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Nuclear envelope-localized torsinA-LAP1 complex regulates hepatic VLDL secretion and steatosis

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Abstract

Deciphering novel pathways regulating liver lipid content has profound implications for understanding the pathophysiology of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis. Recent evidence suggests that the nuclear envelope is a site of regulation of lipid metabolism but there is limited appreciation of the responsible mechanisms and molecular components within this organelle. We showed that conditional hepatocyte deletion of the inner nuclear membrane protein lamina-associated polypeptide 1 (LAP1) caused defective VLDL secretion and steatosis, including intranuclear lipid accumulation. LAP1 binds to and activates torsinA, an AAA+ ATPase that resides in the perinuclear space and continuous main ER. Deletion of torsinA from mouse hepatocytes caused even greater reductions in VLDL secretion and profound steatosis. Both of these mutant mouse lines developed hepatic steatosis and subsequent steatohepatitis on a regular chow diet in the absence of whole-body insulin resistance or obesity. Our results establish an essential role for the nuclear envelope-localized torsinA-LAP1 complex in hepatic VLDL secretion and suggest that the torsinA pathway participates in the pathophysiology of nonalcoholic fatty liver disease.

Authors

Ji-Yeon Shin, Antonio Hernandez-Ono, Tatyana Fedotova, Cecilia Östlund, Michael J. Lee, Sarah B. Gibeley, Chun-Chi Liang, William T. Dauer, Henry N. Ginsberg, Howard J. Worman

AAV8-vectored suprachoroidal gene transfer produces widespread ocular transgene expression

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Abstract

There has been great progress in ocular gene therapy, but delivery of viral vectors to the retinal pigmented epithelium (RPE) and retina can be challenging. Subretinal injection, the preferred route of delivery for most applications, requires a surgical procedure that has risks. Herein we report a novel gene therapy delivery approach, suprachoroidal injection of AAV8 vectors, which is less invasive and could be done in an outpatient setting. Two weeks after suprachoroidal injection of AAV8.GFP in rats, GFP fluorescence covered 18.9% of RPE flat mounts and extended entirely around sagittal and transverse sections in RPE and photoreceptors. After 2 suprachoroidal injections of AAV8.GFP, GFP fluorescence covered 30.5% of RPE flat mounts. Similarly, widespread expression of GFP occurred in nonhuman primate and pig eyes after suprachoroidal injection of AAV8.GFP. Compared with subretinal injection in rats of RGX-314, an AAV8 vector expressing an anti-VEGF Fab, suprachoroidal injection of the same dose of RGX-314 resulted in similar expression of anti-VEGF Fab and similar suppression of VEGF-induced vascular leakage. Suprachoroidal AAV8 vector injection provides a noninvasive outpatient procedure to obtain widespread transgene expression in retina and RPE.

Authors

Kun Ding, Jikui Shen, Zibran Hafiz, Sean F. Hackett, Raquel Lima e Silva, Mahmood Khan, Valeria E. Lorenc, Daiqin Chen, Rishi Chadha, Minie Zhang, Sherri Van Everen, Nicholas Buss, Michele Fiscella, Olivier Danos, Peter A. Campochiaro

cGAS/STING axis mediates a topoisomerase II inhibitor-induced tumor immunogenicity

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Abstract

Checkpoint blockade antibodies have been approved as immunotherapy for multiple types of cancer, but the response rate and efficacy are still limited. There are few immunogenic cell death (ICD)-inducing drugs available that can kill cancer cells, enhance tumor immunogenicity, increase the in vivo immune infiltration, and thereby boosting a tumor response to immunotherapy. So far, the ICD markers have been identified as the few immuno-stimulating characteristics of dead cells, but whether the presence of such ICD markers on tumor cells translates into enhanced antitumor immunity in vivo is still investigational. To identify anticancer drugs that could induce tumor cell death and boost T cell response, we performed drug screenings based on both an ICD reporter assay and T cell activation assay. We identified that teniposide, a DNA topoisomerase II inhibitor, could induce high mobility group box 1 (HMGB1) release and type I interferon signaling in tumor cells, and teniposide-treated tumor cells could activate antitumor T cell response both in vitro and in vivo. Mechanistically, teniposide induced tumor cell DNA damage and innate immune signaling including NF-κB activation and STING-dependent type I interferon signaling, both of which contribute to the activation of dendritic cells and subsequent T cells. Furthermore, teniposide potentiated the antitumor efficacy of anti-PD1 on multiple types of mouse tumor models. Our findings showed that teniposide could trigger tumor immunogenicity, and enabled a potential chemo-immunotherapeutic approach to potentiate the therapeutic efficacy of anti-PD1 immunotherapy.

