AP-1 family transcription factors have been implicated in the control of proliferation, apoptosis and malignant transformation. However, their role in oncogenesis is unclear and no recurrent alterations of AP-1 activities have been described in human cancers. Here, we show that constitutively activated AP-1 with robust c-Jun and JunB overexpression is found in all tumor cells of patients with classical Hodgkin's disease. A similar AP-1 activation is present in anaplastic large cell lymphoma (ALCL), but is absent in other lymphoma types. Whereas c-Jun is up-regulated by an autoregulatory process, JunB is under control of NF-kappa B. Activated AP-1 supports proliferation of Hodgkin cells, while it suppresses apoptosis of ALCL cells. Furthermore, AP-1 cooperates with NF-kappa B and stimulates expression of the cell-cycle regulator cyclin D2, proto-oncogene c-met and the lymphocyte homing receptor CCR7, which are all strongly expressed in primary HRS cells. Together, these data suggest an important role of AP-1 in lymphoma pathogenesis.

Notch signaling controls cell fate decisions of hematopoietic progenitors by inhibiting certain steps of differentiation and inducing either self-renewal or differentiation toward lymphoid or myeloid lineages. In addition, truncated Notch1 alleles could be associated with 10% of all cases of human T lymphoblastic leukemia and, when introduced into mouse bone marrow stem cells, cause T-cell neoplasms. However, functional links between the abundant expression of intact Notch1 and oncogenesis are still lacking. Here we show that Notch1 is highly expressed in B-and T-cell-derived tumor cells of Hodgkin and anaplastic large cell lymphoma. We demonstrate a novel mechanism for the oncogenic capacity of Notch1 by showing that the interaction between intact Notch1 on tumor cells and its ligand Jagged1 dramatically induces proliferation and inhibition of apoptosis in vitro. We further provide evidence that in Hodgkin and anaplastic large cell lymphoma, Jagged1 is expressed in malignant and in bystander cells colocalizing with Notch1-positive tumor cells. Notch1 signaling may therefore be activated in tumor cells by Jagged1 through homotypic or heterotypic cell-cell interactions, and it seems likely that these interactions contribute to lymphomagenesis in vivo. Thus, our data suggest that activated Notch1 signaling plays an important role in the pathobiology of Hodgkin and anaplastic large cell lymphoma and that it might be a potential new target for treatment. ( It has further been demonstrated that HRS cells contain nonfunctional immunoglobulin (Ig) genes, suggesting that they are derived from germinal center cells that should have been negatively selected but were rescued from apoptosis by cellular transforming events. 3,4 Our previous work has provided evidence that constitutive NF-B activity is a survival factor for HRS cells. [5][6][7][8] We have directly manipulated the NF-B system and overexpressed a dominant-negative version of the inhibitor IB␣. HRS cells depleted of constitutive nuclear NF-B reveal decreased proliferation rates, enhanced apoptotic response, and strongly impaired tumor growth in severe combined immunodeficient mice. 7 To investigate molecular alterations of the NF-B/IB system that might be responsible for constitutive NF-B activity, we have analyzed the IB␣ gene. 9 We and others have demonstrated mutations of the IB␣ gene in a subset of HD cases that contribute to constitutive NF-B activation and are involved in the pathogenesis of HD. 9,10 To evaluate additional molecular mechanisms that lead to constitutive NF-B activity in HRS cells, and because activated Notch signaling has been implicated in the regulation of NF-B, 11-13 we analyzed Notch1 gene expression in cultured and primary HRS cells.Notch1 belongs to a family of transmembrane receptors that control cell proliferation and differentiation in response to extracellular ligands expressed on neighboring cells. 14-18 Notch1 has been isolated as a translocation in human acute T-cell lymphoblastic leukemia-lymphoma, 19 and its constitutively ac...

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.

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 Hodgkin's 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.

