Autocrine Stimulation Research Articles

Chemoprevention of pancreatic cancer by inhibition of glutathione-S transferase P1.

Pancreatic cancer is among the most refractory malignancies with poor prognosis. Thus, preventive approaches, in addition to the development of novel therapeutic strategies are essential for this type of cancer. KRAS mutations occur very early in the development of pancreatic cancers and could be targeted for its prevention, yet specific inhibitors for mutated KRAS are lacking. Accordingly, Glutathione-S Transferase p1 (GSTP1), which we recently found to be an autocrine stimulator of mutated KRAS signaling, is predicted to be an alternative target for chemoprevention of pancreatic cancer. In this study, chemopreventive effects of O-Hexadecyl-γ-glutamyl-S-benzyl-cysteinyl-D-phenyl glycine-Ethylester (HGBPE), which we previously synthesized to inhibit GSTP1 activity, was analyzed for its effect on the prevention of a rat pancreatic carcinogenesis model induced by 7,12-dimethyl-benzanthracene (DMBA). Rats administered with DMBA were grouped into five cohorts. In the treated group I, which was treated neither with HGBPE nor vehicle, sequential appearance of precancerous lesions, ductal complexes, and adenocarcinoma was confirmed as previously reported. We also confirmed in this group that mutations of KRAS and expression of GSTP1 simultaneously occurred in the ductal complex. To rats of groups II and IV, HGBPE was administered, and vehicle to those of group III and V. In groups of II and IV, the incidence of both ductal complex and adenocarcinoma were significantly lower than those in groups III and V. These data clearly suggest the efficacy of HGBP as a potential chemopreventive agent for pancreatic cancer.

The C3-like molecule CD109 controls Th1 versus Th17 induction in CD4+ T cells

Abstract Recent workdefined an unexpected and critical role for intracellular/autocrine active complement in human Th1 biology. Specifically, autocrine stimulation of CD46via TCR-induced C3b generation is needed for IFN-γ production by human CD4+ T cells. Thus, CD46-deficient patients are unable to generate normal Th1 responses. Analysis of the CD46-driven gene signature in Th1 cells revealed CD109 as a direct target of CD46. Indeed, T cells from CD46 deficient patients and from healthy donors in which CD46 expression was ablated via CRISPR-Cas9 technology failed to upregulate CD109. CD109 is aGPI-linked surface protein, mostly expressed on non-immune cells, and belongs to the complement C3 protein family. Though CD109 has been reported as a major negative regulator of TGF-β receptor signaling, direct functional activity for of CD109 on T cells remains unexplored. Knock-out of CD109 in human CD4+ T cells with CRISPR-Cas9 technology induced uncontrolled Th1 and Th17 activation upon in vitro stimulation under non-skewing conditions. Similarly, CD4+T cells from Cd109−/− mice displayed augmented Th1 and Th17 in vitro responses and caused significantly more tissue pathology and weight loss in a T cell-transfer colitis model. Surprisingly, on T cells, CD109 does not restrain IFN-γ and/or IL-17 induction via modulation of TGF-β receptor activity. Instead, CD109 engages with a non-canonical costimulatory molecule and controls stemness- and metabolism-related signaling pathways. Together, these data suggest that the complement-related protein CD109 serves as an important and novel molecular switch on CD4+ T-cells, where it regulates the balance between Th1 and Th17-induction pathways.

PDGFB RNA in situ hybridization for the diagnosis of dermatofibrosarcoma protuberans.

Dermatofibrosarcoma protuberans (DFSP) is a spindle cell neoplasm of the skin and superficial soft tissue with a tendency for locally aggressive behavior; metastatic potential coincides with fibrosarcomatous transformation. The vast majority of DFSPs harbor the t(17;22) translocation resulting in a COL1A1-PDGFB fusion that drives autocrine growth stimulation via PDGFB overexpression. Here, we examined the utility of PDGFB RNA chromogenic in situ hybridization (CISH) for the diagnosis of DFSP. A total of 337 tumors represented in whole tissue sections and tissue microarrays, including 37 cases of DFSP and 300 histologically similar spindle cell tumors, were subjected to PDGFB RNA CISH using commercially available probes. PDGFB overexpression was observed by light microscopy in 24 of 26 conventional DFSPs (92%) and 11 of 11 fibrosarcomatous DFSPs (100%). One of two DFSPs negative for PDGFB by RNA CISH was found to harbor an uncommon alternative rearrangement involving PDGFD. All examined cases of histologic mimics were negative for PDGFB overexpression; limited PDGFB expression, not reaching an empirical threshold of greater than 5 puncta or one aggregate of chromogen in more than 25% of cells, was observed in 7 of 300 mimics (2.3%), including desmoplastic melanoma, malignant peripheral nerve sheath tumor, angiosarcoma, and pleomorphic dermal sarcoma. Vascular PDGFB expression was seen in several tumor types. We conclude that PDGFB RNA CISH, with careful interpretation and the use of appropriate thresholds, may serve as a surrogate marker of PDGFB rearrangement and a useful ancillary tool for the diagnosis of DFSP.

