Constitutive NF-κB Signaling Research Articles

Lymphoma-Derived IL-10 Is a Key Immunomodulatory Factor at the Tumor Microenvironment of Activated B-Cell Diffuse Large B-Cell Lymphoma and Influences In Vivo Responses to Immunotherapy

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of aggressive non-Hodgkin's lymphoma with ~40% of patients experiencing refractory or relapsed disease to R-CHOP immunochemotherapy. These higher risk patients often exhibit gene expression profiles that resemble a late germinal center-derived activated B-cell (ABC) phenotype, where diverse specific mutations in genes along BCR and TLR signaling pathways converge into constitutive activation of the NF-κB pathway. Identification of factors that contribute to the survival of malignant ABC-DLBCL cells is crucial for designing specific interventions. In this setting, previous in vitro studies in DLBCL cell lines have evidenced that IL-10 is a direct target of NF-κB signaling that sustains lymphoma cell survival by an auto-stimulatory loop via STAT3 signaling. However, the action of IL-10 could be more complex within the lymphoma microenvironment, as IL-10 can have both stimulatory and inhibitory effects depending on the cellular source of IL-10, the timing of its secretion, and the type of immune cells that receives signals via IL-10 receptor (IL-10R). To explore in vivo the relevance of lymphoma-derived IL-10, we have generated a novel quintuple transgenic mouse model that we call pBIC10, after crossing our previous ABC-DLBCL-like pBIC mice carrying constitutive NF-κB signaling (Pascual et al., Blood 2019) to mice with a floxed Il10 gene for conditional knock-out of IL-10 specifically in malignant ABC-DLBCL cells. As expected, increased intracellular IL-10 signaling was detected in primary murine DLBCL cells from pBIC mice but not in IL-10 deficient pBIC10 mice, which promoted lymphoma cell survival ex vivo as revealed by perturbation studies of the IL-10/JAK1/STAT3 autocrine loop with blocking monoclonal antibodies or selected inhibitors. However, in vivo genetic deletion of lymphoma-derived IL-10 did not offer improved overall survival but, on the contrary, notably accelerated DLBCL progression in pBIC10 mice (Figure 1A, left). A comprehensive integration of multiparametric flow cytometry and transcriptomic analyses using bulk and single-cell RNA-seq of lymphoma cells and the tumor microenvironment coupled with BCR- and TCR-seq, identified lymphoma-derived IL-10 as a key immunomodulator of the DLBCL microenvironment, including unexpected protective paracrine functions in the progression of DLBCL. By comparing IL-10-proficient (pBIC) and -deficient (pBIC10) murine DLBCL models, we observed a multifaceted role of IL-10 produced by DLBCL cells, enabling immune-effector processes (facilitating immune chemotaxis, antigen presentation, and IFNγ responses) that yielded higher percentages of long-lived stem-like CD8 +TCF-1 +PD-1 lo/int T cells and dendritic cells, while restraining PD-1/LAG3-driven T-cell exhaustion and reducing immunosuppression by CD4 +CD25 +FOXP3 + regulatory T (Treg) cells. Thus, depletion of lymphoma-derived IL-10 decreased T-cell cytotoxicity and increased Treg-driven immunosuppression, which collectively accelerated lymphoma development. Consequently, T-cell targeted immune-checkpoint therapy (with anti-PD-1 antibodies) was effective only in IL-10-proficient DLBCL but not in IL-10-deficient pBIC10 mice (Figure 1A, right). Further comparative studies between pBIC and pBIC10 mice showed that tumor vascularization and expression of B-cell calcium channels, which have been previously associated with sensitivity to rituximab, were counteracted by lymphoma-derived IL-10. Accordingly, anti-CD20-based targeting of lymphoma cells (with rituximab surrogate antibodies) showed best in vivo responses in IL-10-deficient DLBCL mice with respect to pBIC mice (Figure 1A, right). Consistent with this preclinical data, analyses of transcriptional IL-10-associated hallmarks to stratify DLBCL patients (Schmitz et al., NEJM 2018) identified better clinical responses to R-CHOP in patients with pBIC10-like DLBCL phenotype (Figure 1B). Collectively, our study provides new functional and mechanistic insights into the role of lymphoma-derived IL-10 in the pathogenesis of DLBCL and the restraint of an immunosuppressed tumor microenvironment, which holds predictive potential as a biomarker for immunotherapy responses in DLBCL.

Pharmacological NF-κB inhibition decreases cisplatin chemoresistance in muscle-invasive bladder cancer and reduces cisplatin-induced toxicities.

