CD30 induces Reed-Sternberg cell-like morphology and chromosomal instability in classic Hodgkin lymphoma cell lines.

Classic Hodgkin lymphoma (cHL) is characterized by multinucleated cells called Reed-Sternberg (RS) cells and genetic complexity. Although CD30 also characterizes cHL cells, its biological roles are not fully understood. In this report, we examined the link between CD30 and these characteristics of cHL cells. CD30 stimulation increased multinucleated cells resembling RS cells. We found chromatin bridges, a cause of mitotic errors, among the nuclei of multinucleated cells. CD30 stimulation induced DNA double-strand breaks (DSBs) and chromosomal imbalances. RNA sequencing showed significant changes in the gene expression by CD30 stimulation. We found that CD30 stimulation increased intracellular reactive oxygen species (ROS), which induced DSBs and multinucleated cells with chromatin bridges. The PI3K pathway was responsible for CD30-mediated generation of multinucleated cells by ROS. These results suggest that CD30 involves generation of RS cell-like multinucleated cells and chromosomal instability through induction of DSBs by ROS, which subsequently induces chromatin bridges and mitotic error. The results link CD30 not only to the morphological features of cHL cells, but also to the genetic complexity, both of which are characteristic of cHL cells.

CD30 stimulation induces multinucleation and chromosomal instability in HTLV-1-infected cell lines.

A recent report indicated involvement of CD30 in progression of human leukemia virus type 1 (HTLV-1) infection, but the exact roles of CD30 in this process remain unclear. This study was conducted to determine the role of CD30 by stimulating CD30 expressed on HTLV-1-infected cell lines with CD30 ligand and observing its effects. CD30 stimulation increased multinucleated cells and inhibited proliferation of HTLV-1-infected cells. This inhibition was recovered by interruption of CD30 stimulation. Chromatin bridges found in multinucleated cells suggested DNA damage. CD30 stimulation triggered DNA double-strand breaks (DSBs) and chromosomal imbalances. CD30 stimulation induced reactive oxygen species (ROS), which induced DSBs. Generation of ROS and multinucleated cells by CD30 was dependent on phosphoinositide 3-kinase. RNA sequencing showed that CD30 stimulation produced significant changes in gene expression profiles, including upregulation of programmed death ligand 1 (PD-L1). Tax, which has also been shown to induce multinucleation and chromosomal instability, failed to induce CD30. These results suggest that induction of CD30, independent of Tax, triggers morphological abnormalities, chromosomal instability, and alteration of gene expression in HTLV-1-infected cells.

Introduction of direct-acting antiviral agents alters frequencies of anti-GPIIb/IIIa antibody-producing B cells in chronic hepatitis C patients with thrombocytopenia.

The pathogenesis of thrombocytopenia in chronic hepatitis C (CHC) conceivably involves autoimmunity; however, the dynamics of autoantibodies and other autoimmune mechanisms remain unclear. In this study, we examined the changes in the frequency of anti-glycoprotein (GP) IIb/IIIa antibody-producing B cells and the levels of plasma B-cell-activating factor (BAFF), a proliferation-inducing ligand (APRIL), and interleukin (IL)-21 following treatment of CHC with direct-acting antiviral agents (DAA). We recruited 28 patients with CHC who underwent treatment with DAA for 8-12 weeks and subsequently tested negative for serum hepatitis C virus RNA. Thirty healthy controls were recruited for comparison. Platelet counts increased significantly (p = .016), and the frequency of anti-GPIIb/IIIa antibody-producing B cells decreased significantly (p = .002) in CHC patients with thrombocytopenia at the end of treatment (EOT) than before DAA treatment (baseline). However, these changes were not observed in CHC patients without thrombocytopenia. Plasma BAFF levels in CHC patients with thrombocytopenia significantly decreased from baseline to EOT (p = .002). Anti-GPIIb/IIIa antibody-producing B cells were positively correlated with plasma BAFF levels in these patients (r = 0.669, p = .039). These results suggest that DAA treatment suppresses the autoimmune response against platelets and improves thrombocytopenia.