Authors

Zining Wang, Jiemin Chen, Jie Hu, Hongxia Zhang, Feifei Xu, Wenzhuo He, Xiaojuan Wang, Mengyun Li, Wenhua Lu, Gucheng Zeng, Penghui Zhou, Peng Huang, Siyu Chen, Wende Li, Liang-ping Xia, Xiaojun Xia

Myocardial infarction triggers cardioprotective antigen-specific T helper cell responses

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Abstract

T cell autoreactivity is a hallmark of autoimmune diseases but can also benefit self-maintenance and foster tissue repair. Herein, we investigated whether heart-specific T cells exert salutary or detrimental effects in the context of myocardial infarction (MI), the leading cause of death worldwide. After screening more than 150 class-II-restricted epitopes, we found that myosin heavy chain alpha (MYHCA) was a dominant cardiac antigen triggering post-MI CD4+ T cell activation in mice. Transferred MYHCA614-629-specific CD4+ T (TCR-M) cells selectively accumulated in the myocardium and mediastinal lymph nodes (med-LN) of infarcted mice, acquired a Treg phenotype with a distinct pro-healing gene expression profile, and mediated cardioprotection. Myocardial Treg cells were also detected in autopsies from patients who suffered a MI. Noninvasive PET/CT imaging using a CXCR4 radioligand revealed enlarged med-LNs with increased cellularity in MI-patients. Notably, the med-LN alterations observed in MI patients correlated with the infarct size and cardiac function. Taken together, the results obtained in our study provide evidence showing that MI-context induces pro-healing T cell autoimmunity in mice and confirms the existence of an analogous heart/med-LN/T cell axis in MI patients.

Authors

Max Rieckmann, Murilo Delgobo, Chiara Gaal, Lotte Büchner, Philipp Steinau, Dan Reshef, Cristina Gil-Cruz, Ellis N. ter Horst, Malte Kircher, Theresa Reiter, Katrin G. Heinze, Hans W.M. Niessen, Paul A.J. Krijnen, Anja M. van der Laan, Jan J. Piek, Charlotte Koch, Hans-Jürgen Wester, Constantin Lapa, Wolfgang R. Bauer, Burkhard Ludewig, Nir Friedman, Stefan Frantz, Ulrich Hofmann, Gustavo Campos Ramos

Plasma deconvolution identifies broadly neutralizing antibodies associated with hepatitis C virus clearance

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Abstract

A vaccine for hepatitis C virus (HCV) is urgently needed. Development of broadly-neutralizing plasma antibodies during acute infection is associated with HCV clearance, but the viral epitopes of these plasma antibodies are unknown. Identification of these epitopes could define the specificity and function of neutralizing antibodies (NAbs) that should be induced by a vaccine. Here, we present development and application of a high-throughput method that deconvolutes polyclonal anti-HCV NAbs in plasma, delineating the epitope specificities of anti-HCV NAbs in acute infection plasma of forty-four humans with subsequent clearance or persistence of HCV. Remarkably, we identified multiple broadly neutralizing antibody (bNAb) combinations that were associated with greater plasma neutralizing breadth and with HCV clearance. These studies have potential to inform new strategies for vaccine development by identifying bNAb combinations in plasma associated with natural clearance of HCV, while also providing a high-throughput assay that could identify these responses after vaccination trials.

