Functional IKK/NF-κB signaling in pancreatic stellate cells is essential to prevent autoimmune pancreatitis.

Pancreatic stellate cells (PSCs) are resident cells in the exocrine pancreas which contribute to pancreatic fibrogenesis and inflammation. Studies on NF-κB in pancreatitis so far focused mainly on the parenchymal and myeloid compartments. Here we show a protective immunomodulatory function of NF-κB in PSCs. Conditional deletion of NEMO (IKKγ) in PSCs leads to spontaneous pancreatitis with elevated circulating IgM, IgG and antinuclear autoantibodies (ANA) within 18 weeks. When further challenged with caerulein, NEMOΔCol1a2 mice show an exacerbated autoimmune phenotype characterized by increased infiltration of eosinophils, B and T lymphocytes with reduced latency period. Transcriptomic profiling shows that NEMOΔCol1a2 mice display molecular signatures resembling autoimmune pancreatitis patients. Mechanistically, we show that PSCΔNEMO cells produce high levels of CCL24 ex vivo which contributes to eosinophil recruitment, as neutralization with a CCL24 antibody abolishes the transwell migration of eosinophils. Our findings uncover an unexpected immunomodulatory role specifically of NF-κB in PSCs during pancreatitis.

Modeling acute graft-versus-host disease (aGVHD) in murine bone marrow transplantation (BMT) models with MHC disparity.

For more than 50years, hematopoietic stem cell transplantation (HSCT) has been the major curative therapy for hematological malignancies and genetic disorders, but its success is limited by the development of graft-versus-host disease (GVHD). GVHD represents a post-transplantation disorder representing the immune-mediated attack of transplant-derived T cells against recipient tissue finally leading to increased morbidity and mortality of the recipient. GVHD develops if donor and recipient are disparate in major or minor histocompatibility antigens (MHC, miHA). Most of the initial knowledge about the biology of GVHD is derived from murine bone marrow transplantation (BMT) models. Of course, GVHD mouse models do not reflect one to one the human situation, but they contribute significantly to our understanding how conditioning and danger signals activate the immune system, enlighten the role of individual molecules, e.g., cytokines, chemokines, death-inducing ligands, define the function of lymphocytes subpopulations for GVHD development and have significant impact on establishing new treatment and prevention strategies used in clinical HSCT. This chapter describes in detail the procedure of allogeneic BMT and the development of GVHD in two commonly used allogeneic murine BMT models (B6→B6.bm1, B6→B6D2F1) with different MHC disparities, which can be used as a basis for advanced studies of GVHD pathology or the development of new treatment strategies.

Usefulness of BATF3 Immunohistochemistry in Diagnosing Classical Hodgkin Lymphoma.

It is well recognized that the AP-1 transcription factor BATF3 is constitutively expressed in Hodgkin/Reed-Sternberg (HRS) cells, but its potential as a diagnostic marker for classical Hodgkin lymphoma (cHL) has not yet been addressed. In this study, we performed immunohistochemistry and analyzed the BATF3 expression in lymphoma cells on 218 lymphoma samples belonging to 14 different lymphoma entities. We observed varying degrees of BATF3 expression in nearly half of the cases (n = 100) with BATF3 expression being a constitutive feature of cHL (n = 53) and anaplastic large cell lymphoma (ALCL). By scoring BATF3 expression (BATF3-score) we observed constitutively high BATF3-scores in cHL and ALCL and low to moderate BATF3-scores in all other entities examined. Western blot analysis confirmed BATF3 protein expression in cell lysates from cHL cell lines (n = 7). Thus, BATF3 can be considered a useful IHC marker for the diagnosis of cHL as it is highly sensitive and sufficiently specific when analyzed by BATF3-scoring.

U-DCS: characterization of the first permanent human dendritic sarcoma cell line.

A dendritic cell sarcoma cell line, U-DCS, was established from a dendritic cell sarcoma in a 53-year-old Caucasian male patient. Since its establishment, U-DCS has maintained stable phenotypic characteristics in vitro and has a doubling time of approximately 2 days under standard culture conditions. U-DCS is growing with typical dendritic cell morphology in tissue and expresses the dendritic cell sarcoma immunophenotypic markers S100 protein, MHCI, MHCII, and vimentin. Expression analysis revealed transcripts for the toll-like receptors TLR3, -4, -9 and DDX58 (RIG-I), but not for TLR2. U-DCS shows functional features of dendritic cells with the ability of phagocytosis and antigen-specific T cell stimulation. Karyotype-, CGH-, and mFISH analysis point to a chromosomal instability and a hypotetraploid karyotype with approximately 130 chromosomes. U-DCS is the first immortalized human dendritic cell sarcoma cell line and has some morphological and functional features of dendritic cells without dependency on growth factors.