What is the context? Production of antiplatelet antibodies is one of the mechanisms underlying thrombocytopenia in patients with chronic hepatitis C.Antiplatelet antibodies against platelet membrane glycoprotein (GP) IIb/IIIa are commonly detected in hepatitis C virus-associated immune thrombocytopenia.Hepatitis C virus elimination by direct-acting antiviral agents (DAA) improves thrombocytopenia in patients with hepatitis C; however, the dynamics of autoantibodies and other autoimmune mechanisms remain unclear.What is new? In this study, we determined whether DAA treatment can alter the autoimmune response against platelets and improve platelet count.The frequency of anti-GPIIb/IIIa antibody-producing B cells decreased significantly from the baseline following DAA treatment in chronic hepatitis C patients with thrombocytopenia.DAA treatment reduced the levels of B-cell-activating factor, a cytokine associated with autoantibody production.What is the impact? The study provides evidence that DAA treatment diminishes the autoimmune response to GPIIb/IIIa and, therefore, improves platelet counts in chronic hepatitis C patients with thrombocytopenia.

The oncogenic driving force of CD30 signaling-induced chromosomal instability in adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATL) develops via stepwise accumulation of gene mutations and chromosome aberrations. However, the molecular mechanisms underlying this tumorigenic process are poorly understood. We previously reported the presence of a biological link between the expression of CD30, which serves as a marker for ATL progression, and the actively proliferating fraction of human T-cell leukemia virus type 1 (HTLV-1)-infected cells that display polylobulation. Here, we demonstrated that CD30 signaling induced chromosomal instability with clonal expansion through DNA double-strand breaks (DSBs) via an increase of intracellular reactive oxygen species. CD30+ ATL cells were composed of subclones with additional genomic aberrations compared with CD30- ATL cells in ATL patients. Furthermore, we found an accumulation of copy number loss of DSB repair-related genes as the disease progressed. Taken together, CD30 expression on ATL cells appears to be correlated with genomic instability, suggesting that CD30 signaling is one of the oncogenic factors of ATL progression with clonal evolution. This study provides new insight into the biological roles of CD30 signaling and could improve our understanding of tumorigenic processes of HTLV-1-infected cells.

B cell-activating factor is involved in thrombocytopenia in patients with liver cirrhosis.

Liver cirrhosis (LC) involves B cells that produce anti-glycoprotein (GP) IIb/IIIa antibodies, found in primary immune thrombocytopenia (ITP). The role of autoimmunity in the pathology of thrombocytopenia in LC was investigated using 25 LC patients with thrombocytopenia, 18 ITP patients, and 30 healthy controls. Anti-GPIIb/IIIa antibody-producing B cells were quantified using enzyme-linked immunospot assay. Platelet-associated and plasma anti-GPIIb/IIIa antibody, plasma B cell-activating factor (BAFF), and a proliferation-inducing ligand (APRIL) levels were measured using enzyme-linked immunosorbent assay. B cell subset fractions and regulatory T cells (Tregs) were quantified using flow cytometry.The number of anti-GPIIb/IIIa antibody-producing B cells was significantly higher in LC patients than in ITP patients and healthy controls (both p < 0.001). Platelet-associated anti-GPIIb/IIIa antibodies were significantly higher in LC patients than in ITP patients and healthy controls (p = 0.002, p < 0.001, respectively). BAFF levels were significantly higher in LC patients than in ITP patients and healthy controls (p = 0.001 and p < 0.001, respectively), and APRIL levels were significantly higher in LC patients than in healthy controls (p < 0.001). Anti-GPIIb/IIIa antibody-producing B cells and platelet-associated anti-GPIIb/IIIa antibodies were positively correlated with BAFF levels in LC patients. LC patients had more naïve B cells and plasmablasts than healthy controls (p = 0.005, p = 0.03, respectively); plasmablasts were positively correlated with BAFF levels. LC patients had similar Tregs levels as ITP patients and healthy controls. Therefore, excessive BAFF production in LC patients with thrombocytopenia is likely associated with autoimmune B cell response, inducing anti-GPIIb/IIIa antibody production.

Differentiation of Hodgkin lymphoma cells by reactive oxygen species and regulation by heme oxygenase-1 through HIF-1α.

