PAK4 Phosphorylates Fumarase and Blocks TGFß-Induced Cell Growth Arrest in Lung Cancer Cells.

The metabolic activity of fumarase (FH) participates in gene transcription linking to tumor cell growth. However, whether this effect is implicated in lung cancer remains unclear. Here, we show TGFß induces p38-mediated FH phosphorylation at Thr 90, which leads to a FH/CSL (also known as RBP-Jκ)/p53 complex formation and FH accumulation at p21 promoter under concomitant activation of Notch signaling; in turn, FH inhibits histone H3 Lys 36 demethylation and thereby promotes p21 transcription and cell growth arrest. In addition, FH is massively phosphorylated at the Ser 46 by PAK4 in non-small cell lung cancer (NSCLC) cells, and PAK4-phosphorylated FH binds to 14-3-3, resulting in cytosolic detention of FH and prohibition of FH/CSL/p53 complex formation. Physiologically, FH Ser 46 phosphorylation promotes tumorigenesis through its suppressive effect on FH Thr 90 phosphorylation-mediated cell growth arrest in NSCLC cells and correlates with poor prognosis in patients with lung cancer. Our findings uncover an uncharacterized mechanism underlying the local effect of FH on TGFß-induced gene transcription, on which the inhibitory effect from PAK4 promotes tumorigenesis in lung cancer. SIGNIFICANCE: Fumarase counteracts CSL via its metabolic activity to facilitate TGFß-induced cell growth arrest, an effect largely blocked by PAK4-mediated phosphorylation of fumarase.

Structural Biology of the TNFα Antagonists Used in the Treatment of Rheumatoid Arthritis.

The binding of the tumor necrosis factor α (TNFα) to its cognate receptor initiates many immune and inflammatory processes. The drugs, etanercept (Enbrel®), infliximab (Remicade®), adalimumab (Humira®), certolizumab-pegol (Cimzia®), and golimumab (Simponi®), are anti-TNFα agents. These drugs block TNFα from interacting with its receptors and have enabled the development of breakthrough therapies for the treatment of several autoimmune inflammatory diseases, including rheumatoid arthritis, Crohn's disease, and psoriatic arthritis. In this review, we describe the latest works on the structural characterization of TNFα-TNFα antagonist interactions related to their therapeutic efficacy at the atomic level. A comprehensive comparison of the interactions of the TNFα blockers would provide a better understanding of the molecular mechanisms by which they neutralize TNFα. In addition, an enhanced understanding of the higher order complex structures and quinary structures of the TNFα antagonists can support the development of better biologics with the improved pharmacokinetic properties. Accumulation of these structural studies can provide a basis for the improvement of therapeutic agents against TNFα for the treatment of rheumatoid arthritis and other autoimmune inflammatory diseases in which TNFα plays an important role in pathogenesis.

Differences in the Activity of Endogenous Bone Morphogenetic Protein Signaling Impact on the Ability of Induced Pluripotent Stem Cells to Differentiate to Corneal Epithelial-Like Cells.

Cornea is a clear outermost layer of the eye which enables transmission of light onto the retina. The transparent corneal epithelium is regenerated by limbal stem cells (LSCs), whose loss/dysfunction results in LSCs deficiency (LSCD). Ex vivo expansion of autologous LSCs obtained from patient's healthy eye followed by transplantation onto the LSCs damaged/deficient eye, has provided a successful treatment for unilateral LSCD. However, this is not applicable to patient with total bilateral LSCD, where LSCs are lost/damaged from both eyes. We investigated the potential of human induced pluripotent stem cell (hiPSC) to differentiate into corneal epithelial-like cells as a source of autologous stem cell treatment for patients with total bilateral LSCD. Our study showed that combined addition of bone morphogenetic protein 4 (BMP4), all trans-retinoic acid and epidermal growth factor for the first 9 days of differentiation followed by cell-replating on collagen-IV-coated surfaces with a corneal-specific-epithelial cell media for an additional 11 days, resulted in step wise differentiation of human embryonic stem cells (hESC) to corneal epithelial progenitors and mature corneal epithelial-like cells. We observed differences in the ability of hiPSC lines to undergo differentiation to corneal epithelial-like cells which were dependent on the level of endogenous BMP signaling and could be restored via the activation of this signaling pathway by a specific transforming growth factor ß inhibitor (SB431542). Together our data reveal a differential ability of hiPSC lines to generate corneal epithelial cells which is underlined by the activity of endogenous BMP signaling pathway. Stem Cells 2018;36:337-348.