Authors

Valerie J. Kinchen, Guido Massaccesi, Andrew I. Flyak, Madeleine C. Mankowski, Michelle D. Colbert, William O. Osburn, Stuart C. Ray, Andrea L. Cox, James E. Crowe Jr., Justin R. Bailey

Degradation of splicing factor SRSF3 contributes to progressive liver disease

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Abstract

Serine rich splicing factor 3 (SRSF3) plays a critical role in liver function and its loss promotes chronic liver damage and regeneration. As a consequence, genetic deletion of SRSF3 in hepatocytes caused progressive liver disease and ultimately led to hepatocellular carcinoma. Here we show that SRSF3 is decreased in human liver samples with non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), or cirrhosis that was associated with alterations in RNA splicing of known SRSF3 target genes. Hepatic SRSF3 expression was similarly decreased and RNA splicing dysregulated in mouse models of NAFLD and NASH. We showed that palmitic acid-induced oxidative stress caused conjugation of the ubiquitin like NEDD8 protein to SRSF3 and proteasome mediated degradation. SRSF3 was selectively neddylated at lysine11 and mutation of this residue (SRSF3-K11R) was sufficient to prevent both SRSF3 degradation and alterations in RNA splicing. Finally prevention of SRSF3 degradation in vivo partially protected mice from hepatic steatosis, fibrosis and inflammation. These results highlight a neddylation-dependent mechanism regulating gene expression in the liver that is disrupted in early metabolic liver disease and may contribute to the progression to NASH, cirrhosis and ultimately hepatocellular carcinoma.

Authors

Deepak Kumar, Manasi Das, Consuelo Sauceda, Lesley G. Ellies, Karina Kuo, Purva Parwal, Mehak Kaur, Lily Jih, Gautam K. Bandyopadhyay, Douglas Burton, Rohit Loomba, Olivia Osborn, Nicholas J.G. Webster

Hotspot SF3B1 mutations induce metabolic reprogramming and vulnerability to serine deprivation

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Abstract

Cancer-associated mutations in the spliceosome gene SF3B1 create a neomorphic protein that produces aberrant mRNA splicing in hundreds of genes, but the ensuing biologic and therapeutic consequences of this missplicing are not well understood. Here we have provided evidence that aberrant splicing by mutant SF3B1 altered the transcriptome, proteome, and metabolome of human cells, leading to missplicing-associated downregulation of metabolic genes, decreased mitochondrial respiration, and suppression of the serine synthesis pathway. We also found that mutant SF3B1 induces vulnerability to deprivation of the nonessential amino acid serine, which was mediated by missplicing-associated downregulation of the serine synthesis pathway enzyme PHGDH. This vulnerability was manifest both in vitro and in vivo, as dietary restriction of serine and glycine in mice was able to inhibit the growth of SF3B1MUT xenografts. These findings describe a role for SF3B1 mutations in altered energy metabolism, and they offer a new therapeutic strategy against SF3B1MUT cancers.

Authors

W. Brian Dalton, Eric Helmenstine, Noel Walsh, Lukasz P. Gondek, Dhanashree S. Kelkar, Abigail Read, Rachael Natrajan, Eric S. Christenson, Barbara Roman, Samarjit Das, Liang Zhao, Robert D. Leone, Daniel Shinn, Taylor Groginski, Anil K. Madugundu, Arun Patil, Daniel J. Zabransky, Arielle Medford, Justin Lee, Alex J. Cole, Marc Rosen, Maya Thakar, Alexander Ambinder, Joshua Donaldson, Amy E. DeZern, Karen Cravero, David Chu, Rafael Madero-Marroquin, Akhilesh Pandey, Paula J. Hurley, Josh Lauring, Ben Park

JMJD3 regulates CD4 T cell trafficking by targeting actin cytoskeleton regulatory gene Pdlim4

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Abstract

Histone H3K27 demethylase, JMJD3 plays a critical role in gene expression and T-cell differentiation. However, the role and mechanisms of JMJD3 in T cell trafficking remain poorly understood. Here we show that JMJD3 deficiency in CD4+ T cells resulted in an accumulation of T cells in the thymus, and reduction of T cell number in the secondary lymphoid organs. We identified PDLIM4 as a significantly down-regulated target gene in JMJD3-deficient CD4+ T cells by gene profiling and ChIP-seq analyses. We further showed that PDLIM4 functioned as an adaptor protein to interact with S1P1 and filamentous actin (F-actin), thus serving as a key regulator of T cell trafficking. Mechanistically, JMJD3 bound to the promoter and gene body regions of Pdlim4 gene and regulated its expression by interacting with zinc finger transcription factor KLF2. Our findings have identified Pdlim4 as a JMJD3 target gene that affects T-cell trafficking by cooperating with S1P1, and provided insights into the molecular mechanisms by which JMJD3 regulates genes involved in T cell trafficking.

