Songqing Na

STAT3 and STAT1 mediate IL-11–dependent and inflammation-associated gastric tumorigenesis in gp130 receptor mutant mice

Deregulated activation of STAT3 is frequently associated with many human hematological and epithelial malignancies, including gastric cancer. While exaggerated STAT3 signaling facilitates an antiapoptotic, proangiogenic, and proproliferative environment for neoplastic cells, the molecular mechanisms leading to STAT3 hyperactivation remain poorly understood. Using the gp130(Y757F/Y757F) mouse model of gastric cancer, which carries a mutated gp130 cytokine receptor signaling subunit that cannot bind the negative regulator of cytokine signaling SOCS3 and is characterized by hyperactivation of the signaling molecules STAT1 and STAT3, we have provided genetic evidence that IL-11 promotes chronic gastric inflammation and associated tumorigenesis. Expression of IL-11 was increased in gastric tumors in gp130(Y757F/Y757F) mice, when compared with unaffected gastric tissue in wild-type mice, while gp130(Y757F/Y757F) mice lacking the IL-11 ligand-binding receptor subunit (IL-11Ralpha) showed normal gastric STAT3 activation and IL-11 expression and failed to develop gastric tumors. Furthermore, reducing STAT3 activity in gp130(Y757F/Y757F) mice, either genetically or by therapeutic administration of STAT3 antisense oligonucleotides, normalized gastric IL-11 expression and alleviated gastric tumor burden. Surprisingly, the genetic reduction of STAT1 expression also reduced gastric tumorigenesis in gp130(Y757F/Y757F) mice and coincided with reduced gastric inflammation and IL-11 expression. Collectively, our data have identified IL-11 as a crucial cytokine promoting chronic gastric inflammation and associated tumorigenesis mediated by excessive activation of STAT3 and STAT1.

Resistance to Experimental Autoimmune Encephalomyelitis and Impaired IL-17 Production in Protein Kinase Cθ-Deficient Mice

The protein kinase Cθ (PKCθ) serine/threonine kinase has been implicated in signaling of T cell activation, proliferation, and cytokine production. However, the in vivo consequences of ablation of PKCθ on T cell function in inflammatory autoimmune disease have not been thoroughly examined. In this study we used PKCθ-deficient mice to investigate the potential involvement of PKCθ in the development of experimental autoimmune encephalomyelitis, a prototypic T cell-mediated autoimmune disease model of the CNS. We found that PKCθ−/− mice immunized with the myelin oligodendrocyte glycoprotein (MOG) peptide MOG35–55 were completely resistant to the development of clinical experimental autoimmune encephalomyelitis compared with wild-type control mice. Flow cytometric and histopathological analysis of the CNS revealed profound reduction of both T cell and macrophage infiltration and demyelination. Ex vivo MOG35–55 stimulation of splenic T lymphocytes from immunized PKCθ−/− mice revealed significantly reduced production of the Th1 cytokine IFN-γ as well as the T cell effector cytokine IL-17 despite comparable levels of IL-2 and IL-4 and similar cell proliferative responses. Furthermore, IL-17 expression was dramatically reduced in the CNS of PKCθ−/− mice compared with wild-type mice during the disease course. In addition, PKCθ−/− T cells failed to up-regulate LFA-1 expression in response to TCR activation, and LFA-1 expression was also significantly reduced in the spleens of MOG35–55-immunized PKCθ−/− mice as well as in in vitro-stimulated CD4+ T cells compared with wild-type mice. These results underscore the importance of PKCθ in the regulation of multiple T cell functions necessary for the development of autoimmune disease.

IRAK4 kinase activity is required for Th17 differentiation and Th17-mediated disease.

Both IL-23- and IL-1-mediated signaling pathways play important roles in Th17 cell differentiation, cytokine production, and autoimmune diseases. The IL-1R-associated kinase 4 (IRAK4) is critical for IL-1/TLR signaling. We show here that inactivation of IRAK4 kinase in mice (IRAK4 KI) results in significant resistance to experimental autoimmune encephalomyelitis due to a reduction in infiltrating inflammatory cells into the CNS and reduced Ag-specific CD4+ T cell-mediated IL-17 production. Adoptive transfer of myelin oligodendrocyte glycoprotein 35–55-specific IRAK4 KI Th17 cells failed to induce experimental autoimmune encephalomyelitis in either wild-type or IRAK4 KI recipient mice, indicating the lack of autoantigen-specific Th17 cell activities in the absence of IRAK4 kinase activity. Furthermore, the absence of IRAK4 kinase activity blocked induction of IL-23R expression, STAT3 activation by IL-23, and Th17 cytokine expression in differentiated Th17 cells. Importantly, blockade of IL-1 signaling by IL-1RA inhibited Th17 differentiation and IL-23-induced cytokine expression in differentiated Th17 cells. The results of these studies demonstrate that IL-1-mediated IRAK4 kinase activity in T cells is essential for induction of IL-23R expression, Th17 differentiation, and autoimmune disease.

