Proinflammatory Cytokine Signaling Research Articles

Abstract LB-277: Characterization of the anti-cancer and immunologic activity of RGX-019, a novel pre-clinical stage humanized monoclonal antibody targeting the MERTK receptor

Abstract MERTK, a receptor tyrosine kinase of the TYRO3/AXL/MERTK (TAM) family, is expressed in innate immune cells including macrophages, dendritic cells and NK cells and is overexpressed in a wide variety of cancers, including leukemia and many solid cancers. Activation of MERTK on cancer cells via ligand binding results in activation of several tumor-promoting signaling pathways, which stimulate proliferation, migration and angiogenesis, and decrease apoptosis and chemosensitivity. Furthermore, activation of MERTK on macrophages drives immune evasion through the promotion of an immune-suppressive M2 phenotype. Pre-clinical and clinical studies have shown promising emerging evidence of anti-tumor efficacy upon modulation of TAM receptor signaling. Herein, we report the pre-clinical characterization of RGX-019, a humanized IgG1 antibody with high affinity and specificity for human MERTK. Surface Plasmon Resonance, cell-based binding assays and competition ELISA demonstrate high affinity/avidity binding for RGX-019 to human MERTK. In contrast, binding to human AXL, human TYRO3, or murine MERTK was not detected. Binding of RGX-019 to the MERTK receptor triggered its rapid internalization and degradation from the surface of human cancer cells within 4 hours and prevented Gas6 induced phosphorylation of AKT, a downstream signal transduction pathway that promotes cell growth and survival. RGX-019 treatment of SKMel5 melanoma cells effectively inhibited colony formation. RGX-019-induced degradation of MERTK in in vitro differentiated human M2 macrophages reduced AKT activation and promoted a pro-inflammatory cytokine signature. Furthermore, a related surrogate murine MERTK antibody significantly inhibited growth of MDA-MB-231 breast cancer tumors, demonstrating single agent anti-tumor efficacy in vivo. These findings demonstrate that RGX-019 is a potent and selective inhibitor of MERTK signaling with a unique mechanism-of-action that potently disrupts MERTK signaling in both cancer cells and immune-suppressive macrophages via MERTK receptor degradation. This activity results in robust repression of cancer cell growth both in vitro and in vivo. Overall, these data support further development of RGX-019 as a cancer therapeutic. Citation Format: Shugaku Takeda, Celia Andreu-Agullo, Subhasree Sridhar, Nils Halberg, Ivo C. Lorenz, Sohail Tavazoie, Isabel Kurth, Masoud Tavazoie. Characterization of the anti-cancer and immunologic activity of RGX-019, a novel pre-clinical stage humanized monoclonal antibody targeting the MERTK receptor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-277.

Nonmuscle Myosin Heavy Chain IIA Recognizes Sialic Acids on Sialylated RNA Viruses To Suppress Proinflammatory Responses via the DAP12-Syk Pathway.

Viral infections induce proinflammatory signaling cascades and inflammatory cytokine production, which is precisely regulated for host benefits. In the current study, we unravel a previously unappreciated role of nonmuscle myosin heavy chain IIA (NMHC-IIA) as a negative regulator in inflammatory responses. We identified that cell surface NMHC-IIA recognized sialic acids on sialylated RNA viruses during early infections and interacted with an immune adaptor DNAX activation protein of 12 kDa (DAP12) to recruit downstream spleen tyrosine kinase (Syk), leading to suppressed virus-triggered proinflammatory responses. More importantly, recognition of sialylated RNA viruses or sialic acid mimics by NMHC-IIA was shown to inhibit lipopolysaccharide (LPS)-induced proinflammatory responses via the DAP12-Syk pathway. These findings uncover a novel negative regulation mechanism of proinflammatory responses and provide a molecular basis to design anti-inflammatory drugs.IMPORTANCE NMHC-IIA, a subunit of nonmuscle myosin IIA (NM-IIA), takes part in diverse physiological processes, including cell movement, cell shape maintenance, and signal transduction. Recently, NMHC-IIA has been demonstrated to be a receptor or factor contributing to viral infections. Here, we identified that NMHC-IIA recognizes sialic acids on sialylated RNA viruses, vesicular stomatitis virus (VSV) and porcine reproductive and respiratory syndrome virus (PRRSV). Upon recognition, NMHC-IIA associates with the transmembrane region of DAP12 to recruit Syk. Activation of the DAP12-Syk pathway impairs the host antiviral proinflammatory cytokine production and signaling cascades. More importantly, sialic acid mimics and sialylated RNA viruses enable the antagonism of LPS-triggered proinflammatory responses through engaging the NMHC-IIA-DAP12-Syk pathway. These results actually support that NMHC-IIA is involved in negative modulation of the host innate immune system, which provides a molecular basis for prevention and control of the sialylated RNA viruses and treatment of inflammatory diseases.

Progesterone receptor membrane component 1 inhibits tumor necrosis factor alpha induction of gene expression in neural cells.