Aneuploidy-inducing gene knockdowns overlap with cancer mutations and identify Orp3 as a B-cell lymphoma suppressor.

Aneuploidy can instigate tumorigenesis. However, mutations in genes that control chromosome segregation are rare in human tumors as these mutations reduce cell fitness. Screening experiments indicate that the knockdown of multiple classes of genes that are not directly involved in chromosome segregation can lead to aneuploidy induction. The possible contribution of these genes to cancer formation remains yet to be defined. Here we identified gene knockdowns that lead to an increase in aneuploidy in checkpoint-deficient human cancer cells. Computational analysis revealed that the identified genes overlap with recurrent mutations in human cancers. The knockdown of the three strongest selected candidate genes (ORP3, GJB3, and RXFP1) enhances the malignant transformation of human fibroblasts in culture. Furthermore, the knockout of Orp3 results in an aberrant expansion of lymphoid progenitor cells and a high penetrance formation of chromosomal instable, pauci-clonal B-cell lymphoma in aging mice. At pre-tumorous stages, lymphoid cells from the animals exhibit deregulated phospholipid metabolism and an aberrant induction of proliferation regulating pathways associating with increased aneuploidy in hematopoietic progenitor cells. Together, these results support the concept that aneuploidy-inducing gene deficiencies contribute to cellular transformation and carcinogenesis involving the deregulation of various molecular processes such as lipid metabolism, proliferation, and cell survival.

Clinical utility of a protein-based oncopanel in patients with end-stage head and neck cancer.

Aim: In a prospective clinical initiative, we selected heavily pretreated head and neck carcinoma patients and assessed the clinical utility of a protein-based oncopanel for identification of potential targetable markers. Patients & methods: Tumor samples of 45 patients were evaluated using a 12-marker immunohistochemistry panel. The primary end point was the prevalence of potentially actionable markers. Results: At least one expressed marker in each case could be identified. We noted a high prevalence of EGFR (80%, 39/45) and MET (57.4%, 28/45). Three patients received oncopanel-based therapy with variable results. Conclusion: Despite the limited number of treated subjects, oncopanel analysis in end-stage head and neck cancer is operationally and technically feasible. Combination with targeted next generation sequencing might provide additional therapy options.

Block of NF-kB signaling accelerates MYC-driven hepatocellular carcinogenesis and modifies the tumor phenotype towards combined hepatocellular cholangiocarcinoma.

Primary liver cancer ranks among the leading causes of cancer death worldwide. Risk factors are closely linked to inflammation, such as viral hepatitis and alcoholic as well as non-alcoholic steatohepatitis. Among the pathways involved in the pathogenesis of malignant liver tumors, dysregulation of NF-κB signaling plays a prominent role. It provides a link between inflammation and cancer. To examine the role of NF-κB in a MYC-induced model of hepatocellular carcinoma we deleted NEMO (IKKγ) specifically from hepatocytes. NEMO deletion accelerated tumor development and shortened survival, suggesting a tumor-suppressive function of NF-κB signaling. We observed increased proliferation, inflammation and fibrosis, as well as activation of MAPK and STAT signaling. Importantly, deletion of NEMO modified the tumor phenotype from hepatocellular carcinoma to combined hepatocellular cholangiocarcinoma. The intrahepatic cholangiocarcinoma tumor component showed increased expression of progenitor markers such as Sox9 and reduced expression of mature hepatic markers such as CPS1. In both cases tumorigenesis was reversible by turning off MYC expression. To our knowledge this is the first mouse model of combined hepatocellular cholangiocarcinoma and may provide insights into the development of this rare malignant tumor.