Authors

Chuntang Fu, Qingtian Li, Jia Zou, Changsheng Xing, Mei Luo, Bingnan Yin, Junjun Chu, Jiaming Yu, Xin Liu, Helen Y. Wang, Rong-Fu Wang

Evidence-based recommendations for energy intake in pregnant women with obesity

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Abstract

BACKGROUND. In women with obesity, excess gestational weight gain (≥270 g/week) occurs in two out of three pregnancies and contributes to metabolic impairments in both mother and baby. To improve obstetrical care, objectively assessed information on energy balance is urgently needed. The objective of this study was to characterize determinants of gestational weight gain in women with obesity. METHODS. This was a prospective, observational study of pregnant women with obesity. The primary outcome was energy intake calculated by the energy intake-balance method. Energy expenditure was measured by doubly-labeled water and whole-room indirect calorimetry and body composition as 3-compartment model by air displacement plethysmography and isotope dilution in early (13-16 weeks) and late pregnancy (35-37 weeks). RESULTS. In pregnant women with obesity (n=54), recommended weight gain (n=8, 15%) during the second and third trimesters was achieved when energy intake was 125±52 kcal/d less than energy expenditure. In contrast, women with excess weight gain (67%) consumed 186±29 kcal/d more than they expended (P<0.001). Energy balance affected maternal adiposity (recommended: -2.5±0.8 kg fat mass, excess: +2.2±0.5, inadequate: -4.5±0.5, P<0.001), but not fetal growth. Weight gain was not related to demographics, activity, metabolic biomarkers, or diet quality. We estimated that energy intake requirements for recommended weight gain during the second and third trimesters were not increased as compared to energy requirements early in pregnancy (34±53 kcal/d, P=0.83). CONCLUSIONS. We here provide the first evidence-based recommendations for energy intake in pregnant women with obesity. Contrary to current recommendations, energy intake should not exceed energy expenditure. FUNDING. This study was funded by the National Institutes of Health (R01DK099175; Redman, U54GM104940 and P30DK072476; Core support). TRIAL REGISTRATION. clinicaltrials.gov: NCT01954342

Authors

Jasper Most, Marshall St Amant, Daniel Hsia, Abby Altazan, Diana Thomas, Anne Gilmore, Porsha Vallo, Robbie Beyl, Eric Ravussin, Leanne Redman

Elastase 3B mutation links to familial pancreatitis with diabetes and pancreatic adenocarcinoma

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Abstract

While improvements in genetic analysis have greatly enhanced our understanding of the mechanisms behind pancreatitis, it continues to afflict many families for whom the hereditary factors remain unknown. Recent evaluation of a patient with a strong family history of pancreatitis sparked us to reexamine a large kindred originally reported over 50 years ago with an autosomal dominant inheritance pattern of chronic pancreatitis, diabetes and pancreatic adenocarcinoma. Whole exome sequencing analysis identified a rare missense mutation in the gene encoding pancreas-specific protease Elastase 3B (CELA3B) that cosegregates with disease. Studies of the mutant protein in vitro, in cell lines and in CRISPR-Cas9 engineered mice indicate that this mutation causes translational upregulation of CELA3B, which upon secretion and activation by trypsin leads to uncontrolled proteolysis and recurrent pancreatitis. Although lesions in several other pancreatitic proteases have been previously linked to hereditary pancreatitis, this is the first known instance of a mutation in CELA3B and a defect in translational control contributing to this disease.

Authors

Paul C. Moore, Jessica T. Cortez, Chester E. Chamberlain, Diana Alba, Amy C. Berger, Zoe Quandt, Alice Chan, Mickie H. Cheng, Jhoanne L. Bautista, Justin Peng, Michael S. German, Mark Anderson, Scott A. Oakes

Molecular profiling stratifies diverse phenotypes of treatment-refractory metastatic castration-resistant prostate cancer