Most patients with muscle-invasive bladder cancer (MIBC) are not cured with platinum chemotherapy. Up-regulation of nuclear factor kappa light-chain enhancer of activated B cells (NF-κB) is a major mechanism underlying chemoresistance, suggesting that its pharmacological inhibition may increase platinum efficacy. NF-κB signaling was investigated in two patient cohorts. The Cancer Genome Atlas (TCGA) was used to correlate NF-κB signaling and patient survival. The efficacy of cisplatin plus the NF-κB inhibitor dimethylaminoparthenolide (DMAPT) versus cisplatin or DMAPT alone was tested in vitro. Xenografted and immunocompetent MIBC mouse models were studied in vivo. Platinum-naive claudin-low MIBC showed constitutive NF-κB signaling and this was associated with reduced disease-specific survival in TCGA patients. Chemotherapy up-regulated NF-κB signaling and chemoresistance-associated genes, including SPHK1, PLAUR, and SERPINE1. In mice, DMAPT significantly improved the efficacy of cisplatin in both models. The combination preserved body weight, renal function, and morphology, reduced musclefatigue and IL-6 serum levels, and did not aggravate immuno-hematological toxicity compared with cisplatin alone. These data provide a rationale for combining NF-κB inhibition with platinum-based chemotherapy and conducting a clinical trial in MIBC patients.

TRAF2 Controls Death Receptor-Induced Caspase-8 Processing and Facilitates Proinflammatory Signaling.

Tumor necrosis factor (TNF) receptor associated factor-2 (TRAF2) knockout (KO) cells were generated to investigate the role of TRAF2 in signaling by TNFR1 and the CD95-type death receptors (DRs) TRAILR1/2 and CD95. To prevent negative selection effects arising from the increased cell death sensitivity of TRAF2-deficient cells, cell lines were used for the generation of the TRAF2 KO variants that were protected from DR-induced apoptosis downstream of caspase-8 activation. As already described in the literature, TRAF2 KO cells displayed enhanced constitutive alternative NFκB signaling and reduced TNFR1-induced activation of the classical NFκB pathway. There was furthermore a significant but only partial reduction in CD95-type DR-induced upregulation of the proinflammatory NFκB-regulated cytokine interleukin-8 (IL8), which could be reversed by reexpression of TRAF2. In contrast, expression of the TRAF2-related TRAF1 protein failed to functionally restore TRAF2 deficiency. TRAF2 deficiency resulted furthermore in enhanced procaspase-8 processing by DRs, but this surprisingly came along with a reduction in net caspase-8 activity. In sum, our data argue for (i) a non-obligate promoting function of TRAF2 in proinflammatory DR signaling and (ii) a yet unrecognized stabilizing effect of TRAF2 on caspase-8 activity.

Mutations of the B-Cell Receptor Pathway Confer Chemoresistance in Primary Cutaneous Diffuse Large B-Cell Lymphoma Leg Type

Primary cutaneous diffuse large B-cell lymphoma, leg type (PCLBCL-LT) preferentially involves the lower limb in elderly subjects. A combination of polychemotherapy and rituximab has improved prognosis. However, about 50% of patients will experience progression or relapse without any predictive biologic marker of therapeutic response. The mutational profile of PCLBCL-LT has highlighted mutations contributing to constitutive NF-κB and B-cell receptor (BCR) signaling pathways but has not demonstrated clinical utility. Therefore, the mutational status of 32 patients with PCLBCL-LT (14 patients with complete durable response and 18 patients with relapsing or refractory disease) was determined with a dedicated lymphopanel. Tumor pairs at diagnosis and relapse or progression were analyzed in 14 relapsing or refractory patients. Patients with PCLBCL-LT harboring one mutation that targets one of the BCR signaling genes, CD79A/B or CARD11, displayed a reduced progression-free survival and specific survival (median 18 months, P= 0.002 and 51 months, P= 0.03, respectively, whereas median duration in the wild-type group was not reached) and were associated with therapeutic resistance (P= 0.0006). Longitudinal analyses revealed that MYD88 and CD79B were the earliest and among the most mutated genes. Our data suggest that evaluating BCR mutations in patients with PCLBCL-LT may help to predict first-line therapeutic response and to select targeted therapies.

Targeting TRAF6 E3 ligase activity with a small-molecule inhibitor combats autoimmunity

Constitutive NF-κB signaling represents a hallmark of chronic inflammation and autoimmune diseases. The E3 ligase TNF receptor-associated factor 6 (TRAF6) acts as a key regulator bridging innate immunity, pro-inflammatory cytokines, and antigen receptors to the canonical NF-κB pathway. Structural analysis and point mutations have unraveled the essential role of TRAF6 binding to the E2-conjugating enzyme ubiquitin-conjugating enzyme E2 N (Ubc13 or UBE2N) to generate Lys63-linked ubiquitin chains for inflammatory and immune signal propagation. Genetic mutations disrupting TRAF6-Ubc13 binding have been shown to reduce TRAF6 activity and, consequently, NF-κB activation. However, to date, no small-molecule modulator is available to inhibit the TRAF6-Ubc13 interaction and thereby counteract NF-κB signaling and associated diseases. Here, using a high-throughput small-molecule screening approach, we discovered an inhibitor of the TRAF6-Ubc13 interaction that reduces TRAF6-Ubc13 activity both in vitro and in cells. We found that this compound, C25-140, impedes NF-κB activation in various immune and inflammatory signaling pathways also in primary human and murine cells. Importantly, C25-140 ameliorated inflammation and improved disease outcomes of autoimmune psoriasis and rheumatoid arthritis in preclinical in vivo mouse models. Hence, the first-in-class TRAF6-Ubc13 inhibitor C25-140 expands the toolbox for studying the impact of the ubiquitin system on immune signaling and underscores the importance of TRAF6 E3 ligase activity in psoriasis and rheumatoid arthritis. We propose that inhibition of TRAF6 activity by small molecules represents a promising novel strategy for targeting autoimmune and chronic inflammatory diseases.