We previously indicated that Hodgkin lymphoma (HL) cells contain a small side population (SP) that differentiate into a large major population (MP) with giant Hodgkin and Reed-Sternberg (H and RS)-like cells. However, its molecular mechanisms are not fully understood. In this study, we found that intracellular reactive oxygen species (ROS) are low in the SP compared to the MP. Hydrogen peroxide induces large H- and RS-like cells in HL cell lines, but induces cell death in unrelated lymphoid cell lines. Microarray analyses revealed the enrichment of upregulated genes under hypoxic conditions in the SP compared to the MP, and we verified that the SP cells are hypoxic. Hypoxia inducible factor (HIF)-1α was preferentially expressed in the SP. CoCl2 , a HIF-1α stabilizer, blunted the effect of hydrogen peroxide. Heme oxygenase-1 (HO-1), a scavenger of ROS, was triggered by HIF-1α. The effect of hydrogen peroxide was inhibited by HO-1 induction, whereas it was promoted by HO-1 knockdown. HO-1 inhibition by zinc protoporphyrin promoted the differentiation and increased ROS. These results stress the unique roles of ROS in the differentiation of HL cells. Immature HL cells are inhibited from differentiation by a reduction of ROS through the induction of HO-1 via HIF-1α. The breakdown of this might cause the accumulation of intracellular ROS, resulting in the promotion of HL cell differentiation.

Impact of Anti-GPIIb/IIIa Antibody-Producing B Cells as a Predictor of the Response to Lusutrombopag in Thrombocytopenic Patients with Liver Disease.

BACKGROUND: To make an accurate estimate of the response to thrombopoietin (TPO) receptor agonists for thrombocytopenia associated with chronic liver disease, we evaluated the influence of antiplatelet autoantibodies on the response to lusutrombopag in thrombocytopenic patients with liver disease. METHODS: A prospective study was conducted at 2 hospitals. Thrombocytopenic patients with liver disease received oral lusutrombopag 3.0 mg once daily for up to 7 days. We analyzed changes in platelet counts from baseline to the maximum platelet count on days 9-14. The definition of clinical response was a platelet count of ≥5 × 104/µL with an increased platelet count of ≥2 × 104/µL from baseline. We assessed the correlation between the response to treatment drug and antiplatelet autoantibodies measured by anti-GPIIb/IIIa antibody-producing B cells. RESULTS: Thirty patients received the trial drug. There were 25 responders and 5 nonresponders. The median change in platelet counts was 3.9 × 104/µL (95% CI 2.8-4.6, p < 0.0001). The correlation between change in platelet counts and the frequency of the anti-glycoprotein IIb/IIIa antibody-producing B cells was moderate (r = 0.414, 95% CI 0.064-0.674, p = 0.023). In multivariate analysis of factors affecting the change in platelet counts, the anti-GPIIb/IIIa antibody-producing B cells were identified as an independent factor (regression coefficient [B] = 0.089; CI 0.021-0.157, p = 0.013). CONCLUSION: Anti-GPIIb/IIIa antibody-producing B cells may be a predictor for TPO receptor agonists in patients with chronic liver disease.

PD-L1/L2 protein levels rapidly increase on monocytes via trogocytosis from tumor cells in classical Hodgkin lymphoma.

In classical Hodgkin lymphoma (cHL)-characterized by the presence of Hodgkin and Reed-Sternberg (HRS) cells-tumor-associated macrophages (TAMs) play a pivotal role in tumor formation. However, the significance of direct contact between HRS cells and TAMs has not been elucidated. HRS cells and TAMs are known to express PD-L1, which leads to PD-1+ CD4+ T cell exhaustion in cHL. Here, we found that PD-L1/L2 expression was elevated in monocytes co-cultured with HRS cells within 1 h, but not in monocytes cultured with supernatants of HRS cells. Immunofluorescence analysis of PD-L1/L2 revealed that their upregulation resulted in membrane transfer called "trogocytosis" from HRS cells to monocytes. PD-L1/L2 upregulation was not observed in monocytes co-cultured with PD-L1/L2-deficient HRS cells, validating the hypothesis that there is a direct transfer of PD-L1/L2 from HRS cells to monocytes. In the patients, both ligands (PD-L1/L2) were upregulated in TAMs in contact with HRS cells, but not in TAMs distant from HRS cells, suggesting that trogocytosis occurs in cHL patients. Taken together, trogocytosis may be one of the mechanisms that induces rapid upregulation of PD-L1/L2 in monocytes to evade antitumor immunity through the suppression of T cells as mediated by MHC antigen presentation.