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Abstract

Metastatic castration-resistant prostate cancer (mCRPC) is a heterogeneous disease with diverse drivers of disease progression and mechanisms of therapeutic resistance. We conducted deep phenotypic characterization of CRPC metastases and patient-derived xenograft (PDX) lines using whole genome RNA sequencing, gene set enrichment analysis and immunohistochemistry. Our analyses revealed five mCRPC phenotypes based on the expression of well-characterized androgen receptor (AR) or neuroendocrine (NE) genes: (i) AR-high tumors (ARPC), (ii) AR-low tumors (ARLPC), (iii) amphicrine tumors composed of cells co-expressing AR and NE genes (AMPC), (iv) double-negative tumors (i.e. AR-/NE-; DNPC) and (v) tumors with small cell or NE gene expression without AR activity (SCNPC). RE1-silencing transcription factor (REST) activity, which suppresses NE gene expression, was lost in AMPC and SCNPC PDX models. However, knockdown of REST in cell lines revealed that attenuated REST activity drives the AMPC phenotype but is not sufficient for SCNPC conversion. We also identified a subtype of DNPC tumors with squamous differentiation and generated an encompassing 26-gene transcriptional signature that distinguished the five mCRPC phenotypes. Together, our data highlight the central role of AR and REST in classifying treatment-resistant mCRPC phenotypes. These molecular classifications could potentially guide future therapeutic studies and clinical trial design.

Authors

Mark P. Labrecque, Ilsa M. Coleman, Lisha G. Brown, Lawrence D. True, Lori Kollath, Bryce Lakely, Holly M. Nguyen, Yu C. Yang, Rui M. Gil da Costa, Arja Kaipainen, Roger Coleman, Celestia S. Higano, Evan Y. Yu, Heather H. Cheng, Elahe A. Mostaghel, Bruce Montgomery, Michael T. Schweizer, Andrew C. Hsieh, Daniel W. Lin, Eva Corey, Peter S. Nelson, Colm Morrissey

Crystal deposition triggers tubule dilation that accelerates cystogenesis in polycystic kidney disease

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Abstract

The rate of disease progression in autosomal-dominant (AD) polycystic kidney disease (PKD) exhibits high intra-familial variability suggesting that environmental factors may play a role. We hypothesized that a prevalent form of renal insult may accelerate cystic progression and investigated tubular crystal deposition. We report that calcium oxalate (CaOx) crystal deposition led to rapid tubule dilation, activation of PKD-associated signaling pathways, and hypertrophy in tubule segments along the affected nephrons. Blocking mTOR signaling blunted this response and inhibited efficient excretion of lodged crystals. This mechanism of “flushing out” crystals by purposefully dilating renal tubules has not previously been recognized. Challenging PKD rat models with CaOx crystal deposition, or inducing calcium phosphate deposition by increasing dietary phosphorous intake, led to increased cystogenesis and disease progression. In a cohort of ADPKD patients, lower levels of urinary excretion of citrate, an endogenous inhibitor of calcium crystal formation, correlated with increased disease severity. These results suggest that PKD progression may be accelerated by commonly occurring renal crystal deposition which could be therapeutically controlled by relatively simple measures.

Authors

Jacob A. Torres, Mina Rezaei, Caroline Broderick, Louis Lin, Xiaofang Wang, Bernd Hoppe, Benjamin D. Cowley, Jr., Vincenzo Savica, Vicente E. Torres, Saeed Khan, Ross P. Holmes, Michal Mrug, Thomas Weimbs

HIV-1 in lymph nodes is maintained by cellular proliferation during antiretroviral therapy

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Abstract

To investigate the possibility that HIV-1 replication in lymph nodes sustains the reservoir during ART, we looked for evidence of viral replication in 5 donors after up to 13 years of viral suppression. We characterized proviral populations in lymph nodes and peripheral blood before and during ART, evaluated the levels of viral RNA expression in single lymph node and blood cells, and characterized the proviral integration sites in paired lymph node and blood samples. Proviruses with identical sequences, identical integration sites, and similar levels of RNA expression were found in lymph nodes and blood samples collected during ART, and no single sequence with significant divergence from the pretherapy population was present in either blood or lymph nodes. These findings show that all detectable persistent HIV-1 infection is consistent with maintenance in lymph nodes by clonal proliferation of cells infected before ART and not by ongoing viral replication during ART.