Beyond TNF: TNF superfamily cytokines as targets for the treatment of rheumatic diseases.

TNF blockers are highly efficacious at dampening inflammation and reducing symptoms in rheumatic diseases such as rheumatoid arthritis, psoriatic arthritis and ankylosing spondylitis, and also in nonrheumatic syndromes such as inflammatory bowel disease. As TNF belongs to a superfamily of 19 structurally related proteins that have both proinflammatory and anti-inflammatory activity, reagents that disrupt the interaction between proinflammatory TNF family cytokines and their receptors, or agonize the anti-inflammatory receptors, are being considered for the treatment of rheumatic diseases. Biologic agents that block B cell activating factor (BAFF) and receptor activator of nuclear factor-κB ligand (RANKL) have been approved for the treatment of systemic lupus erythematosus and osteoporosis, respectively. In this Review, we focus on additional members of the TNF superfamily that could be relevant for the pathogenesis of rheumatic disease, including those that can strongly promote activity of immune cells or increase activity of tissue cells, as well as those that promote death pathways and might limit inflammation. We examine preclinical mouse and human data linking these molecules to the control of damage in the joints, muscle, bone or other tissues, and discuss their potential as targets for future therapy of rheumatic diseases.

Guillain-Barré syndrome in a patient on adalimumab for the treatment of psoriasis

Abstract The use of TNF-α inhibitors for the treatment of moderate to severe psoriasis and psoriatic arthritis is increasingly more frequent. The authors report a case of Guillain-Barré syndrome as a late manifestation of the treatment with adalimumab. Although unusual, this is relevant for professionals who prescribe biologic drugs. We also stress the importance of investigating the past and family medical history regarding demyelinating diseases before starting treatment.

Limited role for transforming growth factor-ß pathway activation-mediated escape from VEGF inhibition in murine glioma models.

BACKGROUND: The vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)-ß pathways regulate key biological features of glioblastoma. Here we explore whether the TGF-ß pathway, which promotes angiogenesis, invasiveness, and immunosuppression, acts as an escape pathway from VEGF inhibition. METHODS: The role of the TGF-ß pathway in escape from VEGF inhibition was assessed in vitro and in vivo and by gene expression profiling in syngeneic mouse glioma models. RESULTS: We found that TGF-ß is an upstream regulator of VEGF, whereas VEGF pathway activity does not alter the TGF-ß pathway in vitro. In vivo, single-agent activity was observed for the VEGF antibody B20-4.1.1 in 3 and for the TGF-ß receptor 1 antagonist LY2157299 in 2 of 4 models. Reduction of tumor volume and blood vessel density, but not induction of hypoxia, correlated with benefit from B20-4.1.1. Reduction of phosphorylated (p)SMAD2 by LY2157299 was seen in all models but did not predict survival. Resistance to B20 was associated with anti-angiogenesis escape pathway gene expression, whereas resistance to LY2157299 was associated with different immune response gene signatures in SMA-497 and GL-261 on transcriptomic profiling. The combination of B20 with LY2157299 was ineffective in SMA-497 but provided prolongation of survival in GL-261, associated with early suppression of pSMAD2 in tumor and host immune cells, prolonged suppression of angiogenesis, and delayed accumulation of tumor infiltrating microglia/macrophages. CONCLUSIONS: Our study highlights the biological heterogeneity of murine glioma models and illustrates that cotargeting of the VEGF and TGF-ß pathways might lead to improved tumor control only in subsets of glioblastoma.

Innate lymphotoxin receptor mediated signaling promotes HSV-1 associated neuroinflammation and viral replication.