Jietacins, azoxy natural products, as novel NF-κB inhibitors: Discovery, synthesis, biological activity, and mode of action.

Deregulation of NF-κB plays an important role in various diseases by controlling cell growth, inflammation, the immune response, and cytokine production. Although many NF-κB inhibitors have been developed, to the best of our knowledge, none of them have been successfully translated into clinical practice as medicines. To overcome this issue, we aimed to develop a new class of NF-κB inhibitors. Previous reports indicated that the N-terminal cysteine is a promising target for NF-κB. Based on this, we first selected 10 natural products or their derivatives from the natural product library that we developed and examined the effect on NF-κB and the viability of cancer cells with constitutively strong NF-κB activity. Among them, we found that an azoxy natural product, jietacin A, with a vinylazoxy group and an aliphatic side chain, reduced cell viability and inhibited nuclear translocation of free NF-κB. In addition, we performed design, synthesis, and biological evaluation of jietacin derivatives for development of a novel NF-κB inhibitor. Of these derivatives, a fully synthesized derivative 25 with vinylazoxy and ynone groups had a potent effect. We clarified the structure-activity relationship of this compound. Jietacin A and 25 also inhibited tumor necrosis factor-α-mediated induction of NF-κB. The NF-κB inhibitory effect depended on the N-terminal cysteine and the neighboring Arg-Ser-Ala-Gly-Ser-Ile (RSAGSI) domain of NF-κB. We also found that 25 inhibited the association between NF-κB and importin α, suggesting inhibition of NF-κB at an early step of nuclear translocation. Overall, this study indicated that the vinylazoxy motif may compose a new class of NF-κB inhibitors, providing further insight for rational drug design and rendering a unique mode of action.

Relationship between response to lusutrombopag and splenic volume.

AIM: To assess the correlation between the efficacy of lusutrombopag and clinical characteristics in patients with chronic liver disease. METHODS: In this retrospective, multicenter study, which conducted at four locations in Japan, 50 thrombocytopenic patients with chronic liver disease were enrolled. All patients received oral lusutrombopag (3.0 mg/d for 7 d) for chronic liver disease. We assessed the increase in platelet count after the trial drug administration. A treatment response was defined as a platelet count ≥ 5 × 104/µL and an increased platelet count ≥ 2 × 104/µL from baseline after drug administration. We evaluated the response to lusutrombopag compared to baseline clinical characteristics in patients with chronic liver disease. RESULTS: The numbers of responders and non-responders were 40 (80.0%) and 10 (20.0%), respectively. The patients were divided into a responder and non-responder group, and we added factors that may correspond to successful treatment with lusutrombopag. Splenic volume and body weight were lower in the responder group than in the non-responder group. White blood cell count and hemoglobin level were higher in responders compared with non-responders. Using a logistic regression model to assess the relationship between response to lusutrombopag and clinical characteristics, multivariate analysis confirmed that splenic volume was an independent factor that predicted the response of platelet counts (P = 0.025; odds ratio = 11.2; 95% confidence interval: 1.354-103.0). Splenic volume negatively correlated to changes in platelet count (r = -0.524, P = 0.001). CONCLUSION: Splenic volume influences the change in platelet counts after administration of lusutrombopag in patients with chronic liver disease.

CD30 Characterizes Polylobated Lymphocytes and Disease Progression in HTLV-1-Infected Individuals.

Purpose: Although expression of CD30 is reported in a subset of adult T-cell leukemia/lymphoma cases, its clinicopathologic significance is poorly understood. We aimed to characterize CD30-positive cells and clarify their tumorigenic role in human T-cell lymphotropic virus type 1 (HTLV-1)-infected cells.Experimental Design: CD30-positive peripheral blood mononuclear cells from individuals with differing HTLV-1 disease status were characterized, and the role of CD30 signaling was examined using HTLV-1-infected cell lines and primary cells.Results: CD30-positive cells were detected in all samples examined, and the marker was coexpressed with both CD25 and CD4. This cell population expanded in accordance with disease progression. CD30-positive cells showed polylobation, with some possessing "flower cell" features, active cycling, and hyperploidy. CD30 stimulation of HTLV-1-infected cell lines induced these features and abnormal cell division, with polylobation found to be dependent on the activation of PI3K. The results thus link the expression of CD30, which serves as a marker for HTLV-1 disease status, to an active proliferating cell fraction featuring polylobation and chromosomal aberrations. In addition, brentuximab vedotin, an anti-CD30 monoclonal antibody conjugated with auristatin E, was found to reduce the CD30-positive cell fraction.Conclusions: Our results indicate that CD30-positive cells act as a reservoir for tumorigenic transformation and clonal expansion during HTLV-1 infection. The CD30-positive fraction may thus be a potential molecular target for those with differing HTLV-1 disease status. Clin Cancer Res; 24(21); 5445-57. ©2018 AACR.