CK1α overexpression correlates with poor survival in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is the fourth leading cause of cancer related deaths worldwide and prognosis in advanced tumor stage still remains poor. Since CK1 isoforms have been reported to be deregulated in several tumor entities CK1 has emerged as a novel drug target in cancer therapy. In this study we set out to investigate whether CK1α might have the potential to serve as prognostic marker. METHODS: CK1α RNA and protein expression levels in healthy and tumor tissue of CRC patients were analyzed using quantitative real-time PCR and Western Blot analysis, respectively. Prognostic relevance was investigated by correlating obtained CK1α expression levels with patients' survival rate generating Kaplan-Meier survival plots. RESULTS: It could be shown that CK1α is overexpressed in colorectal tumor tissue compared to normal tissue and CK1α overexpression in tumor tissue correlates with poor survival in CRC patients. Results become more significant when only considering patients with high-grade tumors, as well as patients assigned to UICC II and UICC III stage. Furthermore, Cox regression analysis revealed that CK1α is an independent prognostic factor. In addition, tumors expressing decreased levels of the kinase reveal positive effects on overall survival when localized in the right colon compared to those in the left side. CONCLUSION: In summary, this study provides evidence for the first time that CK1α RNA levels might serve as prognostic marker for CRC.

CDK9 is a prognostic marker and therapeutic target in pancreatic cancer.

Despite recent advances in diagnosis and therapy, prognosis of pancreatic cancer still remains very poor. Besides valid prognostic markers, novel therapeutic approaches are urgently needed. The family of cyclin-dependent kinases comprises 20 kinases which contribute to malignancy by promoting proliferation, migration, invasion, and apoptotic resistance of cancer cells. In this work, we investigated the role of CDK9 in pancreatic cancer. Immunohistochemical analysis of CDK9 expression in tumor and normal tissue of pancreatic cancer patients revealed an overexpression of CDK9 in pancreatic cancer tissue. In addition, high CDK9 expression in tumor tissue is associated with significantly shortened survival, especially in well-differentiated tumors. Moreover, the therapeutic potential of selective CDK9 inhibition on pancreatic cancer cells was evaluated by analysis of cell viability, long-term survival, and induction of apoptosis and characterized by western blotting and flow cytometry. Pharmacological CDK9 inhibition by SNS-032 drastically reduced cell viability in pancreatic cancer cells and potently suppressed long-term survival. Analyzing the mechanism of action revealed that CDK9 inhibition induced apoptosis and cell cycle arrest in a time-dependent manner by suppression of anti-apoptotic proteins. Furthermore, CDK9 inhibition potently enhances the therapeutic effect of chemotherapeutics in pancreatic cancer cells. In conclusion, we identified CDK9 as a negative prognostic marker in pancreatic cancer. Furthermore, pharmacological CDK9 inhibition is a novel and promising therapeutic approach for pancreatic cancer.

Canonical NF-κB signaling in hepatocytes acts as a tumor-suppressor in hepatitis B virus surface antigen-driven hepatocellular carcinoma by controlling the unfolded protein response.

UNLABELLED: Chronic hepatitis B virus (HBV) infection remains the most common risk factor for hepatocellular carcinoma (HCC). Efficient suppression of HBV viremia and necroinflammation as a result of nucleos(t)ide analogue treatment is able to reduce HCC incidence; nevertheless, hepatocarcinogenesis can occur in the absence of active hepatitis, correlating with high HBV surface antigen (HBsAg) levels. Nuclear factor κB (NF-κB) is a central player in chronic inflammation and HCC development. However, in the absence of severe chronic inflammation, the role of NF-κB signaling in HCC development remains elusive. As a model of hepatocarcinogenesis driven by accumulation of HBV envelope polypeptides, HBsAg transgenic mice, which show no HBV-specific immune response, were crossed to animals with hepatocyte-specific inhibition of canonical NF-κB signaling. We detected prolonged, severe endoplasmic reticulum stress already at 20 weeks of age in NF-κB-deficient hepatocytes of HBsAg-expressing mice. The unfolded protein response regulator binding immunoglobulin protein/78-kDa glucose-regulated protein was down-regulated, activating transcription factor 6, and eIF2α were activated with subsequent overexpression of CCAAT/enhancer binding protein homologous protein. Notably, immune cell infiltrates and liver transaminases were unchanged. However, as a result of this increased cellular stress, insufficient hepatocyte proliferation due to G1 /S-phase cell cycle arrest with overexpression of p27 and emergence of ductular reactions was detected. This culminated in increased DNA damage already at 20 weeks of age and finally led to 100% HCC incidence due to NF-κB inhibition. CONCLUSION: The role of canonical NF-κB signaling in HCC development depends on the mode of liver damage; in the case of HBsAg-driven hepatocarcinogenesis, NF-κB in hepatocytes acts as a critical tumor suppressor by augmenting the endoplasmic reticulum stress response.