Abstract 1879: Pyrazolopyrimidines as novel selective allosteric MALT1 inhibitors with in vivo activity in ABC-DLBCL

Abstract Diffuse large B cell lymphoma (DLBCL) is the most common histological subtype of Non-Hodgkin's lymphoma, comprising 30% to 40% of all newly diagnosed cases. DLBCL is a biologically and clinically diverse disease with more than a dozen subtypes classified by the World Health Organization (WHO). Gene expression profiling groups DLBCL into three molecular subtypes, named according to their cell of origin, and which include germinal center B-cell-like (GCB) DLBCL, activated B-cell-like (ABC) DLBCL and primary mediastinal B cell lymphoma (PMBL). The current standard of care (SoC) is R-CHOP chemo-immunotherapy. Although the SoC is curative in a substantial proportion of patients, 40% of patients, especially in the ABC-DLBCL subtype, do not achieve durable remissions and suffer from progressive disease. In ABC-DLBCL, recurrent mutations in the B-cell receptor (BCR) and Toll-like receptor (TLR) pathways lead to constitutive NF-κB signaling. Mutations in the BCR pathway include gain-of-function mutations of CD79A/B (~20%) and CARD11 (~10%), and loss-of-function mutations of A20 (~25%). In the TLR pathway, MYD88 is commonly mutated (~37%). In recent years, knowledge of these aberrantly regulated pathways and their underlying mutations guided the development and investigation of newer molecular targeted agents, e.g. BTK, SYK, PI3K, PKC and MALT1 inhibitors. Among these strategies, inhibition of MALT1 provides the advantage that it is downstream of most of the reported BCR pathway mutations, including CARD11 mutations, and most of the targeted kinases with respect to potentially emerging resistance. However, no MALT1 inhibitor is in the clinic until now. Here, we report the identification of pyrazolopyrimidines as a new class of allosteric MALT1 inhibitors and their optimization for in vivo. The lead compound shows nanomolar potency in biochemical and cellular MALT1 protease reporter assays. In addition, pyrazolopyrimidines are highly selective for MALT1 and demonstrate differential cell killing of CARD11 mutant ABC-DLBCL cells vs control cells in proliferation assays. Furthermore, we demonstrate in vivo activity of pyrazolopyrimidines in CD79 and CARD11 mutant ABC-DLBCL xenograft models, with the lead compound causing regression in a CARD11 mutant xenograft model. Citation Format: Andreas Weiss, Thomas Radimerski, Daniel Wyss, Rita Andraos, Alexandra Buhles, Dario Sterker, Jean Quancard, Carole Pissot, Oliver Simic, Marc Bigaud, Frederic Bornancin, Achim Schlapbach, Catherine Regnier, Paul McSheehy, Elisabeth Buchdunger, Markus Wartmann, Martin Renatus, Ralf Endres, William Sellers, Francesco Hofmann. Pyrazolopyrimidines as novel selective allosteric MALT1 inhibitors with in vivo activity in ABC-DLBCL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1879.

Intrinsic Plasma Cell Differentiation Defects in B Cell Expansion with NF-κB and T Cell Anergy Patient B Cells.

B cell Expansion with NF-κB and T cell Anergy (BENTA) disease is a novel B cell lymphoproliferative disorder caused by germline, gain-of-function mutations in the lymphocyte scaffolding protein CARD11, which drives constitutive NF-κB signaling. Despite dramatic polyclonal expansion of naive and immature B cells, BENTA patients also present with signs of primary immunodeficiency, including markedly reduced percentages of class-switched/memory B cells and poor humoral responses to certain vaccines. Using purified naive B cells from our BENTA patient cohort, here we show that BENTA B cells exhibit intrinsic defects in B cell differentiation. Despite a profound in vitro survival advantage relative to normal donor B cells, BENTA patient B cells were severely impaired in their ability to differentiate into short-lived IgDloCD38hi plasmablasts or CD138+ long-lived plasma cells in response to various stimuli. These defects corresponded with diminished IgG antibody production and correlated with poor induction of specific genes required for plasma cell commitment. These findings provide important mechanistic clues that help explain both B cell lymphocytosis and humoral immunodeficiency in BENTA disease.