Characterization of LY2228820 Dimesylate, a Potent and Selective Inhibitor of p38 MAPK with Antitumor Activity

p38α mitogen-activated protein kinase (MAPK) is activated in cancer cells in response to environmental factors, oncogenic stress, radiation, and chemotherapy. p38α MAPK phosphorylates a number of substrates, including MAPKAP-K2 (MK2), and regulates the production of cytokines in the tumor microenvironment, such as TNF-α, interleukin-1β (IL-1β), IL-6, and CXCL8 (IL-8). p38α MAPK is highly expressed in human cancers and may play a role in tumor growth, invasion, metastasis, and drug resistance. LY2228820 dimesylate (hereafter LY2228820), a trisubstituted imidazole derivative, is a potent and selective, ATP-competitive inhibitor of the α- and β-isoforms of p38 MAPK in vitro (IC50 = 5.3 and 3.2 nmol/L, respectively). In cell-based assays, LY2228820 potently and selectively inhibited phosphorylation of MK2 (Thr334) in anisomycin-stimulated HeLa cells (at 9.8 nmol/L by Western blot analysis) and anisomycin-induced mouse RAW264.7 macrophages (IC50 = 35.3 nmol/L) with no changes in phosphorylation of p38α MAPK, JNK, ERK1/2, c-Jun, ATF2, or c-Myc ≤ 10 μmol/L. LY2228820 also reduced TNF-α secretion by lipopolysaccharide/IFN-γ–stimulated macrophages (IC50 = 6.3 nmol/L). In mice transplanted with B16-F10 melanoma, tumor phospho-MK2 (p-MK2) was inhibited by LY2228820 in a dose-dependent manner [threshold effective dose (TED)70 = 11.2 mg/kg]. Significant target inhibition (>40% reduction in p-MK2) was maintained for 4 to 8 hours following a single 10 mg/kg oral dose. LY2228820 produced significant tumor growth delay in multiple in vivo cancer models (melanoma, non–small cell lung cancer, ovarian, glioma, myeloma, breast). In summary, LY2228820 is a p38 MAPK inhibitor, which has been optimized for potency, selectivity, drug-like properties (such as oral bioavailability), and efficacy in animal models of human cancer. Mol Cancer Ther; 13(2); 364–74. ©2013 AACR.

OP0159 The initiation, but not the persistence, of experimental spondyloarthritis in HLA-B27/HUβ2M transgenic rats is crucially dependent on the IL-23 axis

Background The pro-inflammatory cytokine IL-17A is a central driver of pathology in human spondyloarthritis (SpA). IL-17A production was originally proposed to be dependent on the upstream cytokine, IL-23. Emerging preclinical and clinical evidence from SpA-related diseases suggest, however, that IL-17A and IL-23 have a partially overlapping but distinct biology. Objectives Here, we aimed to assess to what extend pathogenic IL-17A is dependent on IL-23 in SpA by selectively targeting the IL-23R in the HLA-B27/Huβ2m transgenic rat model of SpA, which we showed previously to be IL-17A-dependent. Methods HLA-B27/Huβ2m tg rats were immunized with low dose heat-inactivated M. tuberculosis/IFA. Rats were treated with a depleting anti-mouse/rat chimeric IL-23R antibody or PBS in a prophylactic (treatment initiation after immunization, before disease onset) or therapeutic (treatment initiation after disease onset) experiment. Clinical measurements included spondylitis and arthritis scores and hind paw swelling (plethysmometry). At the end of the study spleen and lymph nodes were used to assess cytokine expression, serum samples were analyzed for exposure to anti-IL23R. Results In the prophylactic treatment strategy, 58% and 67% of the rats in the control group developed spondylitis and arthritis, respectively. The average arthritis score at the end of the study was 3.9±1.1 and the average hind paw swelling was 0.35±0.09 cm3. Prophylactic treatment with anti-IL-23R completely protected the rats against the development of spondylitis as well as arthritis. In the therapeutic treatment strategy, however, anti-IL23R treatment failed to reduce the incidence or decrease the severity of experimental SpA (fig. 1). With an average increase in arthritis score after the start of treatment of 1.6±2.8 versus 2.1±2.5 and an increase in paw swelling of 0.6±0.7 versus 0.3±0.6 cm3in anti-IL23R treated versus control animals. The differential effect of IL-23R targeting in the initiation phase versus established disease could not be explained by pharmacokinetic differences as serum analyses revealed similar exposure levels. Mechanistically, the expression of presumably downstream effector cytokines such as IL-17A (p<0.05) and IL-22 (p<0.01) was reduced in the popliteal lymph nodes of rats treated prophylactically with anti-IL23R versus controls, with a similar trend in spleen. Accordingly, IL-17A production upon ex vivo re-stimulation was reduced in samples from prophylactically treated rats. In contrast, similar popliteal lymph node expression data in samples from the therapeutic experiment indicate a twofold increase in IL-17A expression and no difference in IL-22 expression in the anti-IL23R treated rats compared to controls. Conclusions IL-17A expression and production is dependent on the IL-23 axis in the initiation phase of experimental SpA but not in established disease. Accordingly, targeting of this axis with an anti-IL23R antibody completely prevented the onset of arthritis and spondylitis in HLA-B27/Huβ2m transgenic rats, but failed to reduce axial and peripheral joint inflammation in established disease. The cellular origin of IL-23-independent IL-17A production in established disease and the relevance to human SpA remains to be further investigated. Disclosure of Interest M. Van Tok: None declared, S. Na Employee of: Eli Lilly and Co, J. Taurog Grant/research support from: AbbVie, Anges, Inc, and Celgene, D. Baeten Grant/research support from: AbbVie, Pfizer, UCB, MSD, Roche, BMS, Novartis, Eli Lilly, Janssen, Glenmark, Boehringer-Ingelheim, Employee of: part-time employee of UCB, L. van Duivenvoorde Grant/research support from: Abbvie, Boehringer-Ingelheim

