2 family members or the cellular or X-Linked inhibitors of apoptosis. In contrast, there was considerable degradation of cFLIP. Moreover, 17-AAG treatment reduced the intracellular levels of molecules that have been shown to be of key importance in HRS cell survival and proliferation, including AKT and the phosphorylated ERK1/2, but with minimal change in total ERK1/2. Cell cycle arrest was observed at G0/G1 or at G2/M phase, and was mediated by reduction in the levels of MDM2, cyclin D1 with cdk4 and cdk6, and cyclin B1. The potential synergy of 17-AAG with conventional chemotherapy and anti-TRAIL death receptor monoclonal antibody, was explored by the simultaneous incubation of HL derived cells with both doxorubicin or antibodies against TRAIL receptors R1 and R2, respectively. The combination of 17-AAG with doxorubicin or anti-TRAIL antibodies was significantly more effective than either agent alone. Based on these data we are conducting a phase II study of 17-AAG in patients with relapsed classical HL.

Constitutive activation of STAT proteins in the HDLM-2 and L540 Hodgkin lymphoma-derived cell lines supports cell survival

Survival and proliferation of Hodgkin lymphoma (HL) cells are influenced by many cytokines produced by different cell types in the lymph node microenvironment. STAT, family of transcription factors, are key mediators of cytokine signaling and their perturbation contributes to various human diseases. Electrophoretic mobility shift and phosphoprotein immunoblotting analyses were used to study STAT activation in HL cell lines. We thus observed high levels of constitutively activated STAT1, 3, 5 and 6 in HDLM-2 and L540 cells, which could be correlated with JAK kinase activation. In contrast KM-H2 cells did not display STAT activation. Preventing constitutive STAT activation by specific JAK kinases inhibitors induced apoptosis of HL cell lines and was associated with a strong decrease in the expression of the anti-apoptotic genes IAP-1, IAP-2, Bcl-xL, Bfl1 and Traf1. Silencing of JAKs by specific siRNAs also induced apoptosis of HL cells. Altogether, these results suggest that aberrant STAT activation in Hodgkin cells may promote cell survival and as a consequence facilitate oncogenic transformation.

The FOXP1 Transcription Factor is Expressed in the Majority of Follicular Lymphomas but is Rarely Expressed in Classical and Lymphocyte Predominant Hodgkin’s Lymphoma

The expression of the FOXP1 forkhead/winged helix transcription factor has been investigated in normal and neoplastic lymphoid tissues using the FOXP1-specific monoclonal antibody, JC12. Using single and double immunoenzymatic staining, FOXP1 expression has been studied in a series of classical and lymphocyte predominant Hodgkin's lymphomas, follicle centre lymphomas and Hodgkin's lymphoma-derived cell lines. Neoplastic cells in the majority of classical and lymphocyte predominant Hodgkin's lymphoma were FOXP1-negative. In two cases of classical Hodgkin's lymphoma, the tumour cells showed mislocalisation of FOXP1 to the cytoplasm, while in one case of lymphocyte predominant Hodgkin's lymphoma, and in the Hodgkin's lymphoma cell line KMH2, scattered tumour cells showed nuclear expression of FOXP1. In contrast, the tumour cells in the majority of follicle centre lymphomas showed strong nuclear FOXP1 reactivity. Double labelling studies of lymphoid tissue indicated that a variable proportion of CD20-positive germinal centre B cells express FOXP1 while the vast majority of CD30-positive cells are FOXP1-negative. The heterogeneity of FOXP1 expression in germinal centre-derived lymphomas, may have more to do with the transforming events underlying these distinct types of lymphoma than with their common origin.

Epigenetic silencing of the immunoglobulin heavy-chain gene in classical Hodgkin lymphoma-derived cell lines contributes to the loss of immunoglobulin expression