Abstract NTOC-085: PPARγ AGONISTS AUGMENT ANTICANCER EFFECTS OF XIAP INHIBITION ON HUMAN GRANULOSA CELL TUMOR–DERIVED CELLS

Abstract BACKGROUND: Ovarian granulosa cell tumors (GCT) are hormonally active cancers characterized by indolent growth and late, invasive relapse. GCT are unusual in that they have an unexplained propensity for late recurrence. ~80% of patients with aggressive or recurrent tumors die from their disease. Aside from surgery the therapeutic options are very limited. We have identified the nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARγ) and the X-linked inhibitor of apoptosis protein (XIAP) as potential specific therapeutic targets. PPARγ is a transcription factor that impedes proliferation and promotes terminal differentiation in granulosa cells (GC), is highly expressed in GCT. Its activity however is transrepressed by constitutive activity of the critical pro-survival NF-κB signaling pathway. Constitutive NF-κB signaling is potentially a consequence of a positive feed-forward loop involving XIAP, which is also highly expressed in GCT. As a potent inhibitor of apoptosis, XIAP is an attractive therapeutic target. We have shown in vitro that inhibiting XIAP releases NF-κB transrepression, and together with PPARγ activation, induces apoptosis. We thus hypothesize that XIAP antagonism sensitizes GCT to pro-apoptotic strategies such as PPARγ activation. METHODS AND RESULTS: We have used stable isotope labeling with amino acids in cell culture (SILAC), a proteomic approach to identify differentially expressed proteins after combined PPARγ activation (rosiglitazone) and XIAP inhibition (Smac mimetic). Using two GCT-derived cell lines, KGN and COV434, we identified 52 proteins which were differentially expressed (by 1.5 fold) with the combined treatment. Upregulated proteins included several involved in metabolic processes including acyl-CoA desaturase (4.50-fold), phosphoglycerate kinase 1 (2.87-fold) and α-enolase (1.75-fold). As PPARγ plays a pivotal role in lipid and glucose metabolism, upregulation of these proteins is a consequence of PPARγ activity being restored. Additionally, we observed downregulation of fascin (-1.65 fold), the main actin-bundling protein associated with cell motility and migration. Overexpression has been observed in metastatic tumors and correlated with clinically aggressive phenotypes, poor prognosis and shorter survival. We used the xCELLigence RTCA system to monitor invasion of KGN cells through a Matrigel® basement membrane in real-time. KGN cells treated either with Vehicle, RGZ/RA or SM alone or combined RGZ/RA/SM were plated in the upper chamber of a CIM-Plate 16 and cell invasion through the Matrigel® membrane was monitored. When compared to vehicle treated cells, we observed that the RGZ/RA/SM-treated KGN cells were (i) 30% less invasive and (ii) demonstrated a delayed onset of invasion by 17 hours towards serum-containing media with and without drugs in the lower chamber, respectively. CONCLUSION: We are currently investigating the mechanism involved in XIAP inhibition abrogating transrepression of PPARγ in GCT-derived cells. This study will improve our understanding of the molecular mechanisms in GCT pathophysiology as well as enable identification of new targets for therapeutic strategies. These observations may also translate to ovarian epithelial cell cancers (EOC) as increased apoptosis was observed in serous EOC-derived cell lines expressing both XIAP and PPARγ. We propose that a combination therapy involving the abrogation of XIAP may be of greater efficacy for the treatment of GCT and potentially other ovarian cancer subtypes. Citation Format: Simon Chu, Dilys TH Leung, Maria Alexiadis and Peter J Fuller. PPARγ AGONISTS AUGMENT ANTICANCER EFFECTS OF XIAP INHIBITION ON HUMAN GRANULOSA CELL TUMOR–DERIVED CELLS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr NTOC-085.

Mass cytometry analysis reveals hyperactive NF Kappa B signaling in myelofibrosis and secondary acute myeloid leukemia.

Myeloproliferative neoplasms (MPNs) feature a malignant clone containing the JAK2 V617F mutation, or another mutation causing dysregulated JAK2 kinase activity. The multiple disease phenotypes of MPNs, and their tendency to transform phenotypically, suggest pathophysiologic heterogeneities beyond a common phenomenon of JAK2 hyperactivation. JAK2 has the potential to activate multiple other signaling molecules, either directly through downstream effectors, or indirectly through induction of target gene expression. We have interrogated myeloproliferative signaling in myelofibrosis (MF) and secondary acute myeloid leukemia (sAML) patient samples using mass cytometry, which allows the quantitative measurement of multiple signaling molecules simultaneously at the single cell level, in cell populations representing a nearly complete spectrum of hematopoiesis. MF and sAML malignant cells demonstrated a high prevalence of hyperactivation of the JAK-STAT, MAP kinase, PI3 kinase, and NFκB signaling pathways. Constitutive NFκB signaling was evident across MF and sAML patients. A supporting GSEA analysis of MF showed many NFκB target genes to be expressed above normal levels in MF patient CD34+ cells. NFκB inhibition suppressed colony formation from MF CD34+ cells. This study indicates that NFκB signaling contributes to human myeloproliferative disease and is abnormally activated in MF and sAML.