Progesterone membrane receptor component 1 (Pgrmc1) is a cytochrome b5-related protein with wide-ranging functions studied most extensively in non-neural tissues. We previously demonstrated that Pgrmc1 is widely distributed in the brain with highest expression in the limbic system. To determine Pgrmc1 functions in cells of these regions, we compared transcriptomes of control siRNA-treated and Pgrmc1 siRNA-treated N42 hypothalamic cells using whole genome microarrays. Our bioinformatics analyses suggested that Pgrmc1 plays a role in immune functions and likely regulates proinflammatory cytokine signaling. In follow-up studies, we showed that one of these cytokines, TNFα, increased expression of rtp4, ifit3 and gbp4, genes found on microarrays to be among the most highly upregulated by Pgrmc1 depletion. Moreover, either Pgrmc1 depletion or treatment with the Pgrmc1 antagonist, AG-205, increased both basal and TNFα-induced expression of these genes in N42 cells. TNFα had no effect on levels of Rtp4, Ifit3 or Gbp4 mRNAs in mHippoE-18 hippocampal control cells, but Pgrmc1 knock-down dramatically increased basal and TNFα-stimulated expression of these genes. P4 had no effect on gbp4, ifit3 or rtp4 expression or on the ability of Pgrmc1 to inhibit TNFα induction of these genes. However, a majority of the top upstream regulators of Pgrmc1 target genes were related to synthesis or activity of steroids, including P4, that exert neuroprotective effects. In addition, one of the identified Pgrmc1 targets was Nr4a1, an orphan receptor important for the synthesis of most steroidogenic molecules. Our findings indicate that Pgrmc1 may exert neuroprotective effects by suppressing TNFα-induced neuroinflammation and by regulating neurosteroid synthesis.

772 Age-associated alterations in pro-inflammatory cytokine signaling as well as UV-induced DNA damage contribute to increased basal cell carcinoma (BCC) tumor burden in Ptch1+/-/SKH-1 mice

Human BCC incidence is age-dependent. We have developed Ptch1+/-/SKH-1 mice as a model of human BCC pathogenesis. These mice progressively develop spontaneous BCCs, suggesting per se age-related effects on cancer induction irrespective of aberrant hedgehog signaling. Here, utilizing cohorts of young (2 months) and aged (> 20 months) mice, we tested the hypothesis that chronologically aged skin is more susceptible to ultraviolet radiation (UVR)-induced skin damage and BCC growth as compared to young adult skin. UVR-induced cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts (6-4PPs) were measured in genomic DNA isolated from skin tissues at 24, 48, and 72h post-irradiation (240 mJ/cm2, single exposure). CPD levels peaked at 24h, decreasing gradually and were virtually undetectable after 72h in all cohorts, irrespective of age and sex. A similar pattern was observed for UV-induced 6-4PP in young cohorts. In contrast, skin 6-4PP levels persisted in aged Ptch1+/-/SKH-1 mice, indicating age-related differences in DNA repair capacity. Moreover, circulating levels of inflammatory cytokines known to be altered in cancer and aging (IL1β, IL6, TNFα) were significantly elevated in both aged male and female Ptch1+/-/SKH-1 mice, as well as in UV-irradiated young cohorts. Aged Ptch1+/-/SKH-1 mice subjected to chronic UVR (180 mJ/cm2, 2x/week, 16 weeks) revealed statistically significant increases in tumor burden (UV-irradiated vs. non-irradiated aged cohorts), while we observed < 2-fold increase in BCC burden in UV-irradiated young cohorts vs. non-irradiated controls. These results demonstrate differential susceptibility to UVR-induced BCCs in young and aged Ptch1+/-/SKH-1 mice such that aging enhances BCC burden in Ptch1-deficient skin, and altered DNA damage responses and systemic pro-inflammatory cytokine signaling may contribute to age-dependent enhancement of BCC tumorigenesis in human populations.

Intestinal Inflammation Differentially Influences Iron Homeostasis in WT versus Hepcidin KO Rats

IntroductionThe chronic anemia associated with IBD in humans may result from trans‐activation of the the iron‐regulatory hormone (Hamp) by pro‐inflammatory cytokines in hepatocytes. High hepcidin decreases serum iron by blunting intestinal iron absorption and impairing release of storage iron, thus decreasing iron delivery to the erythroid marrow and causing anemia. Pharmacological targeting of hepcidin offers an attractive therapeutic approach to mitigate IBD‐associated anemia. Testing of such interventions has mainly utilized mouse models of hereditary hemochromatosis (HH). Collectively, these mice have inappropriately low (or absent) hepcidin expression, and concomitant increases in intestinal iron absorption leading to iron overload. However, since iron metabolism in mice and humans varies notably, development of complementary experimental models is imperative.ObjectiveWe created a novel model of HH, the Hamp KO rat, and utilized the DSS model of colitis for this investigation. We hypothesized that Hamp levels would increase in rats with colitis (due to pro‐inflammatory cytokine signaling), which would then blunt intestinal iron absorption and cause anemia (thus modeling human IBD). We further postulated that iron absorption would be unaffected by inflammation in Hamp KO rats, thus preventing the development of anemia.MethodsIntestinal inflammation was induced by providing DSS (4% w/v) in drinking water for 7 days. Standard bioassays were utilized to track iron loading. Iron absorption was assessed by oral‐intragastric gavage of an 59Fe transport solution.ResultsHamp−/− rats displayed multi‐visceral iron overload, and intestinal iron absorption was increased (≈2.5 fold). DSS treatment caused similar pathological changes in the duodenum and colon of Hamp KO rats and WT littermates. Systemic inflammation was not observed in any experimental animals. DSS exposure decreased TSAT, and serum and splenic nonheme iron in both genotypes. In WT animals, DSS exposure: 1) decreased blood hemoglobin levels by ≈15%; 2) reduced liver hepcidin expression by ≈60%; 3) and increased intestinal iron absorption by &gt;2‐fold. Conversely, in Hamp−/− rats, DSS treatment increased blood hemoglobin levels and decreased iron absorption by ≈2.5‐fold. Moreover, the distribution of absorbed 59Fe in blood and many tissues was different between genotypes after DSS txt.ConclusionsDespite the lack of systemic inflammation, iron homeostasis was perturbed in WT rats by DSS‐induced enteritis. Reduced hepcidin expression likely resulted from anemia and/or iron depletion of serum and spleen. This may explain why iron absorption increased, since a relative state of iron deficiency may have existed after DSS exposure. In the KOs, intestinal inflammation altered iron homeostasis in distinct ways. This differential response to DSS may relate to the iron‐overload phenotype of the KO rats, or alternatively, it could reflect a role for hepcidin in mediating the pathophysiologic response to intestinal inflammation. Future studies in iron‐depleted Hamp KO rats may allow for a better understanding of the role of hepcidin in regulating iron homeostasis in IBD.Support or Funding InformationNIH Grant R01 DK074867 and R01 DK109717 (to JFC)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ruxolitinib Cream Suppresses Th2 Inflammation in Adult Patients With Atopic Dermatitis