Host anti-viral innate immunity plays important roles in the defense against HSV-1 infection. In this study, we find an unexpected role for innate LT/LIGHT signaling in promoting HSV-1 replication and virus induced inflammation in immunocompromised mice. Using a model of footpad HSV-1 infection in Rag1(-/-) mice, we observed that blocking LT/LIGHT signaling with LTßR-Ig could significantly delay disease progression and extend the survival of infected mice. LTßR-Ig treatment reduced late proinflammatory cytokine release in the serum and nervous tissue, and inhibited chemokine expression and inflammatory cells infiltration in the dorsal root ganglia (DRG). Intriguingly, LTßR-Ig treatment restricted HSV-1 replication in the DRG but not the footpad. These findings demonstrate a critical role for LT/LIGHT signaling in modulating innate inflammation and promoting HSV-1 replication in the nervous system, and suggest a new target for treatment of virus-induced adverse immune response and control of severe HSV-1 infection.

Efficacy and safety of pateclizumab (anti-lymphotoxin-α) compared to adalimumab in rheumatoid arthritis: a head-to-head phase 2 randomized controlled study (The ALTARA Study).

INTRODUCTION: Tumor necrosis factor (TNF) and, possibly, lymphotoxin alpha (LTα) signaling contribute to inflammation and rheumatoid arthritis (RA) pathogenesis. Pateclizumab (anti-lymphotoxin- alpha; MLTA3698A) is a humanized monoclonal antibody that blocks and depletes anti-LTα. This phase 2, randomized, head-to-head, active- and placebo-controlled trial examined the safety and efficacy of pateclizumab compared to adalimumab in RA patients with an inadequate response to disease-modifying antirheumatic drugs (DMARD-IR). METHODS: Patients (n = 214) with active RA (≥ 6 swollen and tender joints, C-reactive protein ≥ 10 mg/L) on oral DMARDs were randomized (2:2:1) to receive pateclizumab 360 mg, adalimumab 40 mg, or placebo subcutaneously every 2 weeks. The primary endpoint, 4-variable, 28-joint disease activity score erythrocyte sedimentation rate (DAS28(4)-ESR) response, was evaluated at 12 weeks using an analysis of covariance (ANCOVA) model with adjustments for concomitant DMARD use and geographic region. Secondary efficacy endpoints included American College of Rheumatology (ACR) 20, ACR50, and ACR70 responses at Day 85. Pharmacokinetics, pharmacodynamics, and immunogenicity of pateclizumab were assessed. RESULTS: Pateclizumab reduced the DAS28(4)-ESR response (-1.89) at 12 weeks, however, this did not reach statistical significance compared to placebo (-1.54), while adalimumab (-2.52) differed significantly from both placebo and pateclizumab. Pateclizumab 12-week ACR20, ACR50 and ACR70 response rates (64%, 33%, and 14%) suggested clinical activity but were not statistically significant compared to placebo rates (46%, 24%, and 8%, respectively). CXCL13 serum levels decreased significantly following pateclizumab and adalimumab administration, demonstrating pharmacological target engagement by both drugs. Overall, adverse events (AEs) were comparable among all cohorts. Infections were the most common AE, occurring with comparable frequency in all groups. Serious AEs occurred in 0% of pateclizumab, 5.9% of adalimumab, and 2.3% of placebo patients, with serious infection in 2.3% of adalimumab patients and none in pateclizumab and placebo patients. CONCLUSIONS: Pateclizumab had a good safety profile in patients inadequately responsive to DMARDs, but no statistically significant improvement in RA signs and symptoms after 12 weeks of treatment. Adalimumab demonstrated efficacy and safety comparable to published results in this head-to-head comparison in DMARD-IR RA patients. TRIAL REGISTRATION: ClinicalTrials.gov NCT01225393, Registered 18 October 2010.

Lymphotoxin and TNF: how it all began-a tribute to the travelers.

The journey from the discoveries of lymphotoxin (LT) and tumor necrosis factor (TNF) to the present day age of cytokine inhibitors as therapeutics has been an exciting one with many participants and highs and lows; the saga is compared to that in "The Wizard of Oz". This communication summarizes the contributions of key players in the discovery of the cytokines and their receptors, the changes in nomenclature, and the discovery of the LT family's crucial role in secondary and tertiary lymphoid organs. The remarkable advances in therapeutics are detailed as are remaining problems. Finally, special tribute is paid to two pioneers in the field who have recently passed away: Byron H. Waksman and Lloyd Old.

Controlling herpetic stromal keratitis by modulating lymphotoxin-alpha-mediated inflammatory pathways.