X-linked Dystonia-Parkinsonism patient cells exhibit altered signaling via nuclear factor-kappa B

X-linked Dystonia-Parkinsonism (XDP) is a progressive neurodegenerative disease involving the loss of medium spiny neurons within the striatum. An XDP-specific haplotype has been identified, consisting of seven sequence variants which cluster around the human TAF1 gene, but a direct relationship between any of these variants and disease pathogenesis has not yet been demonstrated. Because the pathogenic gene lesion remains unclear, it has been difficult to predict cellular pathways which are affected in XDP cells. To address that issue, we assayed expression of defined gene sets in XDP vs. control fibroblasts to identify networks of functionally-related transcripts which may be dysregulated in XDP patient cells. That analysis derived a 51-gene signature distinguishing XDP vs. control fibroblasts which mapped strongly to nuclear factor-kappa B (NFκB), a transcription factor pathway also implicated in the pathogenesis of other neurodegenerative diseases, including Parkinson's (PD) and Huntington's disease (HD). Constitutive and TNFα-evoked NFκB signaling was further evaluated in XDP vs. control fibroblasts based on luciferase reporter activity, DNA binding of NFκB subunits, and endogenous target gene transcription. Compared to control cells, XDP fibroblasts exhibited decreased basal NFκB activity and decreased levels of the active NFκB p50 subunit, but increased target gene expression in response to TNFα. NFκB signaling was further examined in neural stem cells differentiated from XDP and control induced pluripotent stem cell (iPSC) lines, revealing a similar pattern of increased TNFα responses in the patient lines compared to controls. These data indicate that an NFκB signaling phenotype is present in both patient fibroblasts and neural stem cells, suggesting this pathway as a site of dysfunction in XDP.

Lymphocyte signaling and activation by the CARMA1-BCL10-MALT1 signalosome.

The CARMA1-BCL10-MALT1 (CBM) signalosome triggers canonical NF-κB signaling and lymphocyte activation upon antigen-receptor stimulation. Genetic studies in mice and the analysis of human immune pathologies unveiled a critical role of the CBM complex in adaptive immune responses. Great progress has been made in elucidating the fundamental mechanisms that dictate CBM assembly and disassembly. By bridging proximal antigen-receptor signaling to downstream signaling pathways, the CBM complex exerts a crucial scaffolding function. Moreover, the MALT1 subunit confers a unique proteolytic activity that is key for lymphocyte activation. Deregulated 'chronic' CBM signaling drives constitutive NF-κB signaling and MALT1 activation, which contribute to the development of autoimmune and inflammatory diseases as well as lymphomagenesis. Thus, the processes that govern CBM activation and function are promising targets for the treatment of immune disorders. Here, we summarize the current knowledge on the functions and mechanisms of CBM signaling in lymphocytes and how CBM deregulations contribute to aberrant signaling in malignant lymphomas.

Activated B cells from BENTA patients display intrinsic differentiation defects and resistance to apoptosis

Abstract We recently characterized a novel, B-cell specific congenital lymphoproliferative disorder termed BENTA (B cell Expansion with NF-κB and T-cell Anergy). BENTA disease is caused by heterozygous, germline gain-of-function mutations in the lymphocyte-specific signaling protein CARD11, triggering constitutive NF-κB signaling. BENTA patients present not only with splenomegaly and remarkable polyclonal immature/naive B cell expansion, but also display signs of primary immunodeficiency, including reduced percentages of class-switched/memory B cells, low serum IgM and poor antibody responses to specific vaccines. Using purified naïve B cells from several BENTA patients, we discovered profound intrinsic defects in plasma cell differentiation and antibody secretion in vitro in response to various stimuli. Although BENTA B cells proliferated slightly more than healthy donor B cells in vitro, they failed to differentiate into short-lived IgDloCD38hi plasmablasts or CD138+ long-lived plasma cells. Consistent with these defects, BENTA B cells exhibited low IgG secretion, and reduced induction of key proteins involved in class switching (IRF4) and late plasma cell commitment (XBP1). On the other hand, activated BENTA B cells exhibited an overwhelming survival advantage in culture, reflecting apoptosis resistance that may be linked to a CARD11-dependent, NF-κB-independent pathway. Together, these results provide new mechanistic insights into our understanding of both B cell lymphocytosis and humoral immunodeficiency in BENTA disease, which may emerge from impairments in cell death and differentiation programs normally triggered by B cell activation.

ESCRT proteins restrict constitutive NF-κB signaling by trafficking cytokine receptors.

Because signaling mediated by the transcription factor nuclear factor κB (NF-κB) is initiated by ligands and receptors that can undergo internalization, we investigated how endocytic trafficking regulated this key physiological pathway. We depleted all of the ESCRT (endosomal sorting complexes required for transport) subunits, which mediate receptor trafficking and degradation, and found that the components Tsg101, Vps28, UBAP1, and CHMP4B were essential to restrict constitutive NF-κB signaling in human embryonic kidney 293 cells. In the absence of exogenous cytokines, depletion of these proteins led to the activation of both canonical and noncanonical NF-κB signaling, as well as the induction of NF-κB-dependent transcriptional responses in cultured human cells, zebrafish embryos, and fat bodies in flies. These effects depended on cytokine receptors, such as the lymphotoxin β receptor (LTβR) and tumor necrosis factor receptor 1 (TNFR1). Upon depletion of ESCRT subunits, both receptors became concentrated on and signaled from endosomes. Endosomal accumulation of LTβR induced its ligand-independent oligomerization and signaling through the adaptors TNFR-associated factor 2 (TRAF2) and TRAF3. These data suggest that ESCRTs constitutively control the distribution of cytokine receptors in their ligand-free state to restrict their signaling, which may represent a general mechanism to prevent spurious activation of NF-κB.