Decreased CK1δ expression predicts prolonged survival in colorectal cancer patients.

Cancers arising from the large intestine or rectum are called colorectal cancer (CRC) and represent the fourth leading cause of cancer-related death worldwide. Since casein kinase 1 (CK1) isoforms are involved in many cellular processes and have been reported to be deregulated in various tumor entities, CK1 has become an interesting drug target. In this study, we examined the potential of CK1δ expression levels in tumor tissue of CRC patients as a prognostic biomarker. We show by quantitative RNA expression analyses that decreased CK1δ expression levels in tumor tissue predict prolonged survival rates. Random sampling of CK1δ stained tumor tissue indicates that CK1δ gene expression corresponds with CK1δ protein expression. Especially in low grade (grade 1, grade 2) and in UICC II/III classified tumors decreased CK1δ RNA levels correlate with significantly improved survival rates when the tumor was located in the right colon. We furthermore found gender-specific differences within these subgroups, revealing most significant increase in overall survival rates in male patients with tumors in right colon expressing low levels of CK1δ RNA. Results become even clearer, when only male patients over 50 years were considered. Together, these findings support the assumption that CK1δ might be a prognostic biomarker for CRC thereby providing an interesting drug target for the development of new therapy concepts.

In vitro-generated MDSCs prevent murine GVHD by inducing type 2 T cells without disabling antitumor cytotoxicity.

Myeloid-derived suppressor cells (MDSCs) inhibit T-cell expansion and functions by versatile mechanisms such as nutrient depletion, nitrosylation, or apoptosis. Since graft-versus-host disease (GVHD) is characterized by the expansion of donor-derived T cells destroying recipient tissue, we analyzed whether MDSCs can be used for GVHD prevention in murine allogeneic bone marrow transplantation models. Transplantation of MDSCs, generated from bone marrow cells by granulocyte-macrophage colony-stimulating factor (GM-CSF)/G-CSF in vitro, inhibited GVHD-induced death and attenuated histologic GVHD, whereas antitumor cytotoxicity of alloantigen-specific T cells was maintained. MDSCs expanded in vivo and invaded lymphatic and GVHD target organs. Major histocompatibility complex class I expression on MDSCs was dispensable for their suppressive capacity. Inhibition of GVHD required the presence of MDSCs during T-cell priming, whereas allogeneic T-cell numbers and homing in lymphoid and GVHD target organs were not considerably affected in MDSC-treated mice. However, MDSCs skewed allogeneic T cells toward type 2 T cells upregulating T helper 2 (Th2)-specific cytokines. Type 2 T-cell induction was indispensable for GVHD prevention since MDSC treatment failed to prevent GVHD when allogeneic STAT6-deficient T cells, which are unable to differentiate into Th2 cells, were transplanted. MDSC-induced Th2 induction might be applicable for GVHD treatment in clinical settings.

Nck adaptor proteins modulate differentiation and effector function of T cells.

Understanding the molecular mechanisms regulating T cell reactivity is required for successful reprogramming of immune responses in medical conditions, characterized by dysfunctions of the immune system. Nck proteins are cytoplasmic adaptors mediating diverse cellular functions, including TCR signaling. By enhancing TCR signal strength, Nck proteins influence thymic selection and regulate the size and sensitivity of the peripheral T cell repertoire. Here, we investigated the contribution of Nck proteins to CD4(+) T cell differentiation and effector function using Nck.T(-/-) mice. Impaired GC formation and reduced Tfh were observed in Nck.T(-/-) mice after immunization with T cell-dependent antigens. Th2/Tfh-related cytokines, such as IL-4, IL-10, and IL-21, were decreased in Nck.T(-/-) mice T cells. Moreover, an increased susceptibility to cell death of Tfh cells in Nck.T(-/-) mice was associated with decreased levels of Akt phosphorylation. As a result of this dysregulation in Tfh cells of Nck.T(-/-) mice, we found impaired production and affinity maturation of antibodies against T cell-dependent antigens. Thus, Nck proteins not only participate in thymic selection and generation of the peripheral T cell repertoire but also are involved in the differentiation and effector functions of CD4(+) T cells.

CK1δ in lymphoma: gene expression and mutation analyses and validation of CK1δ kinase activity for therapeutic application.