Succinate Mediates Tumorigenic Effects via Succinate Receptor 1: Potential for New Targeted Treatment Strategies in Succinate Dehydrogenase Deficient Paragangliomas.

Paragangliomas and pheochromocytomas (PPGLs) are chromaffin tumors associated with severe catecholamine-induced morbidities. Surgical removal is often curative. However, complete resection may not be an option for patients with succinate dehydrogenase subunit A-D (SDHx) mutations. SDHx mutations are associated with a high risk for multiple recurrent, and metastatic PPGLs. Treatment options in these cases are limited and prognosis is dismal once metastases are present. Identification of new therapeutic targets and candidate drugs is thus urgently needed. Previously, we showed elevated expression of succinate receptor 1 (SUCNR1) in SDHB PPGLs and SDHD head and neck paragangliomas. Its ligand succinate has been reported to accumulate due to SDHx mutations. We thus hypothesize that autocrine stimulation of SUCNR1 plays a role in the pathogenesis of SDHx mutation-derived PPGLs. We confirmed elevated SUCNR1 expression in SDHx PPGLs and after SDHB knockout in progenitor cells derived from a human pheochromocytoma (hPheo1). Succinate significantly increased viability of SUCNR1-transfected PC12 and ERK pathway signaling compared to control cells. Candidate SUCNR1 inhibitors successfully reversed proliferative effects of succinate. Our data reveal an unrecognized oncometabolic function of succinate in SDHx PPGLs, providing a growth advantage via SUCNR1.

Ligand based 3D-QSAR model, pharmacophore, molecular docking and ADME to identify potential fibroblast growth factor receptor 1 inhibitors

The FGF/FGFR system may affect tumor cells and stromal microenvironment through autocrine and paracrine stimulation, thereby significantly promoting oncogene transformation and tumor growth. Abnormal expression of FGFR1 in cells is considered to be the main cause of tumorigenesis and a potential target for the treatment of cancer. In this study, a combination of structure-based drug carriers and molecular docking-based virtual screening was used to screen new potential FGFR1 inhibitors. Forty eight known inhibitors were collected to establish 3 D-QSAR models and pharmacophore models, investigate the relationship between the activity and conformation of compounds, and verify the efficiency of pharmacophore. In Accelrys Discovery Studio 2016, the ZINC database was filtered by Lipinski's Rule of Five and SMART's filtration. Then, Hypo01 was used for virtual screening of ZINC database. Compounds with predicted activity values less than 1 μM were molecularly docked with FGFR1 protein crystals, the docking results were observed, and the interaction between compounds and targets was studied. The absorption, distribution, metabolism and excretion (ADME) and toxicity of potential inhibitors were studied, and a compound with new structural scaffolds were obtained. It could be further studied to explore their better therapeutic effects. Communicated by Ramaswamy H. Sarma

Autocrine TGF-β in Cancer: Review of the Literature and Caveats in Experimental Analysis.

Autocrine signaling is defined as the production and secretion of an extracellular mediator by a cell followed by the binding of that mediator to receptors on the same cell to initiate signaling. Autocrine stimulation often operates in autocrine loops, a type of interaction, in which a cell produces a mediator, for which it has receptors, that upon activation promotes expression of the same mediator, allowing the cell to repeatedly autostimulate itself (positive feedback) or balance its expression via regulation of a second factor that provides negative feedback. Autocrine signaling loops with positive or negative feedback are an important feature in cancer, where they enable context-dependent cell signaling in the regulation of growth, survival, and cell motility. A growth factor that is intimately involved in tumor development and progression and often produced by the cancer cells in an autocrine manner is transforming growth factor-β (TGF-β). This review surveys the many observations of autocrine TGF-β signaling in tumor biology, including data from cell culture and animal models as well as from patients. We also provide the reader with a critical discussion on the various experimental approaches employed to identify and prove the involvement of autocrine TGF-β in a given cellular response.