AbstractImmunoglobulin production is impaired in Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (cHL) in spite of functional clonal rearrangements. The presence of “crippling” mutations in coding and regulatory regions, as well as down-regulation of B-cell-specific transcription factors, has been suggested as a potential reason for the lack of immunoglobulin (Ig) chain gene transcription. We have investigated the impact of epigenetic silencing in suppressing Ig heavy (H)-chain expression. Chromatin immunoprecipitation (ChIP) was used to analyze transcription factor binding to octamer motifs present in the IgH regulatory regions. Transcription factors were bound to these motifs in control cell lines, however, they were absent in the cHL-derived cell lines KMH2, L1236, and L428. Ectopic expression of octamer-binding transcription factor (Oct2) and/or B-cell Oct binding protein/Oct-binding factor (BOB.1/OBF.1) did not result in any measurable binding to these sites. Increased histone 3 Lysine 9 (H3-K9) methylation was observed in the promoter region of the IgH locus in L428 and L1236 cells. This is a typical feature of heterochromatic, transcriptionally silent regions. Treatment of cHL-derived cell lines with the DNA demethylating agent 5-aza-2′-deoxycytidine (5-aza-dC) partially reactivated IgH transcription and affected chromatin modifications. Our results suggest an important role of epigenetic silencing in the inhibition of IgH transcription in HRS cells. (Blood. 2004;104:3326-3334)

Unique Polycomb Gene Expression Pattern in Hodgkin's Lymphoma and Hodgkin's Lymphoma-Derived Cell Lines

Human Polycomb-group (PcG) genes play a crucial role in the regulation of embryonic development and regulation of the cell cycle and hematopoiesis. PcG genes encode proteins that form two distinct PcG complexes, involved in maintenance of cell identity and gene silencing patterns. We recently showed that expression of the BMI-1 and EZH2 PcG genes is separated during normal B-cell development in germinal centers, whereas Hodgkin/Reed-Sternberg (H/RS) cells co-express BMI-1 and EZH2. In the current study, we used immunohistochemistry and immunofluorescence to determine whether the binding partners of these PcG proteins are also present in H/RS cells and H/RS-derived cell lines. PcG expression profiles were analyzed in combination with expression of the cell cycle inhibitor p16INK4a, because experimental model systems indicate that p16 is a downstream target of Bmi-1. We found that H/RS cells and HL-derived cell lines co-express all core proteins of the two known PcG complexes, including BMI-1, MEL-18, RING1, HPH1, HPC1, and -2, EED, EZH2, YY1, and the HPC2 binding partner, CtBP. Expression of HPC1 has not been found in normal mature B cells and other malignant lymphomas of B-cell origin, suggesting that the PcG expression profile of H/RS is unique. In contrast to Bmi-1 transgenic mice where p16INK4a is down-regulated, 27 of 52 BMI-1POS cases of HL revealed strong nuclear expression of p16INK4a. We propose that abnormal expression of BMI-1 and its binding partners in H/RS cells contributes to development of HL. However, abnormal expression of BMI-1 in HL is not necessarily associated with down-regulation of p16INK4a.

Selectins and anti-CD15 (Lewis x/a) antibodies transmit activation signals in Hodgkin's lymphoma–derived cell lines

Objective The CD15 (Lewis x) cell surface oligosaccharide moiety is expressed in a variety of normal and tumor cells and recognized by selectins. The detection of CD15 on malignant Hodgkin-Reed-Sternberg (HRS) cells serves as a diagnostic marker of Hodgkin's lymphoma (HL). Retrospective studies suggest that the expression of nonsialylated CD15 molecules on HRS cells has a positive prognostic value while presence of sialylated CD15 may correlate with a poor outcome. However, the relevance of the CD15 antigen expression to the pathobiology of the disease is not clear. In this work, we studied the contribution of CD15 to cell adhesion and the activation of signaling cascades in two HL-derived cell lines, KMH-2 and L428. Methods Immobilized anti-CD15 monoclonal antibodies and recombinant E- and P-selectins were used to activate KMH-2 and L428 cells. Immunoblotting, immunoprecipitation, and the electrophoretic mobility shift assay were performed to detect tyrosine phosphorylation of c-Cbl, c-Jun nuclear translocation, and AP-1 DNA binding. Results Treatment of cells with antibodies against the sialylated and nonsialylated forms of CD15, or with immobilized selectins, induced changes in cell morphology. Tyrosine phosphorylation of c-Cbl, together with tyrosine phosphorylation of multiple protein substrates, was also induced. In addition, binding of the CD15 molecules induced nuclear translocation of c-Jun and an increase in AP-1 DNA binding activity. Conclusions We suggest that CD15 has a dual physiological role, both as an adhesion molecule recognized by selectins and as a regulatory molecule upstream to specific intracellular signaling cascades with implications to the pathogenesis of HL.