The Initiation, but Not the Persistence, of Experimental Spondyloarthritis Is Dependent on Interleukin-23 Signaling

IL-17A is a central driver of spondyloarthritis (SpA), its production was originally proposed to be IL-23 dependent. Emerging preclinical and clinical evidence suggests, however, that IL-17A and IL-23 have a partially overlapping but distinct biology. We aimed to assess the extent to which IL-17A-driven pathology is IL-23 dependent in experimental SpA. Experimental SpA was induced in HLA-B27/Huβ2m transgenic rats, followed by prophylactic or therapeutic treatment with an anti-IL23R antibody or vehicle control. Spondylitis and arthritis were scored clinically and hind limb swelling was measured. Draining lymph node cytokine expression levels were analyzed directly ex vivo, and IL-17A protein was measured upon restimulation with PMA/ionomycin. Prophylactic treatment with anti-IL23R completely protected against the development of both spondylitis and arthritis, while vehicle-treated controls did develop spondylitis and arthritis. In a therapeutic study, anti-IL23R treatment failed to reduce the incidence or decrease the severity of experimental SpA. Mechanistically, expression of downstream effector cytokines, including IL-17A and IL-22, was significantly suppressed in anti-IL23R versus vehicle-treated rats in the prophylactic experiments. Accordingly, the production of IL-17A upon restimulation was reduced. In contrast, there was no difference in IL-17A and IL-22 expression after therapeutic anti-IL23R treatment. Targeting the IL-23 axis during the initiation phase of experimental SpA—but not in established disease—inhibits IL-17A expression and suppresses disease, suggesting the existence of IL-23-independent IL-17A production. Whether IL-17A can be produced independent of IL-23 in human SpA remains to be established.

P059 Ex vivo comparison of baricitinib, upadacitinib, filgotinib and tofacitinib for cytokine signalling in human leukocyte subpopulations

Introduction Baricitinib (BARI), an oral selective Janus kinase (JAK) 1/2 inhibitor, approved in the EU for moderate to severe active RA. Objectives To compare in vitro cellular pharmacology of BARI to upadacitinib (ABT), filgotinib (FILGO), and tofacitinib (TOFA), three JAK inhibitors (JAKis) currently approved or in clinical development. Methods Peripheral blood mononuclear cells from healthy donors (n=6–12) were incubated with different JAKis. After cytokine stimulation, phosphorylated signal transducer and activator of transcription (pSTAT) levels were measured and IC50 calculated in phenotypically gated leukocyte subpopulations. Therapeutic dose relevance of in vitro analysis was assessed using calculated mean concentration-time (CT) profiles over 24u2009hour JAKi-treated subjects (BARI 4u2009mg QD; ABT 15 and 30u2009mg QD; FILGO 100 and 200u2009mg QD; TOFA 5 and 10u2009mg BID). Time above IC50 (T>IC; h/day) and average daily% inhibition of pSTAT formation (%SI) were calculated for each JAKi, cytokine, and cell type. Results Tested cytokines did not signal in all cell types. When signalling was detected, IC50,%SI, and T>IC for a particular JAKi exhibited similar dose dependent inhibition across cell types. For JAK1/3 dependent signalling across 4 cytokines (IL-2, 4, 15, 21), IC50 for ABT and TOFA were more potent than BARI; FILGO was the least potent. Overlaid on CT profiles, this indicated generally higher%SI and longer T>IC for ABT and TOFA compared to BARI and FILGO. For IL-6 (JAK1/2),%SI and T>IC was TOFA>BARI/ABT>FILGO and for IL-10 (JAK1/TYK2),%SI was TOFA>BARI/ABT>FILGO. IFN-γ (JAK1/2) was modulated only by BARI, ABT, and TOFA. IFN-α (JAK1/TYK2) signalling was most potently inhibited by BARI and ABT. FILGO did not appear to modulate GM-CSF signalling (JAK2/2), while%SI and T>IC were similar between BARI and ABT. Conclusions JAKis modulate distinct cytokine pathways to differing degrees and durations over 24u2009hour. BARI and FILGO inhibited JAK1/3 signalling less than ABT and TOFA. No JAKis agent potently or continuously inhibited an individual cytokine signalling pathway throughout dosing interval, implying the varying efficacy and safety profiles of JAKis across disease states. Acknowledgements Study support Eli Lilly and Company and Incyte Corporation. Encore of ACR/ARHP-2017 Annual Scientific Meeting, Nov 4–8, 2016; San Diego, CA, USA. Disclosure of interest None declared