Herpes simplex virus 1 infection of the eye can result in stromal keratitis, a chronic immunoinflammatory lesion that is a significant cause of human blindness. A key to controlling the severity of lesions is to identify cellular and molecular events responsible for tissue damage. This report evaluates the role of lymphotoxin-α, a proinflammatory cytokine that could be involved during stromal keratitis. We demonstrate that after infection, both lymphotoxin-α and lymphotoxin-ß transcripts are detectable at high levels 48 h postinfection, suggesting roles for the secreted homotrimer lymphotoxin-α3 and the membrane-bound lymphotoxin-α1ß2 heterotrimer in stromal keratitis. Using a corneal stromal fibroblast cell line, lymphotoxin-α3 and lymphotoxin-α1ß2 were found to have proinflammatory roles by stimulating production of chemokines. Treatment of mice with a depleting anti-lymphotoxin-α mAb during the clinical phase of the disease significantly attenuated stromal keratitis lesions. In treated mice, expression of proinflammatory molecules and chemokines was reduced, as were numbers of cornea-infiltrating proinflammatory cells, particularly Th1 cells. The protective effect of anti-lymphotoxin-α mAb was highly reduced with a mutant version of the mAb that lacks Fc receptor binding activity, indicating that depletion of lymphotoxin-expressing cells was mainly responsible for efficacy, with LT-α3 contributing minimally to inflammation. These data demonstrate that lymphotoxin-expressing cells, such as Th1 cells, mediate stromal keratitis.

Epigenetic control of cytokine gene expression: regulation of the TNF/LT locus and T helper cell differentiation.

Epigenetics encompasses transient and heritable modifications to DNA and nucleosomes in the native chromatin context. For example, enzymatic addition of chemical moieties to the N-terminal "tails" of histones, particularly acetylation and methylation of lysine residues in the histone tails of H3 and H4, plays a key role in regulation of gene transcription. The modified histones, which are physically associated with gene regulatory regions that typically occur within conserved noncoding sequences, play a functional role in active, poised, or repressed gene transcription. The "histone code" defined by these modifications, along with the chromatin-binding acetylases, deacetylases, methylases, demethylases, and other enzymes that direct modifications resulting in specific patterns of histone modification, shows considerable evolutionary conservation from yeast to humans. Direct modifications at the DNA level, such as cytosine methylation at CpG motifs that represses promoter activity, are another highly conserved epigenetic mechanism of gene regulation. Furthermore, epigenetic modifications at the nucleosome or DNA level can also be coupled with higher-order intra- or interchromosomal interactions that influence the location of regulatory elements and that can place them in an environment of specific nucleoprotein complexes associated with transcription. In the mammalian immune system, epigenetic gene regulation is a crucial mechanism for a range of physiological processes, including the innate host immune response to pathogens and T cell differentiation driven by specific patterns of cytokine gene expression. Here, we will review current findings regarding epigenetic regulation of cytokine genes important in innate and/or adaptive immune responses, with a special focus upon the tumor necrosis factor/lymphotoxin locus and cytokine-driven CD4+ T cell differentiation into the Th1, Th2, and Th17 lineages.

Efficacy of etanercept for the treatment of psoriasis: an overview of the Italian clinical experience from the real-life setting and independent studies.

Etanercept is a dimeric fusion protein consisting of the extracellular portion of tumor necrosis factor (TNF) p75 receptor and human IgG1 Fc fragment, which, similarly to the physiological soluble TNF receptor, is capable of binding TNF-α and lymphotoxin. The mechanism of action of etanercept in psoriatic disease and other approved indications is mediated by the neutralization of soluble TNF-α leading to a modulation of the immune response. Clinical trials have provided robust evidence supporting the sustained efficacy and safety of etanercept in psoriasis, as both intermittent and continuous therapy. The aim of this manuscript was to review the efficacy profile of etanercept in psoriasis according to the clinical experience gained by Italian dermatological centers and derived from the available publications on this topic. Everyday practice confirms that etanercept is a valuable and flexible therapeutic tool for the management of high-need psoriasis patients.

Determination of lymphotoxin-alpha levels in patients with psoriatic arthritis undergoing etanercept treatment.