The prognosis of lymphoid neoplasms has improved considerably during the last decades. However, treatment response for some lymphoid neoplasms is still poor, indicating the need for new therapeutic approaches. One promising new strategy is the inhibition of kinases regulating key signal transduction pathways, which are of central importance in tumorigenesis. Kinases of the CK1 family may represent an attractive drug target since CK1 expression and/or activity are associated with the pathogenesis of malignant diseases. Over the last years efforts were taken to develop highly potent and selective CK1-specific inhibitor compounds and their therapeutic potential has now to be proved in pre-clinical trials. Therefore, we analyzed expression and mutational status of CK1δ in several cell lines representing established lymphoma entities, and also measured the mRNA expression level in primary lymphoma tissue as well as in non-neoplastic blood cells. For a selection of lymphoma cell lines we furthermore determined CK1δ kinase activity and demonstrated therapeutic potential of CK1-specific inhibitors as a putative therapeutic option in the treatment of lymphoid neoplasms.

Effects of altered expression and activity levels of CK1δ and ɛ on tumor growth and survival of colorectal cancer patients.

Colorectal cancer (CRC) is the fourth leading cause of cancer related death worldwide due to high apoptotic resistance and metastatic potential. Because mutations as well as deregulation of CK1 isoforms contribute to tumor development and tumor progression, CK1 has become an interesting drug target. In this study we show that CK1 isoforms are differently expressed in colon tumor cell lines and that growth of these cell lines can be inhibited by CK1-specific inhibitors. Furthermore, expression of CK1δ and ɛ is changed in colorectal tumors compared to normal bowel epithelium, and high CK1ɛ expression levels significantly correlate with prolonged patients' survival. In addition to changes in CK1δ and ɛ expression, mutations within exon 3 of CK1δ were detected in colorectal tumors. These mutations influence ATP binding resulting in changes in kinetic parameters of CK1δ. Overexpression of these mutants in HT29 cells alters their ability to grow anchorage independently. Consistent with these results, these CK1δ mutants lead to differences in proliferation rate and tumor size in xenografts due to changes in gene expression, especially in genes involved in regulation of cell proliferation, cell cycle, and apoptosis. In summary, our results provide evidence that changes in the expression levels of CK1 isoforms in colorectal tumors correlate with patients' survival. Furthermore, CK1 mutants affect growth and proliferation of tumor cells and induce tumor growth in xenografts, leading to the assumption that CK1 isoforms provide interesting targets for the development of novel effective therapeutic concepts to treat colorectal cancer.

FOXO1 downregulation contributes to the oncogenic program of primary mediastinal B-cell lymphoma.

Recently we have shown that the transcription factor FOXO1, highly expressed in B cells, is downregulated in classical Hodgkin lymphoma (cHL). As primary mediastinal B cell lymphoma (PMBL) has similarities with the cHL transcription program we investigated FOXO1 expression in this entity. By using immunohistochemistry we found that FOXO1 was absent or expressed at low levels in 19 of 20 primary PMBL cases. PMBL cell lines reproduce the low FOXO1 expression observed in primary cases. By analyzing gene expression profiling data we found that FOXO1 expression inversely correlated with JAK2 in PMBL cases. Targeting JAK2 activity by the small molecular weight inhibitor TG101348 resulted in upregulation of FOXO1 mRNA and protein expression in MedB-1 and U2940 cell lines, and the MYC inhibitor 10058-F4 increased FOXO1 mRNA in MedB-1 cells. Moreover, in MedB-1 cells FOXO1 expression was strongly upregulated by the inhibitor of DNA methylation 5-aza-2-deoxycytidine and by the histone deacetylase inhibitor trichostatin A. Since FOXO1 promoter was unmethylated, this effect is most likely indirect. FOXO1 activation in the FOXO1-negative Med-B1 cell line led to growth arrest and apoptosis, which was accompanied by repression of MYC and BCL2L1/BCLxL. Thus, FOXO1 repression might contribute to the oncogenic program and phenotype of PMBL.

Recombinant CD95-Fc (APG101) prevents graft-versus-host disease in mice without disabling antitumor cytotoxicity and T-cell functions.