NRG1 fusions in breast cancer

BackgroundNRG1 gene fusions may be clinically actionable, since cancers carrying the fusion transcripts can be sensitive to tyrosine kinase inhibitors. The NRG1 gene encodes ligands for the HER2(ERBB2)-ERBB3 heterodimeric receptor tyrosine kinase, and the gene fusions are thought to lead to autocrine stimulation of the receptor. The NRG1 fusion expressed in the breast cancer cell line MDA-MB-175 serves as a model example of such fusions, showing the proposed autocrine loop and exceptional drug sensitivity. However, its structure has not been properly characterised, its oncogenic activity has not been fully explained, and there is limited data on such fusions in breast cancer.MethodsWe analysed genomic rearrangements and transcripts of NRG1 in MDA-MB-175 and a panel of 571 breast cancers.ResultsWe found that the MDA-MB-175 fusion—originally reported as a DOC4(TENM4)-NRG1 fusion, lacking the cytoplasmic tail of NRG1—is in reality a double fusion, PPP6R3-TENM4-NRG1, producing multiple transcripts, some of which include the cytoplasmic tail. We hypothesise that many NRG1 fusions may be oncogenic not for lacking the cytoplasmic domain but because they do not encode NRG1’s nuclear-localised form. The fusion in MDA-MB-175 is the result of a very complex genomic rearrangement, which we partially characterised, that creates additional expressed gene fusions, RSF1-TENM4, TPCN2-RSF1, and MRPL48-GAB2.We searched for NRG1 rearrangements in 571 breast cancers subjected to genome sequencing and transcriptome sequencing and found four cases (0.7%) with fusions, WRN-NRG1, FAM91A1-NRG1, ARHGEF39-NRG1, and ZNF704-NRG1, all splicing into NRG1 at the same exon as in MDA-MB-175. However, the WRN-NRG1 and ARHGEF39-NRG1 fusions were out of frame. We identified rearrangements of NRG1 in many more (8% of) cases that seemed more likely to inactivate than to create activating fusions, or whose outcome could not be predicted because they were complex, or both. This is not surprising because NRG1 can be pro-apoptotic and is inactivated in some breast cancers.ConclusionsOur results highlight the complexity of rearrangements of NRG1 in breast cancers and confirm that some do not activate but inactivate. Careful interpretation of NRG1 rearrangements will therefore be necessary for appropriate patient management.

Activation of Hepatic Stellate Cells Requires Dissociation of E-Cadherin–Containing Adherens Junctions with Hepatocytes

Hepatic stellate cells (HSCs) are resident mesenchymal cells in the space of Disse interposed between liver sinusoidal endothelial cells and hepatocytes. Thorn-like microprojections, or spines, project out from the cell surface of HSCs, crossing the space of Disse, to establish adherens junctions with neighboring hepatocytes. Although HSC activation is initiated largely from stimulation by adjacent cells, isolated HSCs also activate spontaneously in primary culture on plastic. Therefore, other unknown HSC-initiating factors apart from paracrine stimuli may promote activation. The dissociation of adherens junctions between HSCs and hepatocytes as an activating signal for HSCs was explored, establishing epithelial cadherin (E-cadherin) as an adhesion molecule linking hepatocytes and HSCs. Invivo, following carbon tetrachloride-induced liver injury, HSCs lost their spines and dissociated from adherens junctions in the early stages of injury, and were subsequently activated along with an increase in YAP/TAZ expression. After abrogation of liver injury, HSCs reconstructed their spines and adherens junctions. Invitro, reconstitution of E-cadherin-containing adherens junctions by forced E-cadherin expression quiesced HSCs and suppressed TAZ expression. Additionally, increase of TAZ expression leading to the activation of HSCs by autocrine stimulation of transforming growth factor-β, was revealed as a mechanism of spontaneous activation. Thus, we have uncovered a critical event required for HSC activation through enhanced TAZ-mediated mechanotransduction after the loss of adherens junctions between HSCs and hepatocytes.

Evidence for a protective role of placental growth factor in cardiovascular disease.