Tumor necrosis factor (TNF)-α plays a central role in psoriatic arthritis (PsA). A subgroup of patients with PsA do not respond to anti-TNF-α antibodies but respond to TNF receptor p75-Fc IgG fusion protein (etanercept), which also neutralizes lymphotoxin (LT)-α. It has been suggested that LT-α might be involved in the development of the disease. We determined LT-α serum levels in 15 PsA patients before (T0) and after 3, 6, 9, and 12 months of etanercept therapy (T3, T6, T9, and T12, respectively) and correlated them with their response to treatment. Bath Ankylosing Spondylitis Disease Activity Index, Psoriasis Area Severity Index, Disease Activity Score (DAS28), erythrocyte sedimentation rate (ESR), and C reactive protein (CRP) levels were assessed at the same time points. All patients showed a clinical response at T6, which persisted up to T12; ESR and CRP mean levels significantly decreased at T3 and remained within the normal range up to T12. LT-α levels significantly increased from T3 to T6 and returned to baseline levels at T12. Therefore, the LT-α serum levels do not seem to correlate with clinical and laboratory parameters of the response to etanercept in PsA patients. Further studies are required to better define the role of LT-α and LT-α blockade by etanercept in PsA patients.

Safety, pharmacokinetics, and biologic activity of pateclizumab, a novel monoclonal antibody targeting lymphotoxin α: results of a phase I randomized, placebo-controlled trial.

INTRODUCTION: Pateclizumab (MLTA3698A) is a humanized mAb against lymphotoxin α (LTα), a transiently expressed cytokine on activated B and T cells (Th1, Th17), which are implicated in rheumatoid arthritis (RA) pathogenesis. This study was conducted to assess the safety, tolerability, < NOTE: For clarity and per AMA/S-W Style, please restore the use of Oxford/serial commas (ie: David likes vanilla, strawberry, and chocolate ice cream) throughout. and biologic activity of single and multiple doses of intravenous (IV) or subcutaneous (SC) pateclizumab in RA patients. METHODS: The single ascending dose (SAD) phase in patients with stable RA consisted of six cohorts (4:1 active:placebo at 0.3 mg/kg IV, 1.0 mg/kg IV, 1.0 mg/kg SC, 3.0 mg/kg IV, 3.0 mg/kg SC, and 5.0 mg/kg IV; n = 5/cohort). In the multiple ascending dose (MAD) phase, patients with prespecified RA disease activity received three doses of pateclizumab or placebo (4:1) every 2 weeks (1.0 mg/kg SC, n = 10; 3.0 mg/kg SC, n = 20; or 5.0 mg/kg IV, n = 5). Safety and tolerability were assessed throughout, and clinical activity was determined after three doses (Week 6). RESULTS: We observed no serious adverse events (AEs) or dose-limiting toxicities, and the majority of AEs were mild to moderate. The pharmacokinetic profiles were linear, and clearance was independent of dose. Reductions in levels of serum CXCL13 were observed, supporting the biologic activity of pateclizumab on the LTα pathway. Patients receiving pateclizumab in the 3.0 mg/kg MAD group (3.0 mg/kg SC) demonstrated ACR20, ACR50, and ACR70 response rates at week 6 of 75%, 56% and 25%, respectively, compared with 57%, 29%, and 0% in the placebo group. The median Disease Activity Score in 28 joints, C-reactive protein, reduction was 28% for pateclizumab, versus 8.4% for placebo. CONCLUSIONS: Pateclizumabwas generally well-tolerated in RA patients. Preliminary evidence of clinical activity was observed in active RA patients at the dose level targeted for clinical effect.

Recombinant TNF-binding protein from variola virus as a novel potential TNF antagonist.

Gel-filtration chromatographic separation of the lysate of Sf21 insect cells infected with recombinant baculovirus BVi67 containing the gene for TNF-binding protein (CrmB) of variola virus (VARV) revealed that hTNF-cytotoxicity neutralization activity is associated with a fraction corresponding mainly to high molecular weight proteins (above 500 kDa) and less with fractions corresponding to proteins of 270 or 90 kDa. The recombinant VARV-CrmB protein has been purified by affinity chromatography. Difference in the experimentally determined and estimated (according to amino acid composition) VARV-CrmB molecular weight is due to glycosylation of the recombinant protein expressed in the insect cells. VARV-CrmB neutralizes in vitro the cytotoxic effect of hTNF and hLTalpha, and its TNF-neutralizing activity is two to three orders of magnitude higher compared to the analogous effects of type I and II soluble TNF receptors, comparable with the activity of mAb MAK195, and somewhat lower than the effect of the commercial drug Remicade.