Graft-versus-host disease (GVHD) induced by transplant-derived T cells represents a major complication after allogeneic bone marrow transplantation (BMT). However, these T cells support engraftment, early T-cell immunity, and mediate the graft-versus-tumor (GVT) effect. Cytotoxic effector functions by transplanted T cells are predominantly mediated by the perforin/granzyme and the CD95/CD95L system. APG101, a novel recombinant human fusion protein consisting of the extracellular domain of CD95 and the Fc domain of an IgG1 antibody inhibited CD95L-induced apoptosis without interfering with T-cell function in vitro and was therefore tested for its ability to prevent GVHD in murine BMT models across minor or major histocompatibility barriers. Starting APG101 treatment either 1 day before or 6 days after transplantation effectively reduced clinical GVHD and rescued survival between 60% and 100% if GVHD was CD95L mediated. APG101 did not interfere with the GVT effect, because P815 mastocytoma and most importantly primary Bcr-Abl-transformed B-cell leukemias were completely eradicated by the alloantigen-specific T cells. Phenotype and homing of alloantigen-specific T cells or their perforin/granzyme-mediated cytotoxicity and proliferative capacity were not affected by APG101 treatment suggesting that APG101 therapy might be useful in GVHD prophylaxis without impairing T-cell function and most importantly preserving GVT activity.

Hepatic activation of IKK/NFκB signaling induces liver fibrosis via macrophage-mediated chronic inflammation.

UNLABELLED: Liver damage in humans is induced by various insults including alcohol abuse, hepatitis B/C virus infection, autoimmune or metabolic disorders and, when persistent, leads to development of liver fibrosis. Because the nuclear factor-κB (NF-κB) system is activated in response to several of these stresses, we hypothesized that NF-κB activation in hepatocytes may contribute to fibrosis development. To activate the NF-κB signaling pathway in a time- and cell-type-specific manner in the liver, we crossed transgenic mice carrying the tetracycline-responsive transactivator under the control of the liver activator protein promotor with transgenic mice carrying a constitutively active form of the Ikbkb gene (IKK2 protein [CAIKK2]). Double-transgenic mice displayed doxycycline-regulated CAIKK2 expression in hepatocytes. Removal of doxycycline at birth led to activation of NF-κB signaling, moderate liver damage, recruitment of inflammatory cells, hepatocyte proliferation, and ultimately to spontaneous liver fibrosis development. Microarray analysis revealed prominent up-regulation of chemokines and chemokine receptors and this induction was rapidly reversed after switching off the CAIKK2 expression. Turning off the transgene expression for 3 weeks reversed stellate cell activation but did not diminish liver fibrosis. The elimination of macrophages by clodronate-liposomes attenuated NF-κB-induced liver fibrosis in a liver-injury-independent manner. CONCLUSION: Our results revealed that hepatic activation of IKK/NF-κB is sufficient to induce liver fibrosis by way of macrophage-mediated chronic inflammation. Therefore, agents controlling the hepatic NF-κB system represent attractive therapeutic tools to prevent fibrosis development in multiple chronic liver diseases.

FOXO1 is a tumor suppressor in classical Hodgkin lymphoma.

The FOXO transcription factors control proliferation and apoptosis in different cell types. Their activity is regulated by posttranslational modifications, mainly by the PI3K-PKB pathway, which controls nuclear export and degradation. We show that FOXO1 is highly expressed in normal germinal center B cells as well as in non-Hodgkin lymphomas, including follicular lymphoma, diffuse large B-cell lymphoma, mucosa-associated lymphoid tissue non-Hodgkin lymphoma, B-cell chronic lymphocytic leukemia, and mantle cell lymphoma. In contrast, in 31 of 32 classical Hodgkin lymphoma (cHL) cases, Hodgkin and Reed-Sternberg cells were FOXO1 negative. Neoplastic cells of nodular lymphocyte-predominant Hodgkin lymphoma were negative in 14 of 20 cases. FOXO1 was down-regulated in cHL cell lines, whereas it was expressed in non-Hodgkin lymphoma cell lines at levels comparable with normal B cells. Ectopic expression of a constitutively active FOXO1 induced apoptosis in cHL cell lines and blocked proliferation, accompanied with cell-cycle arrest in the G(0)/G(1) phase. We found that, in cHL cell lines, FOXO1 is inactivated by multiple mechanisms, including constitutive activation of AKT/PKB and MAPK/ERK kinases and up-regulation of microRNAs miR-96, miR-182, and miR-183. These results suggest that FOXO1 repression contributes to cHL lymphomagenesis.