The origin and targeting of mucosa-associated lymphoid tissue lymphomas.

Extranodal mucosa-associated lymphoid tissue (MALT lymphoma) is a distinct clinical-pathological entity that can be distinguished from other lymphomas by a number of unique features, including their location in various extranodal sites, being preceded by chronic inflammatory or infection processes; a characteristic histopathological picture; and the presence of exclusive chromosomal translocations which increase MALT1 proteolytic activity to promote constitutive NF-κB signaling and eventually drive lymphomagenesis. This review explores the major molecular and cellular events that participate in MALT lymphoma pathogenesis, focusing on gastric MALT lymphoma as a model of chronic inflammation-induced tumor development. In addition, the pivotal roles of activated MALT1 protease, its substrate TNFAIP3/A20, and the MyD88 adaptor protein in abnormally triggering downstream NF-κB pathway are overviewed. These new insights provide a mechanistic basis for using novel therapies targeting MALT1 protease or IRAK4 kinase activities. Finally, the putative cellular origin of MALT lymphomas is also discussed. Over the last decade, unraveling the biological complexity of MALT lymphomas has shed light on the fundamental cellular and molecular aspects of the disease that are to be translated into clinical diagnostics and therapy.

A phase I clinical trial of systemically delivered NEMO binding domain peptide in dogs with spontaneous activated B-cell like diffuse large B-cell lymphoma.

Activated B-Cell (ABC) Diffuse Large B-Cell Lymphoma (DLBCL) is a common, aggressive and poorly chemoresponsive subtype of DLBCL, characterized by constitutive canonical NF-κB signaling. Inhibition of NF-κB signaling leads to apoptosis of ABC-DLBCL cell lines, suggesting targeted disruption of this pathway may have therapeutic relevance. The selective IKK inhibitor, NEMO Binding Domain (NBD) peptide effectively blocks constitutive NF-κB activity and induces apoptosis in ABC-DLBCL cells in vitro. Here we used a comparative approach to determine the safety and efficacy of systemic NBD peptide to inhibit constitutive NF-κB signaling in privately owned dogs with spontaneous newly diagnosed or relapsed ABC-like DLBCL. Malignant lymph nodes biopsies were taken before and twenty-four hours after peptide administration to determine biological effects. Intravenous administration of <2 mg/kg NBD peptide was safe and inhibited constitutive canonical NF-κB activity in 6/10 dogs. Reductions in mitotic index and Cyclin D expression also occurred in a subset of dogs 24 hours post peptide and in 3 dogs marked, therapeutically beneficial histopathological changes were identified. Mild, grade 1 toxicities were noted in 3 dogs at the time of peptide administration and one dog developed transient subclinical hepatopathy. Long term toxicities were not identified. Pharmacokinetic data suggested rapid uptake of peptide into tissues. No significant hematological or biochemical toxicities were identified. Overall the results from this phase I study indicate that systemic administration of NBD peptide is safe and effectively blocks constitutive NF-κB signaling and reduces malignant B cell proliferation in a subset of dogs with ABC-like DLBCL. These results have potential translational relevance for human ABC-DLBCL.