Authors

William R. McManus, Michael J. Bale, Jonathan Spindler, Ann Wiegand, Andrew Musick, Sean C. Patro, Michele D. Sobolewski, Victoria K. Musick, Elizabeth M. Anderson, Joshua C. Cyktor, Elias K. Halvas, Wei Shao, Daria Wells, Xiaolin Wu, Brandon F. Keele, Jeffrey M. Milush, Rebecca Hoh, John W. Mellors, Stephen H. Hughes, Steven G. Deeks, John M. Coffin, Mary F. Kearney

Innate and adaptive nasal mucosal immune responses following experimental human pneumococcal colonization

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Abstract

Streptococcus pneumoniae (Spn) is a common cause of respiratory infection, but also frequently colonizes the nasopharynx in the absence of disease. We used mass cytometry to study immune cells from nasal biopsy samples collected following experimental human pneumococcal challenge in order to identify immunological mechanisms of control of Spn colonization. Using 37 markers, we characterized 293 nasal immune cell clusters, of which 7 were associated with Spn colonization. B cell and CD8+CD161+ T cell clusters were significantly lower in colonized than in non-colonized subjects. By following a second cohort before and after pneumococcal challenge we observed that B cells were depleted from the nasal mucosa upon Spn colonization. This associated with an expansion of Spn polysaccharide-specific and total plasmablasts in blood. Moreover, increased responses of blood mucosal associated invariant T (MAIT) cells against in vitro stimulation with pneumococcus prior to challenge associated with protection against establishment of Spn colonization and with increased mucosal MAIT cell populations. These results implicate MAIT cells in the protection against pneumococcal colonization and demonstrate that colonization affects mucosal and circulating B cell populations.

Authors

Simon P. Jochems, Karin de Ruiter, Carla Solórzano, Astrid Voskamp, Elena Mitsi, Elissavet Nikolaou, Beatriz F. Carniel, Sherin Pojar, Esther L. German, Jesús Reiné, Alessandra Soares-Schanoski, Helen Hill, Rachel Robinson, Angela D. Hyder-Wright, Caroline M. Weight, Pascal F. Durrenberger, Robert S. Heyderman, Stephen B. Gordon, Hermelijn H. Smits, Britta C. Urban, Jamie Rylance, Andrea M. Collins, Mark D. Wilkie, Lepa Lazarova, Samuel C. Leong, Maria Yazdanbakhsh, Daniela M. Ferreira

STING activation reprograms tumor vasculatures and synergizes with VEGFR2 blockade

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Abstract

The stimulator of interferon genes (STING) signaling pathway is a critical link between innate and adaptive immunity, and induces anti-tumor immune responses. STING is expressed in vasculatures, but its role in tumor angiogenesis has not been elucidated. Here we investigated STING-induced tumor vascular remodeling and the potential of STING-based combination immunotherapy. Endothelial STING expression was correlated with enhanced T-cell infiltration and prolonged survival in human colon and breast cancer. Intratumoral STING activation with STING agonists (cGAMP or RR-CDA) normalized tumor vasculatures in implanted and spontaneous cancers, but not in STING-deficient mice. These were mediated by upregulation of type I/II interferon genes and vascular stabilizing genes (e.g., Angpt1, Pdgfrb, and Col4a). STING in non-hematopoietic cells is as important as STING in hematopoietic cells to induce a maximal therapeutic efficacy of exogenous STING agonist. Vascular normalizing effects of STING agonists were dependent on type I interferon signaling and CD8+ T cells. Notably, STING-based immunotherapy was maximally effective when combined with VEGFR2 blockade and/or immune checkpoint blockade (αPD-1 or αCTLA-4), leading to complete regression of immunotherapy-resistant tumors. Our data show that intratumoral STING activation can normalize tumor vasculature and the tumor microenvironment, providing a rationale for combining STING-based immunotherapy and anti-angiogenic therapy.

Authors

Hannah Yang, Won Suk Lee, So Jung Kong, Chang Gon Kim, Joo Hoon Kim, Sei Kyung Chang, Sewha Kim, Gwangil Kim, Hong Jae Chon, Chan Kim

Impaired plasma membrane localization of ubiquitin ligase complex underlies 3-M syndrome development

