Franziska Jundt

c-FLIP Mediates Resistance of Hodgkin/Reed-Sternberg Cells to Death Receptor–induced Apoptosis

Resistance to death receptor–mediated apoptosis is supposed to be important for the deregulated growth of B cell lymphoma. Hodgkin/Reed-Sternberg (HRS) cells, the malignant cells of classical Hodgkins lymphoma (cHL), resist CD95-induced apoptosis. Therefore, we analyzed death receptor signaling, in particular the CD95 pathway, in these cells. High level CD95 expression allowed a rapid formation of the death-inducing signaling complex (DISC) containing Fas-associated death domain–containing protein (FADD), caspase-8, caspase-10, and most importantly, cellular FADD-like interleukin 1β–converting enzyme-inhibitory protein (c-FLIP). The immunohistochemical analysis of the DISC members revealed a strong expression of CD95 and c-FLIP overexpression in 55 out of 59 cases of cHL. FADD overexpression was detectable in several cases. Triggering of the CD95 pathway in HRS cells is indicated by the presence of CD95L in cells surrounding them as well as confocal microscopy showing c-FLIP predominantly localized at the cell membrane. Elevated c-FLIP expression in HRS cells depends on nuclear factor (NF)-κB. Despite expression of other NF-κB–dependent antiapoptotic proteins, the selective down-regulation of c-FLIP by small interfering RNA oligoribonucleotides was sufficient to sensitize HRS cells to CD95 and tumor necrosis factor–related apoptosis-inducing ligand–induced apoptosis. Therefore, c-FLIP is a key regulator of death receptor resistance in HRS cells.

Intrinsic inhibition of transcription factor E2A by HLH proteins ABF-1 and Id2 mediates reprogramming of neoplastic B cells in Hodgkin lymphoma

B cell differentiation is controlled by a complex network of lineage-restricted transcription factors. How perturbations to this network alter B cell fate remains poorly understood. Here we show that classical Hodgkin lymphoma tumor cells, which originate from mature B cells, have lost the B cell phenotype as a result of aberrant expression of transcriptional regulators. The B cell–specific transcription factor program was disrupted by overexpression of the helix-loop-helix proteins ABF-1 and Id2. Both factors antagonized the function of the B cell–determining transcription factor E2A. As a result, expression of genes specific to B cells was lost and expression of genes not normally associated with the B lineage was upregulated. These data demonstrate the plasticity of mature human lymphoid cells and offer an explanation for the unique classical Hodgkin lymphoma phenotype.* NOTE: In the version of this article initially published online, the directions to the panels for Figure 6e were incorrect in the legend and text. The legend for this panel should begin as follows: “Immunoblot (top), EMSA (bottom left) and RT-PCR (bottom right)….” The accompanying text should read as follows: “Transfection of L428 cells with a combination of these siRNAs efficiently reduced ABF-1 protein expression (Fig. 6e, top) and resulted in a substantial loss of E2A–ABF-1 DNA-binding activity (Fig. 6e, bottom left). After reduction of ABF-1 expression, we noted considerable downregulation of CSF1R and TCF7 expression and a moderate suppression of GATA3 expression (Fig. 6e, bottom right).” The error has been corrected for the HTML and print versions of the article.

Sp1 as G1 cell cycle phase specific transcription factor in epithelial cells

Sp1 binding sites have been identified in enhancer/promoter regions of several growth and cell cycle regulated genes, and it has been shown that Sp1 is increasingly phosphorylated in G1 phase of the cell cycle. Interactions of Sp1 with proteins involved in control of cell cycle and tumor formation have been reported. Here we show that expression of Sp1 protein predominates in the G1 phase of the cell cycle in epithelial cells. This is achieved by proteasome-dependent degradation. Inhibition of endogeneous Sp1 activity by a dominant-negative Sp1 mutant was associated with a cell cycle arrest in G1 phase, a strongly reduced expression of cyclin D1, the EGF-receptor and increased levels of p27Kip1. We have thus identified Sp1 as an important regulator of the cell cycle in G1 phase.

Aberrant expression of the Th2 cytokine IL-21 in Hodgkin lymphoma cells regulates STAT3 signaling and attracts Treg cells via regulation of MIP-3α

The malignant Hodgkin/Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (HL) are derived from mature B cells, but have lost a considerable part of the B cell-specific gene expression pattern. Consequences of such a lineage infidelity for lymphoma pathogenesis are currently not defined. Here, we report that HRS cells aberrantly express the common cytokine-receptor gamma-chain (gamma(c)) cytokine IL-21, which is usually restricted to a subset of CD4(+) T cells, and the corresponding IL-21 receptor. We demonstrate that IL-21 activates STAT3 in HRS cells, up-regulates STAT3 target genes, and protects HRS cells from CD95 death receptor-induced apoptosis. Furthermore, IL-21 is involved in up-regulation of the CC chemokine macrophage-inflammatory protein-3alpha (MIP-3alpha) in HRS cells. MIP-3alpha in turn attracts CCR6(+)CD4(+)CD25(+)FoxP3(+)CD127(lo) regulatory T cells toward HRS cells, which might favor their immune escape. Together, these data support the concept that aberrant expression of B lineage-inappropriate genes plays an important role for the biology of HL tumor cells.