Ruxolitinib cream, a selective inhibitor of Janus kinases (JAK)-1 and JAK2, potently inhibits proinflammatory cytokine signaling involved in atopic dermatitis (AD) pathogenesis. In a phase 2 trial, 1.5% ruxolitinib cream twice daily (BID) demonstrated efficacy at least comparable to triamcinolone. Rapid decreases in itching were observed. The effects of ruxolitinib cream on Th2 biomarkers and the relationship with clinical measures in this trial are reported below. Sera were collected from 111 patients treated with ruxolitinib cream 0.15% once daily (QD; n=17), 0.5% QD (n=20), 1.5% QD (n=15), 1.5% BID (n=18), triamcinolone BID (n=19), and vehicle BID (n=22). Serum immunoglobulin E (IgE) and thymus and activation-regulated chemokine (TARC/CCL17) were analyzed at baseline and Week 8. Differences between vehicle and each treatment arm were determined using mixed-model repeated measures; significance was conferred at P<0.05. Application of ruxolitinib cream 1.5% BID for 8 weeks significantly (P=0.0095) reduced TARC/CCL17 levels in circulation versus vehicle-treated patients. Additionally, total serum IgE levels were reduced in patients treated with 1.5% ruxolitinib cream (QD or BID). No material differences were observed with 0.15% QD or 0.5% QD ruxolitinib. Efficacy analyses by baseline TARC/CCL17 (≤522 vs >522 pg/mL) and total IgE subgroups (<200 vs ≥200 kU/L) were performed for percentage changes from baseline in Eczema Area and Severity Index (EASI). Levels of TARC/CCL17 and IgE at baseline did not predict ruxolitinib cream treatment response (percentage reductions in EASI). Treatment of patients with AD with ruxolitinib cream inhibited select Th2 biomarkers, suggesting its ability to modulate disease course.

Neuroinflammation and central PI3K/Akt/mTOR signal pathway contribute to bone cancer pain.

BackgroundPain is one of the most common and distressing symptoms suffered by patients with progression of cancer; however, the mechanisms responsible for hyperalgesia are not well understood. Since the midbrain periaqueductal gray is an important component of the descending inhibitory pathway controlling on central pain transmission, in this study, we examined the role for pro-inflammatory cytokines of the periaqueductal gray in regulating mechanical and thermal hyperalgesia evoked by bone cancer via phosphatidylinositide 3-kinase (PI3K)–mammalian target of rapamycin (mTOR) signals.MethodsBreast sarcocarcinoma Walker 256 cells were implanted into the tibia bone cavity of rats to induce mechanical and thermal hyperalgesia. Western blot analysis and ELISA were used to examine PI3K/protein kinase B (Akt)/mTOR and pro-inflammatory cytokine receptors and the levels of interleukin (IL)-1β, IL-6, and tumor necrosis factor-alpha (TNF-α).ResultsProtein expression levels of p-PI3K/p-Akt/p-mTOR were amplified in the periaqueductal gray of bone cancer rats, and blocking PI3K–mTOR pathways in the periaqueductal gray attenuated hyperalgesia responses. In addition, IL-1β, IL-6, and TNF-α were elevated in the periaqueductal gray of bone cancer rats, and expression of their respective receptors (namely, IL-1R, IL-6R, and tumor necrosis factor receptor (TNFR) subtype TNFR1) was upregulated. Inhibition of IL-1R, IL-6R, and TNFR1 alleviated mechanical and thermal hyperalgesia in bone cancer rats, accompanied with downregulated PI3K–mTOR.ConclusionsOur data suggest that upregulation of pro-inflammatory cytokine signal in the periaqueductal gray of cancer rats amplifies PI3K–mTOR signal in this brain region and alters the descending pathways in regulating pain transmission, and this thereby contributes to the development of bone cancer-induced pain.