Placental growth factor (PlGF) is a mitogen for endothelial cells, but it can also act as a proinflammatory cytokine. Because it promotes early stages of plaque formation in experimental models of atherosclerosis and was implicated in epidemiological associations with risk of cardiovascular disease (CVD), PlGF has been attributed a pro-atherogenic role. Here, we investigated whether PlGF has a protective role in CVD and whether elevated PlGF reflects activation of repair processes in response to vascular stress. In a population cohort of 4742 individuals with 20 years of follow-up, high baseline plasma PlGF was associated with increased risk of cardiovascular death, myocardial infarction, and stroke, but these associations were lost or weakened when adjusting for cardiovascular risk factors known to cause vascular stress. Exposure of cultured endothelial cells to high glucose, oxidized low-density lipoprotein (LDL) or an inducer of apoptosis enhanced the release of PlGF. Smooth muscle cells and endothelial cells treated with PlGF small interference RNA demonstrated that autocrine PlGF stimulation plays an important role in vascular repair responses. High expression of PlGF in human carotid plaques removed at surgery was associated with a more stable plaque phenotype and a lower risk of future cardiovascular events. When adjusting associations of PlGF with cardiovascular risk in the population cohort for plasma soluble tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor-2, a biomarker of cellular stress, a high PlGF/TRAIL receptor-2 ratio was associated with a lower risk. Our findings provide evidence for a protective role of PlGF in CVD.

BIOM-43. GLUTAMATE RECEPTOR AND GLUTAMINE METABOLISM PROFILING BY GENE EXPRESSION ANALYSIS AMONG PATIENTS WITH HIGH GRADE GLIOMA (HGG)

Abstract BACKGROUND Glutaminolysis and excessive glutamate production are among the metabolic hallmarks of HGG. In addition to a probable role in causing seizures, autocrine stimulation of metabotropic and ionotropic (NMDA, AMPA, and kainate) receptors are capable of activating intracellular signaling pathways that are tumorigenic and contribute to drug and radiation resistance. While hints of a survival benefit from glutamate receptor targeting have occasionally emerged from the clinic, no single strategy has consistently demonstrated efficacy. Hence, we sought to characterize the magnitude of glutamate receptor overexpression and its diversity across a population of HGG patients. METHODS A set of 41 genes related to glutamate metabolism was selected from the literature. RNA gene counts for TCGA glioblastoma multiforme (N=163) and Grade 3 glioma samples (Astrocytoma=82, Oligodendroglioma=47, Oligoastrocytoma=39) were downloaded from https://portal.gdc.cancer.gov/. Annotation on subtypes and PFS values were obtained from PMID: 24120142 and 26061751. Gene expression normalization as FPKM and hierarchical clustering were performed using R-3.6.0 genes. RESULTS A heatmap with hierarchical clustering for glutamate and glutamine-related genes for the TCGA GBM and grade III glioma samples cohort was generated including colored annotation for the subtype and progression free survival. The graph shows a rough separation into two groups, with grade III gliomas and proneural samples tentatively clustering together and showing higher expression for most of the glutamate related genes. The magnitude of glutaminolysis-related gene over-expression varied significantly across the cohort suggesting a wide variety in the extent of glutamine addiction. CONCLUSION Specific glutamate receptors are commonly upregulated in HGG patients, however the heterogeneity underlying this phenomenon suggests that uniform drug strategies are unlikely to succeed in a diverse population. However, gene expression analysis has utility to identify specific glutamate receptor and glutamine profiles and to inform the treatment strategies for discrete subsets of patients using drugs that are already in the armamentarium.

Osteopontin Links Myeloid Activation and Disease Progression in Systemic Sclerosis

SummaryProgressive lung fibrosis is a major cause of mortality in systemic sclerosis (SSc) patients, but the underlying mechanisms remain unclear. We demonstrate that immune complexes (ICs) activate human monocytes to promote lung fibroblast migration partly via osteopontin (OPN) secretion, which is amplified by autocrine monocyte colony stimulating factor (MCSF) and interleukin-6 (IL-6) activity. Bulk and single-cell RNA sequencing demonstrate that elevated OPN expression in SSc lung tissue is enriched in macrophages, partially overlapping with CCL18 expression. Serum OPN is elevated in SSc patients with interstitial lung disease (ILD) and prognosticates future lung function deterioration in SSc cohorts. Serum OPN levels decrease following tocilizumab (monoclonal anti-IL-6 receptor) treatment, confirming the connection between IL-6 and OPN in SSc patients. Collectively, these data suggest a plausible link between autoantibodies and lung fibrosis progression, where circulating OPN serves as a systemic proxy for IC-driven profibrotic macrophage activity, highlighting its potential as a promising biomarker in SSc ILD.