Role of exosomes as a proinflammatory mediator in the development of EBV-associated lymphoma.

Epstein-Barr virus (EBV) causes various diseases in the elderly, including B-cell lymphoma such as Hodgkin's lymphoma and diffuse large B-cell lymphoma. Here, we show that EBV acts in trans on noninfected macrophages in the tumor through exosome secretion and augments the development of lymphomas. In a humanized mouse model, the different formation of lymphoproliferative disease (LPD) between 2 EBV strains (Akata and B95-8) was evident. Furthermore, injection of Akata-derived exosomes affected LPD severity, possibly through the regulation of macrophage phenotype in vivo. Exosomes collected from Akata-lymphoblastoid cell lines reportedly contain EBV-derived noncoding RNAs such as BamHI fragment A rightward transcript (BART) micro-RNAs (miRNAs) and EBV-encoded RNA. We focused on the exosome-mediated delivery of BART miRNAs. In vitro, BART miRNAs could induce the immune regulatory phenotype in macrophages characterized by the gene expressions of interleukin 10, tumor necrosis factor-α, and arginase 1, suggesting the immune regulatory role of BART miRNAs. The expression level of an EBV-encoded miRNA was strongly linked to the clinical outcomes in elderly patients with diffuse large B-cell lymphoma. These results implicate BART miRNAs as 1 of the factors regulating the severity of lymphoproliferative disease and as a diagnostic marker for EBV+ B-cell lymphoma.

Trogocytosis of ligand-receptor complex and its intracellular transport in CD30 signalling.

BACKGROUND INFORMATION: CD30, which is characteristically expressed in classical Hodgkin lymphoma (cHL), is thought to transduce signals by ligation of trimerised CD30 ligand (CD30L) on the surface of surrounding cells and recruitment of downstream molecules. In this report, we propose a new mechanism for CD30 signalling by its ligand. We prepared two stable transformants, CHO cells expressing CD30L fused to mCherry and HeLa cells expressing CD30 fused to GFP. RESULTS: Co-culture of these cells triggered clustering of CD30 and CD30L at the cellular interface, formation of multiple CD30L-CD30 complexes, internalisation of these complexes with a portion of the plasma membrane into the HeLa cells, and intracellular transport to the lysosomal compartment. The internalisation process was significantly inhibited by actin polymerisation inhibitors. The CD30L-CD30 interaction was found to trigger active signalling processes, as measured by Ca2+ influx, and similar mechanisms were observed using cHL cell lines. CONCLUSIONS: These results suggest that CD30 extracts CD30L from CD30L-expressing cells by actin-mediated trogocytosis, resulting in the generation of signalosomes, intracellular signalling, lysosomal degradation and a subsequent refractory phase. We postulate that similar processes may operate in tumours endogenously expressing CD30. These observations thus provide new insights into our understanding of the biological roles of CD30 in normal and malignant cells and, in particular, in cHL. SIGNIFICANCE: This study suggests a novel model of CD30 signalling that provides new insights into the biological roles of CD30 and other members of this family in normal and malignant cells.

CD30 Induces Heat Shock Protein 90 and Signal Integration in Classic Hodgkin Lymphoma Cells.