Exogenous interleukin-1α signaling negatively impacts acquired chemoresistance and alters cell adhesion molecule expression pattern in colorectal carcinoma cells HCT116

Proinflammatory cytokine and chemokine signaling from the tumor microenvironment is thought to be crucial for developing and sustaining colorectal cancer by regulating a multitude of pathways associated with a variety of cellular mechanisms. Among these pathways there is acquired chemoresistance, which is usually a major obstacle in the way towards successful chemotherapeutic treatment of advanced colorectal cancer cases. Despite of an emerging body of data published on the role of cytokine signaling network in cancer, little is known about the effects of the upstream cytokine interleukin-1α (IL-1α) signaling to the cancer cells. In this study we have shown that the increase in exogenous IL-1α signaling increases chemosensitivity of both chemosensitive and chemoresistant colorectal cancer cell lines, treated with a widely used cytotoxic antimetabolite 5-fluorouracil (5-FU). This was a result of increased cell death but not of the changes in 5-FU-induced cell cycle arrest. Noticeably, combined exogenous IL-1α and 5-FU treatment had significant effects on the expression of cell adhesion molecules, suggesting a decrease in adhesion-dependent chemoresistance and, on the other hand, an increase in metastatic potential of the cells. These results lead to a conclusion that modulation of IL-1 receptor activity could have applications as a part of combination therapy for advanced and highly metastatic colorectal cancers.

MafA Expression Preserves Immune Homeostasis in Human and Mouse Islets.

Type 1 (T1D) and type 2 (T2D) diabetes are triggered by a combination of environmental and/or genetic factors. Maf transcription factors regulate pancreatic beta (β)-cell function, and have also been implicated in the regulation of immunomodulatory cytokines like interferon-β (IFNβ1). In this study, we assessed MAFA and MAFB co-expression with pro-inflammatory cytokine signaling genes in RNA-seq data from human pancreatic islets. Interestingly, MAFA expression was strongly negatively correlated with cytokine-induced signaling (such as IFNAR1, DDX58) and T1D susceptibility genes (IFIH1), whereas correlation of these genes with MAFB was weaker. In order to evaluate if the loss of MafA altered the immune status of islets, MafA deficient mouse islets (MafA−/−) were assessed for inherent anti-viral response and susceptibility to enterovirus infection. MafA deficient mouse islets had elevated basal levels of Ifnβ1, Rig1 (DDX58 in humans), and Mda5 (IFIH1) which resulted in reduced virus propagation in response to coxsackievirus B3 (CVB3) infection. Moreover, an acute knockdown of MafA in β-cell lines also enhanced Rig1 and Mda5 protein levels. Our results suggest that precise regulation of MAFA levels is critical for islet cell-specific cytokine production, which is a critical parameter for the inflammatory status of pancreatic islets.

Genomic and Proteomic Profiling of AF10-Fusion Oncoproteins Reveal Mechanisms of Leukemogenesis and Actionable Targets

The AF10/MLLT10 gene is recurrently involved in chromosomal rearrangements in human leukemia. AF10 rearrangements are linked to a poor prognosis in AML and T-ALL, underscoring the need to identify targeted therapies for AF10-fusion positive leukemia. Defining the molecular mechanisms of oncogenesis mediated by AF10-fusion proteins (AF10-FPs) may unravel novel actionable targets in leukemias with AF10-gene rearrangements. Towards this end, we established tetracycline (Tet)-inducible models of MLL-AF10 and CALM-AF10 AML and performed RNA-seq in AML cells treated with doxycycline (Dox) compared to vehicle treated counterparts. Since Dox treatment completely abrogates AF10-fusion gene expression from the Tet-regulated promoter, these models can be used to characterize the transcriptional landscape of potential AF10-FP target genes. We observed that among transcripts significantly downregulated upon Dox treatment, 168 genes were common in both the MLL-AF10 Tet-Off or CALM-AF10 Tet-Off conditions, indicating a high overlap between potential transcriptional targets of these distinct AF10-FPs. Expectedly, this list included genes previously implicated in leukemogenesis including Hoxa cluster genes, Meis1, Flt3, Mecom, Cd34, Gfi1b, Eya1 and Nkx2-3. Importantly, in addition to these well-characterized genes, we identified a number of novel pathways that were downregulated in the AF10-FP Tet-Off state. The most striking molecular signature of potential AF10-FP-regulated genes emerging from these analyses were factors involved in innate immunity and pro-inflammatory cytokine signaling. Prominent drivers of these molecular signatures included genes of the Jak/Stat and NFkB signaling pathways as well as Interferon response genes. We confirmed that AF10-FPs strongly activated Jak-Stat and NFkB signaling by performing Western blotting for key factors involved in these pathways. Since pro-inflammatory cytokines have been shown to play a role in AML cell survival, we tested the impact of cytokine depletion on murine AF10-FPs-driven AML cells. Proliferation assays demonstrated that AF10-FP-transformed cells could survive significantly better in cytokine-free medium compared to those transformed with other oncogenes such as MLL-AF9, which were completely dependent on cytokines for survival and proliferation in vitro. These results suggest that activation of cytokine signaling may contribute to increased survival of AF10-FP-driven AML cells.Next, we performed proteomic studies in which affinity-purified epitope-tagged AF10-FPs were evaluated for interacting proteins using Mass Spectrometry (MS). While studies on MLL-AF10 fusion are ongoing, our studies revealed that the strongest interactor of the CALM-AF10 fusion protein was the Janus kinase protein Jak1. We confirmed this finding by immunoprecipitation experiments in CALM-AF10 AML cells using a Jak1-specific antibody. Given the role of JAK1 in cytokine-mediated pro-inflammatory signaling, our findings indicate that CALM-AF10 may activate this pathway through direct recruitment of the Jak1 kinase. We sought to directly test the role of JAK1 in AF10-FP-mediated leukemogenesis. For this, we transformed bone marrow stem and progenitor cells from Jak1 floxed mice with the CALM-AF10 fusion. Deletion of Jak1 using Cre-recombinase in CALM-AF10 AML significantly reduced their proliferation in vitro. Furthermore, Jak1 deletion led to a highly significant reduction in the number of colony forming units (CFUs) from CALM-AF10 AML cells, with a particularly striking decrease in the number of blast-like colonies. We also observed a significant increase in differentiation of CALM-AF10 AML cells following Jak1 deletion, demonstrating that Jak1 activity is important for maintaining the CALM-AF10 leukemia cells in an undifferentiated state. Importantly, these results were recapitulated with two different small-molecule JAK1 inhibitors itacitinib and filgotinib that are being tested in clinical trials for a variety of human diseases. Treatment of CALM-AF10 AML cells with these selective JAK1 inhibitors led to a significant, dose-dependent decrease in proliferation accompanied by growth arrest and apoptosis. Taken together, our studies demonstrate that AF10 fusions activate pro-inflammatory signaling by co-opting the Jak-Stat pathway, presenting a potential therapeutic target in AF10-fusion-driven AML. DisclosuresLevine:Janssen: Consultancy, Honoraria; Celgene: Consultancy, Research Funding; Qiagen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Prelude: Research Funding; Loxo: Consultancy, Equity Ownership; Imago: Equity Ownership; C4 Therapeutics: Equity Ownership; Novartis: Consultancy; Gilead: Honoraria; Isoplexis: Equity Ownership; Epizyme: Patents & Royalties; Roche: Consultancy, Research Funding. Deshpande:A2A Pharma: Membership on an entity's Board of Directors or advisory committees; Salgomed Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Role of Exercise Activity in Alleviating Neuropathic Pain in Diabetes via Inhibition of the Pro-Inflammatory Signal Pathway.