Cannabinoid receptor subtype influence on neuritogenesis in human SH-SY5Y cells

Human SH-SY5Y neuroblastoma cells stably expressing exogenous CB1 (CB1XS) or CB2 (CB2XS) receptors were developed to investigate endocannabinoid signaling in the extension of neuronal projections. Expression of cannabinoid receptors did not alter proliferation rate, viability, or apoptosis relative to parental SH-SY5Y. Transcripts for endogenous cannabinoid system enzymes (diacylglycerol lipase, monoacylglycerol lipase, α/β-hydrolase domain containing proteins 6 and 12, N-acyl phosphatidylethanolamine-phospholipase D, and fatty acid amide hydrolase) were not altered by CB1 or CB2 expression. Endocannabinoid ligands 2-arachidonoylglycerol (2-AG) and anandamide were quantitated in SH-SY5Y cells, and diacylglycerol lipase inhibitor tetrahydrolipstatin decreased 2-AG abundance by 90% but did not alter anandamide abundance. M3 muscarinic agonist oxotremorine M, and inhibitors of monoacylglycerol lipase and α/β hydrolase domain containing proteins 6 &12 increased 2-AG abundance. CB1 receptor expression increased lengths of short (<30 μm) and long (>30 μm) projections, and this effect was significantly reduced by tetrahydrolipstatin, indicative of stimulation by endogenously produced 2-AG. Pertussis toxin, Gβγ inhibitor gallein, and β-arrestin inhibitor barbadin did not significantly alter long projection length in CB1XS, but significantly reduced short projections, with gallein having the greatest inhibition. The rho kinase inhibitor Y27632 increased CB1 receptor-mediated long projection extension, indicative of actin cytoskeleton involvement. CB1 receptor expression increased GAP43 and ST8SIA2 mRNA and decreased ITGA1 mRNA, whereas CB2 receptor expression increased NCAM and SYT mRNA. We propose that basal endogenous production of 2-AG provides autocrine stimulation of CB1 receptor signaling through Gi/o, Gβγ, and β-arrestin mechanisms to promote neuritogenesis, and rho kinase influences process extension.

Pentraxin 3 inhibits fibroblast growth factor 2 induced osteoclastogenesis in rheumatoid arthritis

BackgroundSynovial fibroblasts (SFs) act as key effector cells mediating synovial inflammation and joint destruction in rheumatoid arthritis (RA). Fibroblast growth factor 2 (FGF2) and its receptors (FGFRs) play important roles in RASF-mediated osteoclastogenesis. Pentraxin 3 (PTX3) is a soluble pattern recognition receptor with nonredundant roles in inflammation and innate immunity. PTX3 is produced by various cell types, including SFs and is highly expressed in RA. However, the role of PTX3 in FGF2-induced osteoclastogenesis in RA and the underlying mechanism have been poorly elucidated. MethodsWe first determined the expression of FGF2 and RANKL in synovial tissue and synovial fluid of RA patients. We then examined the effect of PTX3 on RASF osteoclastogenesis induced by endogenous and exogenous FGF2 in isolated RASF cells treated with FGF2 and/or recombinant PTX3 (rPTX3). Thirdly, we analyzed the effect of PTX3 on FGF2 binding to FGFR-1 and HSPG receptors on RASFs. Lastly, we evaluated joint morphology after injection of rPTX3 into collagen-induced arthritis (CIA) mice. ResultsFGF2 was confirmed to be highly expressed in both synovial tissue and synovial fluid of RA patients. FGF2 promoted cell proliferation and increased the expressions of RANKL and ICAM-1 and RANKL/OPG to induce osteoclastogenesis in RASF, while anti-FGF2 neutralized this effect. PTX3 significantly inhibited FGF2-induced RASF cell growth and osteoclastogenesis by preventing the interaction of 125I-FGF2 and FGFRs on the same cells. In addition, administration of rPTX3 significantly ameliorated cartilage and bone destruction in mice with CIA. ConclusionsPTX3 exhibited an inhibitory effect on the autocrine and paracrine stimulation of FGF2 on SFs, and ameliorated bone destruction in CIA mice. PTX3 may be implicated in bone destruction in RA, which may provide theoretical evidence and potential therapeutic targets for RA treatment.

Endothelin-1 in portal hypertension: The intricate role of hepatic stellate cells.