Differential Eµ enhancer activity and expression of BOB.1/OBF.1, Oct2, PU.1, and immunoglobulin in reactive B-cell populations, B-cell non-Hodgkin lymphomas, and Hodgkin lymphomas

It has previously been demonstrated that in cultured and in situ tumour cells of classical Hodgkin lymphoma (cHL), the immunoglobulin (Ig) promoter is inactive and its transcription factors Oct2 and/or BOB.1/OBF.1 are down‐regulated. In this study, the analysis of these transcription factors has been extended to a broad spectrum of B‐cell malignancies and the findings have been related to the situation in normal B‐cells of various differentiation stages and to the expression of Ig. Furthermore, an additional Ig transcription factor, PU.1, recently described to be absent from cHL, and a further regulatory element of the Ig gene, the intronic Eµ enhancer, have been studied. BOB.1/OBF.1 and Oct2 were present in all B‐cells expressing Ig, whereas PU.1 proved to be absent from late B‐cell differentiation stages and from a subset of germinal centre B‐cells. Interestingly, there were several normal (eg germinal centre centroblasts and monocytoid B‐cells) and malignant B‐cell populations (eg a proportion of diffuse large B‐cell lymphomas, DLBCLs) that were Ig‐negative, despite their BOB.1/OBF.1 and Oct2 expression. This study further shows that absence of PU.1 alone, as well as inactivation of the intronic Eµ enhancer, is not sufficient to down‐regulate Ig transcription. Taken together, the simultaneous absence of PU.1, Oct2, and/or BOB.1/OBF.1 is unique to Hodgkin and Reed–Sternberg (HRS) cells and cannot be detected in normal B‐cell subsets or B‐cell non‐Hodgkin lymphomas (B‐NHLs). This supports the concept that the down‐regulation of Ig in cHL does not reflect a physiological situation, but a defect probably closely linked to the pathogenesis of cHL. Copyright

Aberrant expression of Notch1 interferes with the B-lymphoid phenotype of neoplastic B cells in classical Hodgkin lymphoma

Plasticity of committed mouse B cells has been demonstrated by inactivation of the B-cell commitment transcription factor PAX5, resulting in loss of the B-cell phenotype and differentiation into various hematopoietic lineages. Furthermore, mature mouse B cells could be reprogrammed into macrophages by overexpression of myeloid-specific transcription factors. Here, we report that aberrant activity of the transmembrane receptor, Notch1, interferes with the B-lymphoid phenotype of mature human germinal center-derived B cells in Hodgkin lymphoma, so called Hodgkin and Reed–Sternberg cells. They have lost the B-cell phenotype despite their mature B-cell origin. Notch1 remodels the B-cell transcription factor network by antagonizing the key transcription factors E2A and early B-cell factor (EBF). Through this mechanism, B lineage-specific genes were suppressed and B lineage-inappropriate genes were induced. We provide evidence that absence of the Notch inhibitor Deltex1 contributes to deregulated Notch activity in Hodgkin and Reed–Sternberg cells. These data suggest that Notch activation interferes with dedifferentiation of neoplastic B cells in Hodgkin lymphoma.

Pathogenic Long-Lived Plasma Cells and Their Survival Niches in Autoimmunity, Malignancy, and Allergy

Long-lived plasma cells survive in a protected microenvironment for years or even a lifetime and provide humoral memory by establishing persistent Ab titers. Long-lived autoreactive, malignant, and allergen-specific plasma cells are likewise protected in their survival niche and are refractory to immunosuppression, B cell depletion, and irradiation. Their elimination remains an essential therapeutic challenge. Recent data indicate that long-lived plasma cells reside in a multicomponent plasma cell niche with a stable mesenchymal and a dynamic hematopoietic component, both providing essential soluble and membrane-bound survival factors. Alternative niches with different hematopoietic cell components compensate fluctuations of single cell types but may also harbor distinct plasma cell subsets. In this Brief Review, we discuss conventional therapies in autoimmunity and multiple myeloma in comparison with novel drugs that target plasma cells and their niches. In the future, such strategies may enable the specific depletion of pathogenic plasma cells while leaving the protective humoral memory intact.