Hyperalgesia and allodynia are commonly observed in patients with diabetic neuropathy. The treatment and management of painful peripheral neuropathy is important in these patients. The purpose of this study was to examine the role of exercise in modulating neuropathic pain induced by diabetes. Diabetes was induced in rats by an intraperitoneal injection of streptozotocin (STZ). Control rats received saline injections. Groups included control rats without exercise (NT-control, n = 12), control rats with exercise (EX-control, n = 16), STZ rats without exercise (NT-STZ, n = 18), and STZ rats with exercise (EX-STZ, n = 22). Rats in EX groups ran on a treadmill 4 days/week for 5 weeks beginning from the week of STZ administration. Mechanical hypersensitivity (mechanical paw withdrawal thresholds [PWTs]) and glucose levels were tested weekly. Then, enzyme-linked immunoassay and Western blot analysis were used to determine the levels of pro-inflammatory cytokines (PICs) and their receptors in sensory nerves. PWTs were significantly increased after 4-5 weeks of exercise in STZ rats ( p < .05 vs. NT-STZ rats). Inhibition of neuropathic pain by exercise in STZ rats was accompanied by decreases in interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α levels and downregulated expression of their receptors. Furthermore, blocking individual PIC receptors elevated PWTs to a greater degree in STZ rats ( p < .05 vs. control rats). Overall, our data suggest that exercise can play a role in improving neuropathic pain induced by STZ and that PIC signaling is a part of the mechanism involved in this effect.

IκBζ is a key transcriptional regulator of IL-36–driven psoriasis-related gene expression in keratinocytes

Proinflammatory cytokine signaling in keratinocytes plays a crucial role in the pathogenesis of psoriasis, a skin disease characterized by hyperproliferation and abnormal differentiation of keratinocytes and infiltration of inflammatory cells. Although IL-17A and TNFα are effective therapeutic targets in psoriasis, IL-36 has recently emerged as a proinflammatory cytokine. However, little is known about IL-36 signaling and its downstream transcriptional responses. Here, we found that exposure of keratinocytes to IL-36 induced the expression of IκBζ, an atypical IκB member and a specific transcriptional regulator of selective NF-κB target genes. Induction of IκBζ by IL-36 was mediated by NF-κB and STAT3. In agreement, IL-36-mediated induction of IκBζ was found to be required for the expression of various psoriasis-related genes involved in inflammatory signaling, neutrophil chemotaxis, and leukocyte activation. Importantly, IκBζ-knockout mice were protected against IL-36-mediated dermatitis, accompanied by reduced proinflammatory gene expression, decreased immune cell infiltration, and a lack of keratinocyte hyperproliferation. Moreover, expression of IκBζ mRNA was highly up-regulated in biopsies of psoriasis patients where it coincided with IL36G levels. Thus our results uncover an important role for IκBζ in IL-36 signaling and validate IκBζ as an attractive target for psoriasis therapy.

Abstract A17: Bromodomain and extraterminal proteins regulate PD-L1/PD-1 signaling in breast cancer.