Attacking MALT1 for ABC-DLBCL therapy

Diffuse large B-cell lymphoma (DLBCL) comprises the largest group of non-Hodgkin lymphoma (NHL). DLBCL can be classified according to their cellular origin and patients with the activated B-cell (ABC) subtype have an inferior prognosis relative to those with the germinal center B-cell (GCB) subtype. Constitutive anti-apoptotic NF-κB signaling is a hallmark of ABC-DLBCL. NF-κB activation is caused by chronic active B-cell receptor (BCR) signaling or constitutive MYD88 signaling. Oncogenic mutations that contribute to pathogenesis of ABC-DLBCL tumors have been identified in critical components of both pathways (Figure ​(Figure1)1) [1]. Figure 1 Oncogenic mechanisms and target directed therapeutic approaches in ABC-DLBCL Target directed approaches for ABC-DLBCL therapy have largely focused on the inhibition of upstream protein kinase [2]. Chronic BCR signaling engages the adaptors CD79A and CD79B in a Syk-dependent mechanism. Syk is constitutively active in many B-cell lymphomas and a clinical phase I/II trial using the Syk inhibitor fostamatinib disodium (FosD, AstraZeneca) shows some response also in DLBCL patients. However, most oncogenic mutations in ABC-DLBCL occur further downstream revealing that Syk may not be an optimal target. Downstream of CD79A/B, Btk and PKCβ bridge proximal BCR signaling events to the CARMA1 (CARD11)-BCL10-MALT1 (CBM) complex. Over 20% of ABC-DLBCL tumors carry oncogenic mutations in CD79A/B. Indeed, the irreversible Btk inhibitor ibrutinib (PCI-32765, Pharamcyclics) and the panPKC inhibitor sotrastaurin (STN) are inhibiting the outgrowth of CD79 mutant ABC-DLBCL in preclinical models [3, 4]. Furthermore, positive clinical responses in a phase II trial were reported in relapsed/refractory DLBCL with the selective PKCβ inhibitor enzastarin (LY317615, Eli Lilly) [5]. However, none of these potential drugs is able to target ABC-DLBCL tumors with lesions downstream of PKCβ or in parallel pathways, such as CARMA1 of MYD88, respectively. Downstream of PKCβ the CBM subunit MALT1 has attracted great attention as a potential therapeutic target [6]. MALT1 serves a dual role in NF-κB signaling in response to antigen receptor stimulation. MALT1 is an adaptor that recruits and activates the IκB kinase (IKK) complex, the gatekeeper of canonical NF-κB. In addition, MALT1 is a paracaspase with a caspase-like proteolytic activity that is required for full NF-κB signaling and survival of ABC-DLBCL cells. MALT1 cleaves and thereby inactivates negative regulators of canonical NF-κB, such as the tumor suppressor A20 and the NF-κB subunit RelB which both counteract pro-survival functions of canonical IKK/NF-κB signaling. Two classes of MALT1 inhibitors have now been identified that effectively and selectively inhibit the growth of ABC- but not GCB-DLBCL in vitro and in vivo [7, 8]. Interestingly, the compounds are inhibiting MALT1 by two very different mechanisms. Fontan et al have identified a structurally new small molecule inhibitor (MI-2) that is covalently modifying catalytic center of MALT1 [7]. MI-2 was tolerated in mice at the effective dose without obvious signs of toxicity. Usually, irreversible inhibitors require optimal pharmacokinetic properties for clinical development, but recent advances for instance on the irreversible Btk inhibitor ibrutinib reveal that a clinical use may be possible. In a parallel study, we have identified the phenothiazines-derivatives (PD) thioridazine, mepazine and promazine as reversible MALT1 inhibitors [8]. PD are not targeting the active site of MALT1, but exhibit a non-competitive, allosteric mode of action. Mepazine, thioridazine and promazine have a long medical history as antipsychotics and sedatives drugs used for the treatment of psychiatric disorders. Well-established toxicokinetics and pharmacokinetics suggest that targeting MALT1 for cancer therapy by this class of compounds may be safe and feasible. Further, medicinal chemistry could be used to generate novel PD that are more potent MALT1 inhibitors, while reducing their neurological effects. Taken together, both studies demonstrate that MALT1 inhibition is a promising strategy for the treatment of ABC-DLBCL. In fact, targeting MALT1 may possess some advantages over the inhibition of upstream protein kinases. MALT1 inhibition also affects survival of CARMA1 mutant ABC-DLBCL. Further, with an occurrence of 29% the MYD88 mutation L265P is the most frequent oncogenic mutation in ABC-DLBCL. 65% of the MYD88-mutant ABC-DLBCL tumors carry additional mutations in CARMA1 or CD79A/B and MALT1 inhibitors are toxic to ABC-DLBCL with aberrant activation of both pathways [7, 8]. Thus, MALT1 inhibition indeed holds great promises for the treatment of the majority of ABC-DLBCL. Research on ABC-DLBCL provides a paradigm for the power of using advanced diagnostic tools (e.g. gene expression profiling and genome sequencing) to classify lymphoma entities and to identify specific oncogenic lesions. In parallel the generation of target directed therapeutics will promote the development of more personalized treatment protocols. Because of the various oncogenic lesions and the possibilities of drug resistances, there cannot be enough ‘smart’ drugs for ABC-DLBCL therapy. In the future, combinatorial treatment protocols may certainly enhance anti-tumor efficacy of the single agents that are currently in preclinical and/or clinical development.

P100 Deficiency Is Insufficient for Full Activation of the Alternative NF-κB Pathway: TNF Cooperates with p52-RelB in Target Gene Transcription

BackgroundConstitutive activation of the alternative NF-κB pathway leads to marginal zone B cell expansion and disorganized spleen microarchitecture. Furthermore, uncontrolled alternative NF-κB signaling may result in the development and progression of cancer. Here, we focused on the question how does the constitutive alternative NF-κB signaling exert its effects in these malignant processes.Methodology/Principal FindingsTo explore the consequences of unrestricted alternative NF-κB activation on genome-wide transcription, we compared gene expression profiles of wild-type and NF-κB2/p100-deficient (p100−/−) primary mouse embryonic fibroblasts (MEFs) and spleens. Microarray experiments revealed only 73 differentially regulated genes in p100−/− vs. wild-type MEFs. Chromatin immunoprecipitation (ChIP) assays showed in p100−/− MEFs direct binding of p52 and RelB to the promoter of the Enpp2 gene encoding ENPP2/Autotaxin, a protein with an important role in lymphocyte homing and cell migration. Gene ontology analysis revealed upregulation of genes with anti-apoptotic/proliferative activity (Enpp2/Atx, Serpina3g, Traf1, Rrad), chemotactic/locomotory activity (Enpp2/Atx, Ccl8), and lymphocyte homing activity (Enpp2/Atx, Cd34). Most importantly, biochemical and gene expression analyses of MEFs and spleen, respectively, indicated a marked crosstalk between classical and alternative NF-κB pathways.Conclusions/SignificanceOur results show that p100 deficiency alone was insufficient for full induction of genes regulated by the alternative NF-κB pathway. Moreover, alternative NF-κB signaling strongly synergized both in vitro and in vivo with classical NF-κB activation, thereby extending the number of genes under the control of the p100 inhibitor of the alternative NF-κB signaling pathway.