Targeted depletion of lymphotoxin-alpha-expressing TH1 and TH17 cells inhibits autoimmune disease.

Uncontrolled T helper type 1 (T(H)1) and T(H)17 cells are associated with autoimmune responses. We identify surface lymphotoxin-alpha (LT-alpha) as common to T(H)0, T(H)1 and T(H)17 cells and employ a unique strategy to target these subsets using a depleting monoclonal antibody (mAb) directed to surface LT-alpha. Depleting LT-alpha-specific mAb inhibited T cell-mediated models of delayed-type hypersensitivity and experimental autoimmune encephalomyelitis. In collagen-induced arthritis (CIA), preventive and therapeutic administration of LT-alpha-specific mAb inhibited disease, and immunoablated T cells expressing interleukin-17 (IL-17), interferon-gamma and tumor necrosis factor-alpha (TNF-alpha), whereas decoy lymphotoxin-beta receptor (LT-betaR) fusion protein had no effect. A mutation in the Fc tail, rendering the antibody incapable of Fcgamma receptor binding and antibody-dependent cellular cytotoxicity activity, abolished all in vivo effects. Efficacy in CIA was preceded by a loss of rheumatoid-associated cytokines IL-6, IL-1beta and TNF-alpha within joints. These data indicate that depleting LT-alpha-expressing lymphocytes with LT-alpha-specific mAb may be beneficial in the treatment of autoimmune disease.

Long-term course of demyelinating neuropathies occurring during tumor necrosis factor-alpha-blocker therapy.

OBJECTIVE: To report the long-term follow-up (mean, 41 months; range, 25-55 months) of patients with demyelinating neuropathy occurring after tumor necrosis factor-alpha (TNF-alpha) blocker treatment (infliximab [Remicade], etanercept [Enbrel], and adalimumab [Humira]). BACKGROUND: Demyelinating neuropathy is a rare adverse event of anti-TNF-alpha therapy. Improvement usually occurs after drug interruption and/or in association with usual treatments for demyelinating neuropathies. DESIGN: Case report with review of the previously published cases. SETTING: University hospital in Le Kremlin-Bicêtre, France: tertiary reference center for peripheral neuropathies and national reference center for rare peripheral neuropathies (www.nnerf.fr). PATIENTS: Five patients (4 men, mean age, 47 years) who developed a demyelinating neuropathy during anti-TNF-alpha therapy. MAIN OUTCOME MEASURE: Development of neuropathy. RESULTS: Neuropathy developed early (8 months) after treatment introduction. Various clinical patterns were encountered, including pure sensory neuropathy. Immunomodulating treatments were always required for neuropathy control. Chronic demyelinating neuropathy developed either after change of anti-TNF-alpha drug or spontaneously after treatment discontinuation without any drug reintroduction. CONCLUSION: Influence of anti-TNF-alpha treatment continuation on the long-term course of neuropathy is variable, suggesting that anti-TNF-alpha treatment withdrawal is not always necessary for neuropathy control.

[B-cell-targeted therapy in the treatment of autoimmune diseases]. / B-Zell-gerichtete Therapie bei Autoimmunerkrankungen.

In recent years a growing body of evidence suggests a more central role for B-cells in the pathogenesis of several autoimmune diseases, apart from being the precursors of autoantibody-producing plasma cells. B-lymphocytes play an important role in the pathogenesis of various autoimmune diseases. In particular, the introduction of rituximab, a depleting antibody targeting CD20+ B cells and its clinical efficacy in rheumatoid arthritis, systemic lupus erythematosus, vasculitis and multiple sclerosis has stimulated further B-cell-targeted therapies. Other biologicals targeting CD20 are under clinical investigation. New strategies include targeting further B-cell surface markers such as CD22, as well as blocking B-cell-activating factors or their receptors.