Abstract Introduction: Breast tumors are massively infiltrated immune cells of both lymphoid and myeloid origin that exert an anti-tumor pressure to limit cancer progression. Tumor cells can escape immune clearance by expressing several molecules that constitute the immune checkpoints. Antibodies targeting the PD-L1/PD-1 pathway are being evaluated clinically for several cancers and show early success. However, the realization that not all patients respond well to immunotherapy suggests other modalities could be combined to improve efficacy. The bromodomain and extraterminal (BET) proteins BRD2, BRD3 and BRD4 are epigenetically acting co-regulators of transcription, and are critical for cancer proliferation and metastasis. Little is known about the molecular mechanisms that regulate PD-L1 and PD-1 expression. BRD4 is known to bind directly to the PD-L1 promoter; its targeting suppresses PD-L1 expression while increasing CD8+ T cell activity to limit progression in ovarian tumor models. Hypotheses: BET proteins regulate PD-L1 and PD-L2 expression in breast tumors and PD-1 in CD8+ T cells. BET proteins are effectors of proinflammatory cytokine signaling, regulating PD-L1/PD-1 signaling in the breast tumor microenvironment. Methods: We used different cellular models of breast cancer. We also collected human peripheral blood mononuclear cells matched with mammary adipose tissue from mammoplasty patients at Boston Medical Center to assess expression of immune checkpoint proteins in the breast microenvironment. Pan-BET protein inhibition was performed by JQ1 treatment. Alternatively, BET proteins were individually and specifically depleted by siRNA. PD-1 and PD-L1 expression were determined by qRT-PCR and flow cytometry. Cytokines and chemokines present in the blood or secreted by the mammary adipose tissue were profiled by multiplexed antibody capture assay. Results: BET proteins regulate PD-L1 expression by breast cancer cells. BRD2 and BRD4 expression correlate with PD-1 in activated circulating T cells and BET inhibition ablates activation-induced PD-1 expression. Co-culture of breast cancer cells with activated circulating T cells enhances expression of immune checkpoint proteins in both compartments. BET protein inhibition rescues these phenotypes. Conditioned media from mammary adipose tissue increase PD-1 in PBMCs. Peripheral and mammary adipose tissue inflammatory signatures associate with PD-1 expression in PBMCs, suggesting that local inflammation in the breast tumor microenvironment contributes to diminishing anti-tumor immune responses. Conclusion: BET proteins regulate the PD-L1/PD-1 pathway in breast cancer. Next-generation BET protein inhibitors may combine well with PD-1 or PD-L1-targeted immunotherapies in difficult-to-treat cancers. Personalized BET profiles could inform individual patient responses to BET proteins and immune checkpoint inhibitors. Therapeutic approaches that treat the microenvironment should be leveraged to maximize efficacy of checkpoint inhibitor approaches for specific cancers. Citation Format: Guillaume Andrieu, Gerald V. Denis. Bromodomain and extraterminal proteins regulate PD-L1/PD-1 signaling in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A17.

Dual Inhibition of TYK2 and JAK1 for the Treatment of Autoimmune Diseases: Discovery of (( S)-2,2-Difluorocyclopropyl)((1 R,5 S)-3-(2-((1-methyl-1 H-pyrazol-4-yl)amino)pyrimidin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)methanone (PF-06700841).

Cytokine signaling is an important characteristic of autoimmune diseases. Many pro-inflammatory cytokines signal through the Janus kinase (JAK)/Signal transducer and activator of transcription (STAT) pathway. JAK1 is important for the γ-common chain cytokines, interleukin (IL)-6, and type-I interferon (IFN) family, while TYK2 in addition to type-I IFN signaling also plays a role in IL-23 and IL-12 signaling. Intervention with monoclonal antibodies (mAbs) or JAK1 inhibitors has demonstrated efficacy in Phase III psoriasis, psoriatic arthritis, inflammatory bowel disease, and rheumatoid arthritis studies, leading to multiple drug approvals. We hypothesized that a dual JAK1/TYK2 inhibitor will provide additional efficacy, while managing risk by optimizing selectivity against JAK2 driven hematopoietic changes. Our program began with a conformationally constrained piperazinyl-pyrimidine Type 1 ATP site inhibitor, subsequent work led to the discovery of PF-06700841 (compound 23), which is in Phase II clinical development (NCT02969018, NCT02958865, NCT03395184, and NCT02974868).

Visceral leishmaniasis: A novel nuclear envelope protein ‘nucleoporins-93 (NUP-93)’ from Leishmania donovani prompts macrophage signaling for T-cell activation towards host protective immune response