Notch receptors expressed on hematopoietic stem cells interact with their ligands on bone marrow stromal cells and thereby control cell fate decisions and survival. We recently demonstrated that Notch signaling is involved in proliferation and survival of B cell-derived tumor cells of classic Hodgkin disease and described a novel mechanism for the oncogenic capacity of Notch. In this study we investigated whether Notch signaling is involved in the tight interactions between neoplastic plasma cells and their bone marrow microenvironment, which are essential for tumor cell growth in multiple myeloma (MM). Here we demonstrate that Notch receptors and their ligand Jagged1 are highly expressed in cultured and primary MM cells, whereas nonneoplastic counterparts show low to undetectable levels of Notch. Functional data indicate that ligandinduced Notch signaling is a growth factor for MM cells and suggest that these interactions contribute to myelomagenesis in vivo.

IntroductionAlthough organic and inorganic arsenic-containing compounds are environmental toxins, they have been used for more than 100 years for the treatment of human diseases. 1,2 Salvarsan, active against syphilis, 3 or melarsoprol, still used for the treatment of African sleeping sickness, 4 are examples of organic arsenicals. Fowler solution, which was used for the treatment of chronic myeloid leukemia 5 (CML) or psoriasis, 6 is an example of inorganic arsenic. Most of these drugs have been replaced by others, as it became clear that arsenic causes diverse pathologies and increases the risk of cancer. 7,8 Since the discovery that arsenic trioxide (As 2 O 3 ) is an efficient drug for the treatment of acute promyelocytic leukemia 9 (APL), As 2 O 3 was reintroduced in current therapeutic concepts. Initial clinical studies focused on the treatment of hematopoietic malignancies. Currently, the action of arsenic on many other tumor entities is under investigation. 10,11 Depending on the cell type and the applied concentrations of arsenic, diverse cellular effects are observed (for a recent review, see Miller et al 12 ). Low concentrations can induce differentiation or cell cycle arrest, whereas high concentrations can induce apoptosis. Mechanisms leading to induction of apoptosis were extensively studied. An important role was attributed to arsenic-triggered degradation of specific proteins with prosurvival function. The strongest correlation was found with the degradation of the promyelocytic leukemia/retinoic acid receptor-␣ (PML/RAR-␣) protein in APL, 13 but also degradation of the viral Tax protein in human T-cell lymphotropic virus (HTLV)-induced T-cell leukemia has been reported. 14 However, these mechanisms cannot explain the induction of apoptosis in many other cell types. As a general finding, all 3 groups of mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK), p38, and Jun Nterminal kinase (JNK), are activated in response to arsenic. 15,16 This activation may be responsible for the carcinogenic effects of arsenic, but MAPK activation is not required for arsenic-induced apoptosis. 17 Furthermore, the generation of reactive oxygen species (ROS) has been implicated in arsenic-mediated apoptosis. 18 The biologic effects of arsenic may be attributed to structural and functional alterations of critical cellular proteins by its reactivity with sulfhydryl groups. 19 The resulting loss of function of specific enzymes, including kinases and phosphatases, functionally alters diverse signaling pathways. For example, arsenicmediated inhibition of MAPK phosphatases, which contain cysteines in their catalytic pocket, induces MAPK activation. 16 Arsenic exerts an opposite effect on the IB kinase (IKK) complex. 20 The IKK complex, which is composed of the 2 catalytic IKK␣ and IKK␤ subunits and the regulatory IKK␥/NEMO component, is the central mediator of nuclear factor-B (NF-B)-inducing stimuli. 21 Activation of the IKK complex results in phosphorylation of IBs, which subsequently ar...