Structure of the Nuclear Factor κB-inducing Kinase (NIK) Kinase Domain Reveals a Constitutively Active Conformation

NF-κB-inducing kinase (NIK) is a central component in the non-canonical NF-κB signaling pathway. Excessive NIK activity is implicated in various disorders, such as autoimmune conditions and cancers. Here, we report the first crystal structure of truncated human NIK in complex with adenosine 5'-O-(thiotriphosphate) at a resolution of 2.5 Å. This truncated protein is a catalytically active construct, including an N-terminal extension of 60 residues prior to the kinase domain, the kinase domain, and 20 residues afterward. The structure reveals that the NIK kinase domain assumes an active conformation in the absence of any phosphorylation. Analysis of the structure uncovers a unique role for the N-terminal extension sequence, which stabilizes helix αC in the active orientation and keeps the kinase domain in the catalytically competent conformation. Our findings shed light on the long-standing debate over whether NIK is a constitutively active kinase. They also provide a molecular basis for the recent observation of gain-of-function activity for an N-terminal deletion mutant (ΔN324) of NIK, leading to constitutive non-canonical NF-κB signaling with enhanced B-cell adhesion and apoptosis resistance.

Abstract LB-181: Piperlongumine inhibits growth and survival of human Burkitt lymphoma by downregulating NF-κB, c-Myc and LMP1

Abstract Piperlongumine (PL), a natural product constituent of the fruit of the Long pepper, Piper longum, has well documented anti-inflammatory and cancer inhibiting properties. The latter include evidence that PL selectively kills solid tumor cells, but spares their normal counterparts. Because it has not been established whether PL can also inhibit hematological malignancies of the B-lymphocyte lineage, we here decided to evaluate the efficacy with which PL suppresses growth and survival of human non-Hodgkin's Burkitt lymphoma (BL) cells in vitro. We found that PL curbed cell growth (IC50 values of PL for 24h treatment: DG-75; 8.5uM, Daudi; 2.84uM, Raji; 7.7uM and Ramos; 4.5uM) and caused Caspase-3-dependent apoptosis of BL cells in a concentration- and time-dependent manner. PL strongly suppressed constitutive NF-κB signaling by inhibition of IκBα degradation and subsequent nuclear translocation of p65 as well as interruption of NF-κB binding to its consensus DNA sequence. PL also disrupted the expression of NF-κB target genes such as E2F1, c-Myb and GADD45B. c-Myc, transcription factor important for BL development and survival, was remarkably reduced after treatment with PL. In EBV-positive BL cell lines that express high level of LMP1 (e.g., Daudi, Raji), PL efficiently repressed LMP1, a known activator of NF-κB. Considering that NF-κB, c-Myc and LMP1 play important roles in the development and in vivo maintenance of B-lineage and other types of blood cells neoplasms, our findings strongly suggest that PL may afford new approaches to the chemoprevention and treatment of blood cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-181. doi:1538-7445.AM2012-LB-181

IκB Kinases Modulate the Activity of the Androgen Receptor in Prostate Carcinoma Cell Lines

Enhanced nuclear localization of nuclear factor κB (NF-κB) in prostate cancer (PCa) samples and constitutive NF-κB signaling in a class of PCa cell lines with low androgen receptor (AR) expression (PC3 and DU-145) imply an important role of the IκB kinase (IKK)/NF-κB system in PCa. However, most PCa and PCa cell lines depend on the activity of the AR, and the role of NF-κB in these AR-expressing PCa remains unclear. Here, we demonstrate that inhibition of NF-κB signaling by the IKK inhibitor BMS345541 reduced proliferation and increased apoptosis in AR-expressing PCa cell lines. Furthermore, AR activity and target gene expression were distinctively reduced, whereas AR protein levels remained unaltered on BMS345541 treatment. Similar effects were observed particularly after small interfering RNA (siRNA)-mediated knockdown of IKK1, but not by siRNA-mediated suppression of IKK2. Moreover, IKK1 overexpression augmented 5α-dihydrotestosterone-induced nuclear AR translocation, whereas nuclear AR was reduced by IKK1 knockdown or BMS345541. However, because IKK1 also enhances the activity of a chronically nuclear AR mutant, modulation of the subcellular distribution seems not to be the only mechanism by which IKK1 enhances AR activity. Finally, reduced in vivo AR phosphorylation after BMS345541 treatment and in vitro AR phosphorylation by IKK1 or IKK2 imply that AR constitutes a novel IKK target. Taken together, our data identify IKK1 as a potentially target structure for future therapeutic intervention in PCa.