The shift of macrophage and T-cell repertoires towards proinflammatory cytokine signalling ensures the generation of host-protective machinery that is otherwise compromised in cases of the intracellular Leishmania parasite. Different groups have attempted to restore host protective immunity. These vaccine candidates showed good responses and protective effects in murine models, but they generally failed during human trials. In the present study, we evaluated the effect of 97 kDa recombinant nucleoporin-93 of Leishmania donovani (rLd-NUP93) on mononuclear cells in healthy and treated visceral leishmaniasis (VL) patients and on THP-1 cell lines. rLd-NUP93 stimulation increased the expression of the early lymphocyte activation marker CD69 on CD4+ and CD8+ T cells. The expression of the host protective pro-inflammatory cytokines IFN-γ, IL-12 and TNF-α was increased, with a corresponding down-regulation of IL-10 and TGF-β upon rLd-NUP93 stimulation. This immune polarization resulted in the up-regulation of NF-κB p50 with scant expression of SMAD-4. Augmenting lymphocyte proliferation upon priming with rLd-NUP93 ensured its potential for activation and generation of strong T-cell mediated immune responses. This stimulation extended the leishmanicidal activity of macrophages by releasing high amounts of reactive oxygen species (ROS). Further, the leishmanicidal activity of macrophages was intensified by the elevated production of nitric oxide (NO). The fact that this antigen was earlier reported in circulating immune complexes of VL patients highlights its antigenic importance. In addition, in silico analysis suggested the presence of MHC class I and II-restricted epitopes that proficiently trigger CD8+ and CD4+ T-cells, respectively. This study reported that rLd-NUP93 was an effective immunoprophylactic agent that can be explored in future vaccine design.

Immunomodulatory Action of Substituted 1,3,4-Thiadiazines on the Course of Myocardial Infarction.

This review focuses on the biological action of the compounds from the group of substituted 1,3,4-thiadiazines on stress response and myocardial infarction. The aim of this review is to propose the possible mechanisms of action of 1,3,4-thiadiazines and offer prospectives in the development of new derivatives as therapeutic agents. It is known, that compounds that have biological effects similar to those used as antidepressants can down-regulate the secretion of proinflammatory cytokines, up-regulate the release of anti-inflammatory ones and affect cell recruitment, which allows them to be considered immunomodulators as well. The results of pharmacological evaluation, in silico studies, and in vivo experiments of several compounds from the group of substituted 1,3,4-thiadiazines with antidepressant properties are presented. It is proposed that the cardioprotective effects of substituted 1,3,4-thiadiazines might be explained by the peculiarities of their multi-target action: the ability of the compounds to interact with various types of receptors and transporters of dopaminergic, serotonergic and acetylcholinergic systems and to block the kinase signal pathway PI3K-AKT. The described effects of substituted 1,3,4-thiadiazines suggest that it is necessary to search for a new agents for limiting the peripheral inflammatory/ischemic damage through the entral mechanisms of stress reaction and modifying pro-inflammatory cytokine signaling pathways in the brain.

Abstract B58: The role of epigenetic reader and alternative mRNA splicing variant MBD2_v2 in IL6 signaling in prostate cancer

Abstract Background: African American men (AAM) have a 60% higher risk of being diagnosed with prostate cancer and a 2 to 3 times greater risk of dying from the disease compared to European American men (EAM). We previously reported evidence of molecular underpinnings for these disparities from high-throughput gene expression analysis of prostate cancer (PCa) specimens. The data showed that proinflammatory cytokine signaling factors, including IL6 and TGFB1, are significantly over-represented in PCa specimens from AAM. The limitation of that study, however, is that it did not investigate the potential molecular diversity for high-grade PCa, and it did not consider adjacent noncancer tissue. Methods: We conducted a pilot RNA-sequencing study of high-grade PCa, GS greater or equal to 7(4+3), and matched non-cancer adjacent prostate tissue specimens from AAM and EAM. We also studied the molecular biology of PCa cell lines derived from AAM and EAM tumors. Results: According to our genomic analysis, IL6 was once again relevant to our research, this time upregulated in PCa specimens from EAM, but also higher in the stromal compartment in AAM. Also, we noted that whether or not a PCa cell line expresses IL6 appears to be linked to TP53 mutation status. Moreover, cell lines derived from AAM (MDA-PCa-2b and RC77T), which are TP53 wild-type, did not express IL6 and showed an increased stem cell-like phenotype in response to IL6 treatment that was dose dependent. PCa cell lines that are TP53 mutant and expressed IL6 did not respond to exogenous IL6 treatment. We uncovered that in responsive PCa cell lines, IL6 treatment induced mRNA and protein expression of the epigenetic reader methyl CpG binding domain protein 2 (MBD2), more specifically the alternative mRNA splicing variant MBD2_v2. Further investigation validated that this short isoform promotes self-renewal and expansion of PCa stem cell-like cells. Conclusion: The outcomes suggest that there may be a dual nature for IL6 signaling in PCa that remains to be understood, yet contributes to the molecular diversity of high-grade prostate cancer and race disparities. Citation Format: Emily Girsch, Bin Bao, Cristina Mitrea, Wael A. Sakr, Gregory Dyson, Isaac Powell, Aliccia Bollig-Fischer. The role of epigenetic reader and alternative mRNA splicing variant MBD2_v2 in IL6 signaling in prostate cancer [abstract]. In: Proceedings of the Tenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2017 Sep 25-28; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2018;27(7 Suppl):Abstract nr B58.