Portal hypertension is pathologically defined as increase of portal venous pressure, mainly due to chronic liver diseases such as fibrosis and cirrhosis. In fibrotic liver, activated hepatic stellate cells increase their contraction in response to endothelin-1 (ET-1) via autocrine and paracrine stimulation from liver sinusoidal endothelial cells and injured hepatocytes. Clinical studies are limited with ET receptor antagonists in cirrhotic patients with portal hypertension. Hence, studies are needed to find molecules that block ET-1 synthesis. Accumulation of extracellular matrix proteins in the perisinusoidal space, tissue contraction, and alteration in blood flow are prominent during portal hypertension. Therefore, novel matrix modulators should be tested experimentally as well as in clinical studies. Specifically, tumor necrosis factor-α, transforming growth factor-β1, Wnt, Notch, rho-associated protein kinase 1 signaling antagonists, and peroxisome proliferator-activated receptor α and γ, interferon-γ and sirtuin 1 agonists should be tested elaborately against cirrhosis patients with portal hypertension.

Abstract 562: Novel combination therapy for treating TNBC using LIFR and HDAC Inhibitors

Abstract Background: Triple-negative breast cancer (TNBC) lacks targeted therapies and represents a disproportional share of the breast cancer (BC) mortality rate. TNBC exhibits autocrine stimulation of the LIF/LIFR axis and overexpression of LIF is associated with poorer relapse-free survival in BC patients. Histone deacetylase inhibitors (HDACIs) are emerging as promising multifunctional agents in TNBC to elicit cytotoxic actions. Recent studies have shown that cancer cells elicit feedback activation of leukemia inhibitory factor receptor (LIFR) which in turn curtails response to HDACIs. We developed a first-in-class inhibitor of LIFR, EC359 that directly interacts with LIFR and effectively blocks LIFR downstream signaling. The objective of this study is to examine the therapeutic efficacy of combination therapy using preclinical and patient-derived xenograft (PDX) models. Methods: We tested utility of combination therapy using multiple HDACIs that are currently in clinical trails along with EC359. The effect of combination therapy was evaluated using MTT, invasion, colony formation, and Caspase3/7 assays. Mechanistic studies were performed using Western blotting, qRT-PCR, and STAT3 reporter assays. The efficacy of combination therapy in vivo was examined using xenograft, PDX, and patient-derived explant (PDEx) models. Results: Immunohistochemical analyses of breast tumors using tissue microarrays revealed significant expression of LIFR in TNBC tissues. Treatment of TNBC model cells with four different HDACIs increased the expression of LIFR. LIFR inhibitor EC359 at nM concentration is additive to HDACIs in reducing cell viability. Knockdown of LIFR or treatment with EC359 significantly enhanced the efficacy of HDACIs in reducing the cell viability, colony formation ability, and invasiveness as well as promoted apoptosis compared to monotherapy in TNBC model cells. On the contrary, treatment with STAT3 inhibitor requires µM concentrations to reduce the cell viability of TNBC cells and is not additive to HDACIs. Mechanistic studies utilizing STAT3 reporter gene assays and biochemical studies using multiple TNBC model cells exhibited activation of the LIFR signaling pathway upon HDACIs treatment but was attenuated by EC359 therapy. Treatment of human TNBC utilizing PDEx assays showed that EC359 enhanced the ability of HDACIs to decrease proliferation (Ki-67 positivity) compared to monotherapy. Using TNBC xenografts and PDX models, we demonstrated that EC359 treatment enhanced the ability of HDACIs to reduce in vivo tumor growth compared to monotherapy. Conclusions: Our results suggest that the combination therapy of HDACIs and EC359 provides therapeutic utility in overcoming the limitation of feedback activation of LIFR observed in the treatment of HDACIs in treating TNBC. Supported by DOD BCRP grant W81XWH-18-1-0016 (R.K. Vadlamudi; K.J. Nickisch) Citation Format: Suryavathi Viswanadhapalli, Mengxing Li, Bindu Santhamma, Uday P. Pratap, Yiliao Luo, Junhao Liu, Kristin A. Altwegg, Xiaonan Li, Ahmed Gulzar, Hui Yan, Zhenming Xu, Andrew Brenner, Gangadhara R. Sareddy, Manjeet K. Rao, Rajeshwar R. Tekmal, Hareesh B. Nair, Klaus J. Nickisch, Ratna K. Vadlamudi. Novel combination therapy for treating TNBC using LIFR and HDAC Inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 562.