Notch is an essential upstream regulator of NF-κB and is relevant for survival of Hodgkin and Reed-Sternberg cells.

A major pathogenetic mechanism in classical Hodgkin lymphoma (cHL) is constitutive activation of canonical nuclear factor-κB (NF-κB) p50/p65 signaling, controlling lymphoma cell proliferation and survival. Recently, we demonstrated that aberrant Notch1 activity is a negative regulator of the B cell program in B cell-derived Hodgkin and Reed–Sternberg (HRS) cells. Despite abundant evidence for a complex context-dependent cross talk between Notch and NF-κB signaling in hematopoietic cells, it is unknown whether these pathways interact in HRS cells. Here, we show that Notch-signaling inhibition in HRS cells by the γ-secretase inhibitor (GSI) XII results in decreased alternative p52/RelB NF-κB signaling, interfering with processing of the NF-κB2 gene product p100 into its active form p52. As a result, expression of Notch and NF-κB target genes is reduced, and survival of HRS cells is impaired. Stimulation of alternative NF-κB signaling in the Hodgkin cell line L540cy by activation of the CD30 receptor rescued GSI-mediated loss of cell viability and apoptosis induction. Our data reveal that Notch is an essential upstream regulator of alternative NF-κB signaling and indicate cross talk between both the pathways in HRS cells. Therefore, we suggest that targeting the Notch pathway is a promising therapeutic option in cHL.

Notch signaling in leukemias and lymphomas.

Aberrant Notch activation is linked to cancer since 1991 when mammalian Notch1 was first identified as part of the translocation t(7;9) in a subset of human T-cell acute lymphoblastic leukemias (T-ALL). Since then oncogenic Notch signaling has been found in many solid and hematopoietic neoplasms. Depending on tumor type Notch interferes with differentiation, proliferation, survival, cell-cycle progression, angiogenesis, and possibly self-renewal. In hematopoietic neoplasms, recent findings indicate an important role of Notch for T-ALL induction and progression and the pathogenesis of human T- and B-cell-derived lymphomas. Notch signaling has been identified as a potential new therapeutic target in these hematopoietic neoplasms. This review will focus on the most recent findings on Notch signaling in leukemias and lymphomas and its potential role in the maintenance of malignant stem cells.

Notch inhibition blocks multiple myeloma cell-induced osteoclast activation

We have read with interest the paper by Ning et al. reporting a correlation between the cotransplantation of mesenchymal stem cells (MSCs) in allogeneic haemapoietic stem cell transplantation and a higher recurrence rate of malignant haematologic diseases. The authors concluded their paper by suggesting that ‘the use of MSCs must be handled with extreme caution before a large-scale trial is performed’. Recently, several studies have reported the ability of MSCs, when co-cultured with activated peripheral blood mononuclear cells or directly activated by exogenous Interleukin 10, to modulate membrane bound and soluble human leukocyte antigen-G (HLA-G) antigens. HLAG antigens are nonclassical HLA-class I molecules characterized by tolerogenic and anti-inflammatory functions. In particular, both membrane and soluble HLA-G molecules have been demonstrated to inhibit Natural killer and CD8þ T cell mediated cytolysis, CD4þ T lymphocyte proliferation and dendritic cell maturation. Furthermore, the expression of HLA-G antigens has been associated with the induction of regulatory T cells. Overall, it is currently accepted that HLA-G molecules, by direct and indirect mechanisms, can inhibit innate and adaptive immune responses. The production of sHLA-G molecules by MSCs has suggested, in addition to other mechanisms, a rationale for the immunomodulatory properties of MSCs in preventing graft versus host disease (GVHD). Several researches have demonstrated that HLA-G modulation represents a beneficial event in organ transplantations, autoimmune diseases and pregnancy where the downregulation of the immune response is essential for a positive outcome. In contrast, the presence of HLA-G antigens has been associated with clinical negative consequences in cancer and viral infections where the tolerogenic function of these molecules permits the mutated/infected cells to avoid the innate and adaptive immune responses. In particular, the HLA-G expression by cancer tissues and the relationship between plasma sHLA-G levels and cancer development confirm the role of HLA-G molecules in sustaining the immune escape of cancer cells. The association between the cotransplantation of MSCs and the development of malignant haematologic diseases reported by Ning et al., could be related to the functional ability of HLA-G molecules, on the one hand, to counteract GVHD but on the other to permit the relapse of the disease. This hypothesis underlines the necessity of further studies to analyze plasma sHLA-G concentrations in MSCs of cotransplanted patients in a longitudinal follow-up. The detection of a significant correlation between sHLA-G concentrations, GVHD prevention and relapse rate could identify a possible cutoff in sHLA-G plasma levels responsible for the occurrence of these two phenomena.