Abstract 4064: Spatial re-distribution of natural killer cells targets drug resistance and anti-tumor response, ex-vivo

Abstract Background: It is now clear that response or resistance to therapy is contingent on the tumor microenvironment, which is comprised of malignant cells, normal stroma, and immune landscape; attributes that are unique to each individual patient. This is particularly true for emerging anticancer drugs, such as immune checkpoint inhibitors, which recalibrate the body's own immune defense largely by modulating exhaustion of cytotoxic lymphocytes including T cells and natural killer (NK) cells. However, clinical response to therapy varies enormously. There is a critical gap in our understanding for the mechanisms that drive response or resistance to conventional drugs and immunotherapies at the individual patient level. Methods: Here, we used in-vitro and in-vivo experiments to study the role of NK cells in models of drug tolerance. Next, we employed (CANscriptTM), a clinically-validated ex-vivo tumor model that recreates and preserves the native, patient tumor microenvironment, which integrates an algorithm-driven method to predict clinical response to therapy (M-Score). Utilizing tissue from patients diagnosed with luminal, HER2 positive, and triple-negative (ER- PR- HER2-) breast cancers (N=10), we studied spatial heterogeneity of the tumor-immune contexture, M-Score, and phenotypic alterations under pressure of conventional standard-of-care regimens and immunotherapies including immune-checkpoint inhibitors. To do this, we used a comprehensive panel of immunological assays to evaluate changes in cytotoxic lymphocytes by flow cytometry, immunohistochemistry and cytokine profiling (i.e. CD56, MHC class 1A/B, NKG2D/C, CD8, CD3, PD-1, CTLA-4, TIM-3, LAG-3, 4-1BB, granzyme A/B). Results: We identified that drug resistance is associated to downregulation of activating proteins on NK cells including NKG2D/C, reducing their cytotoxic capability. Furthermore, tumor response, predicted by M-Score, correlates to a unique pattern of spatially-distributed NK cells, which associated a pro-inflammatory cytokine signature from the tumor microenvironment. Interestingly, we determined that therapy-induced expression of exhaustion biomarkers on NK cell exhaustion inversely correlated to the expression of cytotoxic granzyme B in the tumor microenvironment. Conclusions: Taken together, these data demonstrate an integral role that NK cells contribute to resistance and the antitumor response to therapy including both conventional and immuno-modulatory drugs. We further demonstrate that CANscriptTM can be harnessed to study response and resistance of the innate and adaptive immune system. Such an advance in our preclinical methods to study anticancer drugs at the individual patient level can help guide treatment decisions for clinicians while simultaneously functioning as a platform to study clinical efficacy of novel and emerging agents. Citation Format: Munisha Smalley, Basava U. Shanthappa, Hans Gertje, Mark Lawson, Manjusha Biswas, Saravanan Thiyagarajan, Biswanath Majumder, D. C. Doval, Anurag Mehta, Neyaz Alam, Nabendu Murmu, S. P. Somashekhar, Aaron Goldman. Spatial re-distribution of natural killer cells targets drug resistance and anti-tumor response, ex-vivo [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 4064.

SOCS-1 Suppresses Inflammation Through Inhibition of NALP3 Inflammasome Formation in Smoke Inhalation-Induced Acute Lung Injury.

Smoke inhalation leads to acute lung injury (ALI), a devastating clinical problem associated with high mortality rates. Suppressor of cytokine signaling-1 (SOCS-1) is a negative regulator of proinflammatory cytokine signaling. We have found that adenoviral gene transfer of SOCS-1 ameliorates smoke inhalation-induced lung injury in C57BL/6 mice. We also found that the release of adenosine triphosphate (ATP) was increased post smoke exposure, while oxidized ATP, an inhibitor of purinergic P2X7 receptor, suppressed smoke-induced NALP3 inflammasome assembly, caspase-1 activation, and K+ efflux. Similar to oxidized ATP, high protein level of SOCS-1 dampened the formation of NALP3 inflammasome and the activation of caspase-1 and IL-1β induced by smoke exposure in mouse alveolar macrophages. In conclusion, SOCS-1 relieves smoke inhalation-induced pulmonary inflammation and injury by inhibiting NALP3 inflammasome formation.

The long noncoding RNA Blnc1 orchestrates homeostatic adipose tissue remodeling to preserve metabolic health

ObjectiveLong noncoding RNAs (lncRNAs) are emerging as powerful regulators of adipocyte differentiation and gene expression. However, their significance in adipose tissue metabolism and physiology has not been demonstrated in vivo. We previously identified Blnc1 as a conserved lncRNA regulator of brown and beige adipocyte differentiation. In this study, we investigated the physiological role of Blnc1 in thermogenesis, adipose remodeling and systemic metabolism. MethodsWe generated fat-specific Blnc1 transgenic and conditional knockout mouse strains and investigated how adipocyte Blnc1 levels are causally linked to key aspects of metabolic health following diet-induced obesity. We performed studies using cultured adipocytes to establish cell-autonomous role of Blnc1 in regulating adipocyte gene programs. ResultsBlnc1 is highly induced in both brown and white fats from obese mice. Fat-specific inactivation of Blnc1 impairs cold-induced thermogenesis and browning and exacerbates obesity-associated brown fat whitening, adipose tissue inflammation and fibrosis, leading to more severe insulin resistance and hepatic steatosis. On the contrary, transgenic expression of Blnc1 in adipose tissue elicits the opposite and beneficial metabolic effects, supporting a critical role of Blnc1 in driving adipose adaptation and homeostatic remodeling during obesity. Mechanistically, Blnc1 cell-autonomously attenuates proinflammatory cytokine signaling and promotes fuel storage in adipocytes through its protein partner Zbtb7b. ConclusionsThis study illustrates a surprisingly pleiotropic and dominant role of lncRNA in driving adaptive adipose tissue remodeling and preserving metabolic health.