VEGF is an autocrine/paracrine neuroprotective factor for injured retinal ganglion neurons

Vascular endothelial growth factor-A (VEGF) is the angiogenic factor promoting the pathological neovascularization in age-related macular degeneration (AMD) or diabetic macular edema (DME). Evidences have suggested a neurotrophic and neuroprotective role of VEGF, albeit in retina, cellular mechanisms underlying the VEGF neuroprotection remain elusive. Using purified adult retinal ganglion cells (RGCs) in culture, we demonstrated here that VEGF is released by RGCs themselves to promote their own survival, while VEGF neutralization by specific antibodies or traps drastically reduced the RGC survival. These results indicate an autocrine VEGF neuroprotection on RGCs. In parallel, VEGF produced by mixed retinal cells or by mesenchymal stem cells exerted a paracrine neuroprotection on RGCs. Such neuroprotective effect was obtained using the recombinant VEGF-B, suggesting the involvement of VEGF-R1 pathway in VEGF-elicited RGC survival. Finally, glaucomatous patients injected with VEGF traps (ranibizumab or aflibercept) due to either AMD or DME comorbidity, showed a significant reduction of RGC axon fiber layer thickness, consistent with the plausible reduction of the VEGF autocrine stimulation of RGCs. Our results provide evidence of the autocrine neuroprotective function of VEGF on RGCs is crucially involved to preserve injured RGCs such as in glaucomatous patients.

4308 DEEP-PRIMED IL-15 SUPERAGONIST IMPROVES ANTIVIRAL EFFICACY OF HIV-SPECIFIC CD8+ T-CELLS IN HUMANIZED MOUSE MODEL

OBJECTIVES/GOALS: HIV-specific CD8+ T-cells play a critical role in partially controlling viral replication in infected-individuals, but ultimately fail to eliminate infection. Enhancing these T-cell responses through lymphocyte engineering approaches has the potential as a novel therapy capable of achieving durable control or eradication of infection. METHODS/STUDY POPULATION: IL-15 Superagonist (IL-15SA) potently supports the in vivo persistence and antiviral activity of adoptively transferred CD8+ T-cells. The Deep-PrimingTM technology platform, developed by Torque, allows for loading of immunomodulators onto the surface of T-cells via electrostatic ‘nanogels’, which slowly release to deliver sustained autocrine immune stimulation without the harmful effects of systemic exposure. Here, we investigate the impact of IL-15SA Deep-Priming on HIV-specific CD8+ T-cells in a humanized mouse model of HIV infection. Humanized mice were generated by engrafting NOD-scid-IL2Rgnull mice with memory CD4+ T-cells isolated from an ARV-suppressed HIV+ donor. An autologous HIV-specific Cytotoxic T-Lymphocyte (CTL) clone was isolated, and killing potential confirmed. Four weeks post humanization, mice were infected with HIV and received an infusion of unmodified HIV-Specific CTLs, or IL-15SA Deep-Primed HIV-specific CTLs (CTL-DP). T-cell numbers and plasma viral loads were quantified weekly by flow cytometry and qRT-PCR. RESULTS/ANTICIPATED RESULTS: Mice receiving unmodified CTLs trended toward reduced viral loads compared to the No Treatment condition, while mice receiving CTL-DP saw significant, 2-Log10 reductions in VL (p &lt; 0.01). At 41 days post-infection 100% (5/5) of the No Treatment, 66.7% (4/6) of the CTL treatment, and 16.7% (1/6) of CTL-DP treatment mice had detectable viremia. IL-15SA Deep-Priming increased CTL expansion and persistence in peripheral blood which correlated with improved CD4+T-cell preservation. DISCUSSION/SIGNIFICANCE OF IMPACT: Here we demonstrate the first in vivo analysis of IL-15SA Deep-Priming of HIV-Specific CTLs. These data suggest that Deep-Priming of patient T-cells can enhance in vivo function and persistence, leading to improved viral suppression; a significant advancement in the field of HIV cure research. CONFLICT OF INTEREST DESCRIPTION: Austin Boesch, Thomas Andresen, and Douglas Jones are employees of Torque. Darrell Irvine is a co-founder of Torque and Chairman of Torque’s Scientific Advisory Board.

Regulatory mechanisms for natriuretic peptide signalling in sheep granulosa cells.