Abstract B235: Characterization of LY2228820 dimesylate, a potent and selective inhibitor of p38 MAPK with antitumor activity.

p38α mitogen-activated protein kinase (MAPK) is activated by cancer cells in response to environmental factors, such as oncogenic stress, radiation, and chemotherapy. p38α MAPK phosphorylates a number of substrates, including MAPKAP-K2 (MK2), and regulates the production/message stability of cytokines produced in the tumor microenvironment, such as TNFα, IL1 β, IL-6 and IL-8. p38α MAPK is activated and highly expressed in human cancers and may play a role in tumor growth, invasion and metastasis. LY2228820 dimesylate is a tri-substituted imidazole derivative that is a potent and ATP-competitive inhibitor of the α and β isoforms of p38 MAPK in vitro (IC50 = 5.3 nM and 3.2 nM, respectively). This compound displays > 1000-fold selectivity for p38α MAPK versus 179 other kinases tested (including p38δ and γ isoforms). In cell-based assays, LY2228820 dimesylate also potently and selectively inhibits phosphorylation of MK2 (Thr334) in TNFα-stimulated HeLa cells (IC50 = 8.1 nM) and anisomycin-induced mouse RAW264.7 macrophages (IC50 = 35.3 nM) with no changes in phosphorylation of p38α MAPK, JNK, ERK1/2, c-jun, ATF2 or cMyc at concentrations up to 10μM. LY2228820 dimesylate also reduces TNFα secretion by LPS/IFNγ-stimulated macrophages (IC50 = 6.3 nM). In mice transplanted with B16-F10 melanomas, phospho-MK2 was effectively inhibited by LY2228820 dimesylate in tumors in a dose-dependent manner (TMED70 = 19.4 mg/kg). Significant target inhibition (>40% inhibition of phospho-MK2) was maintained for approx. 4–8hrs following a single 10mg/kg oral dose. In a broad range of xenograft models (A-549 NSCLC, SK-OV-3 Ovarian, U-87MG Glioma, MDA-MB-468 Breast), LY2228820 dimesylate demonstrates significant tumor growth delay. In summary, LY2228820 dimesylate is a novel, potent and selective inhibitor of p38 MAPK with anti-tumor activity. Further studies are ongoing to determine its molecular mechanism(s) of action, potential for combination with standard-of-care agents, and potential clinical activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B235.

Abstract A237: Elevated Mnk kinase activity is associated with malignant progression and reduced patient survival in human prostate cancer and can be therapeutically inhibited in human prostate cancer cells by the novel, orally bioavailable Mnk inhibitor cercosporamide.

In a wide variety of malignancies, progressive disease and reduced patient survival have been linked to increased function of eukaryotic translation initiation factor 4E (eIF4E), which selectively enhances the translation of potent growth and survival factors and oncoproteins (e.g. c-myc, VEGF, cyclin D1, Mcl-1, MMP-9, etc.). Recent published reports have shown that the oncogenic activity of eIF4E is critically dependent on the activity of the MNK kinases (Map kinase interacting kinase 1 and 2), which phosphorylate eIF4E at S209 (p4ES209). Increased p4ES209 has been linked to a variety of advanced cancers, notably head and neck and non-small cell lung cancers. Herein, we report that p4ES209 is significantly elevated in human prostate cancer (CaP) tissues (n=133) and associated with reduced patient survival. p4ES209 is elevated In both low grade (Gleason score ≤ 6) and high grade (Gleason score ≥ 7) CaP vs. adjacent normal prostate tissue (BPH) (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A237.