Previous studies report deregulation of multiple signaling pathways in classic Hodgkin lymphoma (cHL) cells. However, the mechanisms of how these pathways are integrated are not fully understood. Herein, we show involvement of cHL hallmark antigen CD30 in this process. CD30 facilitates phosphorylation of heat shock factor 1, activates heat shock promoter element, and induces heat shock protein (HSP) 90. CD30 repression and subsequent inhibition of HSP90 suppresses NF-κB, extracellular signal-regulated kinase, AKT, and STAT pathways in cHL cell lines. Thus, CD30-mediated induction of HSP90 appears to serve as a central hub for integration of intracellular signaling in cHL cells. We also show that CD30 induces HSP90 through phosphorylation of heat shock factor 1 via c-Jun N-terminal kinase in cHL cells. Although anaplastic large-cell lymphoma (ALCL) also is associated with CD30 overexpression, our experiments reveal that HSP90 induction in ALCL-bearing nucleophosmin-anaplastic lymphoma kinase (ALK) does not depend on CD30 but instead on ALK via c-Jun N-terminal kinase. Together, these results highlight a novel role for CD30 in mediating integration of signaling pathways of cHL cells while being replaced in this function by ALK in ALCL cells.

Immature platelet fraction measurement is influenced by platelet size and is a useful parameter for discrimination of macrothrombocytopenia.

BACKGROUND: Reticulated platelets (RPs) as measured using flow cytometry are useful parameters of thrombopoiesis; however, difficulties remain with standardization between laboratories. On the other hand, immature platelet fraction (IPF) measurement, as determined using an automated hematology analyzer, is simple, reproducible, and displays a good correlation with RP, although specific factors may affect its value. We previously noticed that a small proportion of patients exhibit extremely high IPF values that do not correlate with flow cytometrically measured RP. OBJECTIVES: We investigated the mechanism of the aberrant increase in IPF values of different types of macrothrombocytopenia. PATIENTS/METHODS: IPF, RP, and other platelet indexes were analyzed using samples from 15 congenital macrothrombocytopenic patients from 12 families, 150 immune thrombocytopenic patients, and 27 normal individuals. We further monitored the change in IPF values and morphology during platelet agglutination. RESULTS: IPF values were about five times higher in MYH9 disorders (IPF 48.6 ± 1.9%) and about twice as high in other macrothrombocytopenias (IPF 18.4 ± 2.1%) than in immune thrombocytopenic patients with similar platelet counts (IPF 9.2 ± 0.3%). We then examined changes in IPF values during ethylenediaminetetraacetic acid- and macroglobulinemia-induced platelet agglutination. The IPF value significantly increased in a time-dependent manner along with the formation of platelet clumps and was strongly influenced by a few tiny platelet aggregates. CONCLUSIONS: These results suggested that IPF values are influenced by platelet size. Furthermore, IPF could be a useful and convenient parameter for screening of macrothrombocytopenia, which presents with a disproportionately high IPF value.

Brefeldin A exerts differential effects on anaplastic lymphoma kinase positive anaplastic large cell lymphoma and classical Hodgkin lymphoma cell lines.

To obtain further insights into the biological differences of anaplastic lymphoma kinase positive anaplastic large cell lymphoma (ALK+ ALCL) and classical Hodgkin lymphoma (HL), we screened microbial culture filtrates to search for compounds that would exert a significantly greater effect on the viability of ALK+ ALCL cell lines compared to HL cell lines and identified Brefeldin A (BFA) as a suitable candidate. BFA inhibited phosphorylation of ALK and its downstream molecule, signal transducer and activator of transcription 3 (STAT3), one of the central pathways for the survival of ALK+ ALCL cells. In HL cell lines BFA did not affect CD30 expression or constitutive nuclear factor (NF)-κB activity, both of which are critical for HL cell survival. BFA induced disruption of the Golgi apparatus in ALK+ ALCL cell lines, which was accompanied by a decrease in active ADP-ribosylation factor 1 (ARF1), whereas BFA had no significant effect on these parameters in HL cell lines. These results add extra insights into the biological distinction between ALK+ ALCL and HL cells and highlight the Golgi apparatus as a target for the treatment of ALK+ ALCL.

Activation of Akt involves resistance to NF-κB inhibition and abrogation of both triggers synergistic apoptosis in lung adenocarcinoma cells.