Natriuretic peptides (NPs) have been reported to have critical roles in follicular development and oocyte maturation in rodents. This study aimed to extend our current understanding of NP-mediated signalling pathways and mechanisms of action in the follicles of a monovulatory species. Ovine granulosa cells (GCs) and theca cells (TCs) were cultured under conditions designed to allow gonadotrophin-stimulated cell differentiation. Gene expression analysis was performed by qualitative (q)PCR for NPs and NPRs (between 16 and 96 h of culture) and VEGF120 and VEGF164 (between 16 and 144 h of culture). A qualitative analysis of the production of NP/NPR family members and NP ligand/receptor associations was carried out utilising a highly sensitive immunological approach known as 'proximity ligation assay' (PLA). All NPRs were observed in GCs, while NPRA was absent in TCs. In GCs, gene expression of NPRA, NPRB and NPRC was apparent but only active BNP and CNP and not ANP, were detected. Also in GCs, ANP but not CNP was able to significantly (P < 0.05) reduce oestradiol and increase (P < 0.05) progesterone. Inhibition of VEGF164 by ANP and CNP (P < 0.01) after 48 h of culture preceded up-regulation of VEGF120 by ANP (P < 0.01) after 144 h, but not CNP. Taken together, these findings appear to demonstrate that NP responsiveness in the GC compartment of sheep follicles is multi-facilitated, utilising both autocrine and paracrine stimulation pathways.

The C3-like molecule CD109 controls Th1 versus Th17 induction in CD4+ T cells

Abstract Recent work defined an unexpected and critical role for intracellular/autocrine active complement in human Th1 biology. Specifically, autocrine stimulation of CD46 via TCR-induced C3b generation is needed for IFN-γ production by human CD4+T cells. Analysis of the CD46-driven gene signature in Th1 cells revealed CD109, a C3-like surface protein known to regulate TGF-β signaling, as a direct target of CD46. We confirmed that T cells from patients with CD46 deficiency, which suffer from recurrent infection due to reduced Th1 responses, and from healthy donors knocked-out for CD46 via CRISPR-Cas9 technology failed to upregulate CD109. CD109 is expressed on keratinocytes, endothelial and stem cells and overexpression is connected with cancer but its function on T cells remains unexplored. In vitro activation of mouse Cd109−/− CD4+ T-cells showed a strong increase of Th1- and Th17-related cytokines compare to their WT counterpart. In line with this observation, Cd109−/− mouse CD4+ T cells showed an increase in IL-17A+ and IFN-γ+/IL-17A+ T cell numbers in vivo and caused significantly more weight loss and disease pathology in a T cell-transfer colitis model. Although CD109 is known to regulate TGF-β signaling, our RNA-sequencing analysis of activated Cd109−/− mouse T cells revealed that this molecule is restraining Th17 induction rather via impact on cell cycle and apoptosis related pathways, which we are investigating now. Together, these data suggest that the complement-related protein CD109 serves as an important and novel molecular switch on CD4+ T-cells, where it regulates the balance between Th1 and Th17-induction pathways.

Serum IP-10 and IL-17 from Kawasaki disease patients induce calcification-related genes and proteins in human coronary artery smooth muscle cells in vitro

BackgroundKawasaki disease (KD) is one of the major causes of heart disease and vasculitis in children under 5 years old in the world. Clinical evidence has shown that coronary artery calcification may develop in KD patients, however the mechanism has not been elucidated. Previous studies have found that interferon-γ-inducible protein (IP)-10 and interleukin (IL)-17 can be elevated and may play a role in KD development and coronary artery lesion formation. The purpose of this in vitro study was to investigate the possible role of plasma circulating IP-10 and IL-17 of KD patients in vascular calcification development and its underlying mechanism.ResultHuman coronary artery smooth muscle cells (HCASMCs) were used in this study. We found that HCASMCs treated with IP-10/IL-17-containing KD serum and co-treated with IP-10/IL-17 recombinant proteins could induce a phenotype that may promote vascular calcification by the bone morphogenetic protein (BMP) 6 autocrine effect. Moreover, the BMP6 autocrine stimulation in IP-10/IL-17 co-treated HCASMCs could upregulate the smad1/5-runx2 signaling activation, thus increasing the expression of bone matrix-related proteins, i.e., osteopontin, osteocalcin, and alkaline phosphatase.ConclusionsThe presented in vitro results provided new insights into the comprehension of the pathogenesis of vascular calcification in SMCs in KD progression. Although additional in vivo experimental models should be completed to confirm the in vivo relevance of these in vitro findings, the results related to the autocrine role of BMP6 may provide a new direction for theranostic drug development to treat KD.