The immunosuppressive macrolide rapamycin and its derivative everolimus (SDZ RAD, RAD) inhibit the mammalian target of rapamycin (mTOR) signaling pathway. In this study, we provide evidence that RAD has profound antiproliferative activity in vitro and in NOD/SCID mice in vivo against Hodgkin lymphoma (HL) and anaplastic large cell lymphoma (ALCL) cells. Moreover, we identified 2 molecular mechanisms that showed how RAD exerts antiproliferative effects in HL and ALCL cells. RAD down-regulated the truncated isoform of the transcription factor CCAAT enhancer binding protein ␤ (C/ EBP␤), which is known to disrupt terminal differentiation and induce a transformed phenotype. Furthermore, RAD inhibited constitutive nuclear factor B (NF-B) activity, which is a critical sur- IntroductionHodgkin lymphoma (HL) and anaplastic large cell lymphoma (ALCL) share morphologic and immunophenotypic markers in a subgroup of cases although they are biologically distinct entities. 1 Therefore, pathologic diagnosis is sometimes difficult to achieve and these cases are classified as "gray-zone lymphomas." 2 Moreover, in both entities novel therapeutic options are needed, as curative therapy of HL is compromised by a high risk of long-term complications, and anaplastic lymphoma kinase (ALK)-negative ALCL still has a very unfavorable prognosis with current treatment strategies. [3][4][5] The macrocyclic lactone everolimus (SDZ, RAD) RAD is a rapamycin derivative with potent immunosuppressive and antiproliferative properties. [6][7][8][9][10] It is further known to inhibit growth factor-driven cell proliferation of hematopoietic and nonhematopoietic cells. 6,10 In addition, RAD is a potent inhibitor of human Epstein-Barr virus (EBV)-transformed B lymphocytes in vitro and in vivo, arresting cell-cycle progression and increasing the apoptotic rate of EBV ϩ B cells. 10 Therefore, it has been suggested that RAD might be effective in the prevention and treatment of human posttransplant lymphoproliferative disorders. 10 Here, we investigated whether RAD inhibits tumor cell proliferation of HL and ALCL. We show that RAD significantly inhibits proliferation of HL and ALCL cells in vitro and arrests cell-cycle progression in G 0 /G 1 . Furthermore, we demonstrate that in vivo, RAD markedly suppresses tumor cell proliferation of HL and ALCL cells, xenotransplanted into NOD/SCID mice. Our data suggest that RAD might be used in combination chemotherapy for the treatment of HL and ALCL. Moreover, we studied the mechanisms of proliferation arrest mediated by the mammalian target of rapamycin (mTOR) inhibitor RAD to identify the molecular targets in HL and ALCL. The mTOR pathway controls the translation initiation machinery in response to nutrients and growth factors thereby coordinating cell growth with cell division. 11 A transcription factor that is a critical target of mTOR is the CCAAT enhancer binding protein (C/EBP)␤. 11-13 C/EBP␤ has previously been identified as an essential downstream target in tumors expressing activated cyclin D1. 14 Our dat...

Multiple myeloma (MM) is a lethal human cancer characterized by a clonal expansion of malignant plasma cells in bone marrow. Mouse models of human MM are technically challenging and do not always recapitulate human disease. Therefore, new mouse models for MM are needed. Mineral-oil induced plasmacytomas (MOPC) develop in the peritoneal cavity of oil-injected BALB/c mice. However, MOPC typically grow extramedullary and are considered poor models of human MM. Here we describe an in vivo-selected MOPC315 variant, called MOPC315.BM, which can be maintained in vitro. When injected i.v. into BALB/c mice, MOPC315.BM cells exhibit tropism for bone marrow. As few as 104 MOPC315.BM cells injected i.v. induced paraplegia, a sign of spinal cord compression, in all mice within 3–4 weeks. MOPC315.BM cells were stably transfected with either firefly luciferase (MOPC315.BM.Luc) or DsRed (MOPC315.BM.DsRed) for studies using noninvasive imaging. MOPC315.BM.Luc cells were detected in the tibiofemoral region already 1 hour after i.v. injection. Bone foci developed progressively, and as of day 5, MM cells were detected in multiple sites in the axial skeleton. Additionally, the spleen (a hematopoietic organ in the mouse) was invariably affected. Luminescent signals correlated with serum myeloma protein concentration, allowing for easy tracking of tumor load with noninvasive imaging. Affected mice developed osteolytic lesions. The MOPC315.BM model employs a common strain of immunocompetent mice (BALB/c) and replicates many characteristics of human MM. The model should be suitable for studies of bone marrow tropism, development of osteolytic lesions, drug testing, and immunotherapy in MM.