OBJECTIVES: Although nuclear factor (NF)-κB and phosphoinositide 3-kinase (PI3K)-Akt-mTOR comprise key pathways, their interrelationship in lung cancer cell survival is poorly understood and needs further analyses. MATERIALS AND METHODS: We examined the activation of the NF-κB and Akt-mTORC1-p70 S6 kinase (S6K) pathways and the effect of inhibitors for NF-κB, mTORC1, and Akt using fresh lung adenocarcinoma cells. RESULTS: The cases used for this study showed constitutive NF-κB activity; however, all cases but one showed resistance to NF-κB inhibition. Further examination revealed that the resistant cases were also active in the Akt-mTORC1-S6K pathway. These cases were insensitive to mTORC1 inhibition but sensitive to Akt inhibition. Akt inhibition recovered sensitivity to NF-κB inhibition and dual inhibition showed a synergistic effect on apoptosis induction. CONCLUSION: These results indicate that the activation of Akt involves resistance to NF-κB inhibition and both pathways synergistically support the survival of lung adenocarcinoma cells. The results also indicate that inhibition of the mTORC1-S6K pathway does not inhibit the survival of these cells.

Molecularly-targeted Strategy and NF-κB in lymphoid malignancies.

Molecularly-targeted therapy is a promising strategy for the treatment of cancer. Nuclear factor (NF)-κB is a transcription factor that is constitutively activated in various lymphoid malignancies and may therefore be a good therapeutic target. Lymphoid malignancies arise from different stages of normal lymphocyte differentiation and acquire distinct pathways for constitutive NF-κB activation. However, no NF-κB inhibitor has yet been successfully applied in clinical medicine. This review focuses on the concept of molecularly-targeted therapeutics with small molecule drugs, molecular mechanisms of constitutive NF-κB activation in lymphoid malignancies, and the development of NF-κB inhibitors. A future perspective regarding the development of NF-κB inhibitors is also included.

Incidence and clinical significance of aberrant T-cell marker expression on diffuse large B-cell lymphoma cells.

INTRODUCTION: Aberrant expression of T-cell markers is occasionally observed in B-cell lymphomas. We conducted a retrospective study to establish its incidence and to determine its relationship with clinical features of patients with diffuse large B-cell lymphoma (DLBCL). PATIENTS AND METHODS: We reviewed DLBCL patients diagnosed between January 2002 and April 2009. Patients fulfilled the following criteria: (1) age >18 years, (2) HIV negative, (3) B-cell lymphoma confirmed by restricted expression of surface immunoglobulin light chains by flow cytometry (FCM). Aberrant T-cell marker expression (ATCME) was defined as positivity for CD2, CD3, CD4, CD7, and/or CD8 on DLBCL cells by FCM. Phenotyping was also performed by immunohistochemistry (IHC). Patients were grouped according to positive or negative ATCME and their clinical features including survival were compared. RESULTS: Of 150 patients, 11 (7.3%) showed ATCME; CD2 and CD7 were most often expressed. ATCME was less often detected and the signal was weaker using IHC. There were no statistically significant differences in clinical features between the two groups. CONCLUSIONS: FCM may be useful to detect ATCME in a small amount of lymphoma cells. The mechanism responsible for ATCME, and whether it contributes in any way to the pathogenesis of B-cell neoplastic transformation, requires clarification.

Combined inhibition of NF-κB and Bcl-2 triggers synergistic reduction of viability and induces apoptosis in melanoma cells.

Constitutive activation of nuclear factor κB (NF-κB) characterizes melanoma cells. To explore the molecular mechanism of melanoma cell survival by constitutive NF-κB activation, we used the NF-κB inhibitor dehydroxymethylepoxyquinomicin (DHMEQ), which directly binds to NF-κB. DHMEQ abrogated constitutive NF-κB activity, which included RelA (p65)/p50 in melanoma cell lines G361 and HMV-II; however, the reduction of the viability was marginal. Expression of c-FLIP was not observed in the melanoma cell lines tested, and DHMEQ could not repress the expression of the Bcl-2 family proteins Bcl-2 and Bcl-xL. Concomitant treatment with DHMEQ and the inhibitor of antiapoptotic Bcl-2 family proteins, GX15-070, triggered synergistic reduction of the viability and induced apoptosis of G361 cells. These results indicate that abrogation of the NF-κB pathway alone is not sufficient to suppress the survival of melanoma cells. The NF-κB and the antiapoptotic Bcl-2 pathways cooperatively support the survival, and the dual targeting triggers synergistic reduction of the viability and induces apoptosis of melanoma cells.