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Abstract

3-M primordial dwarfism is an inherited disease characterized by severe pre- and postnatal growth retardation and by mutually exclusive mutations in three genes, CUL7, OBSL1, and CCDC8. The mechanism underlying 3-M dwarfism is not clear. We showed here that CCDC8, derived from a retrotransposon Gag protein in placental mammals, exclusively localized on the plasma membrane and was phosphorylated by CK2 and GSK3. Phosphorylation of CCDC8 resulted in its binding first with OBSL1, and then CUL7, leading to the membrane assembly of the 3-M E3 ubiquitin ligase complex. We identified LL5β, a plasma membrane protein that regulates cell migration, as a substrate of 3-M ligase. Wnt inhibition of CCDC8 phosphorylation or patient-derived mutations in 3-M genes disrupted membrane localization of the 3-M complex and accumulated LL5β. Deletion of Ccdc8 in mice impaired trophoblast migration and placental development, resulting in intrauterine growth restriction and perinatal lethality. These results identified a mechanism regulating cell migration and placental development that underlies the development of 3-M dwarfism.

Authors

Pu Wang, Feng Yan, Zhijun Li, Yanbao Yu, Scott E. Parnell, Yue Xiong

Gene loci associated with insulin secretion in islets from non-diabetic mice

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Abstract

Genetic susceptibility to type 2 diabetes is primarily due to β-cell dysfunction. However, a genetic study to directly interrogate β-cell function ex vivo has never been previously performed. We isolated 233,447 islets from 483 Diversity Outbred (DO) mice maintained on a Western-style diet, and measured insulin secretion in response to a variety of secretagogues. Insulin secretion from DO islets ranged >1,000-fold even though none of the mice were diabetic. The insulin secretory response to each secretagogue had a unique genetic architecture; some of the loci were specific for one condition, whereas others overlapped. Human loci that are syntenic to many of the insulin secretion QTL from mouse are associated with diabetes-related SNPs in human genome-wide association studies. We report on three genes, Ptpn18, Hunk and Zfp148, where the phenotype predictions from the genetic screen were fulfilled in our studies of transgenic mouse models. These three genes encode a non-receptor type protein tyrosine phosphatase, a serine/threonine protein kinase, and a Krϋppel-type zinc-finger transcription factor, respectively. Our results demonstrate that genetic variation in insulin secretion that can lead to type 2 diabetes is discoverable in non-diabetic individuals.

Authors

Mark P. Keller, Mary E. Rabaglia, Kathryn L. Schueler, Donnie S. Stapleton, Daniel M. Gatti, Matthew Vincent, Kelly A. Mitok, Ziyue Wang, Takanao Ishimura, Shane P. Simonett, Christopher H. Emfinger, Rahul Das, Tim Beck, Christina Kendziorski, Karl W. Broman, Brian S. Yandell, Gary A. Churchill, Alan D. Attie

Lysophosphatidic acid-induced YAP/TAZ activation promotes developmental angiogenesis by repressing Notch ligand Dll4

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Abstract

Lysophosphatidic acid (LPA) is a potent lipid mediator with various biological functions mediated through six G protein-coupled receptors (GPCRs), LPA1–6. Previous studies have demonstrated that LPA-Gα12/Gα13 signaling plays an important role in embryonic vascular development. However, the responsible LPA receptors and underlying mechanisms are poorly understood. Here, we show a critical role of LPA4 and LPA6 in developmental angiogenesis. In mice, Lpa4;Lpa6 double knockout (DKO) embryos were lethal due to global vascular deficiencies, and endothelial cell (EC)-specific Lpa4;Lpa6 DKO retinas had impaired sprouting angiogenesis. Mechanistically, LPA activated the transcriptional regulators YAP and TAZ through LPA4/LPA6-mediated Gα12/Gα13-Rho-ROCK signaling in ECs. YAP/TAZ knockdown increased β-catenin- and Notch intracellular domain (NICD)-mediated endothelial expression of the Notch ligand delta-like 4 (DLL4). Fibrin gel sprouting assay revealed that LPA4/LPA6, Gα12/Gα13, or YAP/TAZ knockdown consistently blocked EC sprouting, which was rescued by a Notch inhibitor. Of note, the inhibition of Notch signaling also ameliorated impaired retinal angiogenesis in EC-specific Lpa4;Lpa6 DKO mice. Overall, these results suggest that the Gα12/Gα13-coupled receptors LPA4 and LPA6 synergistically regulate endothelial Dll4 expression through YAP/TAZ activation. This could in part account for the mechanism of YAP/TAZ-mediated developmental angiogenesis. Our findings provide a novel insight into the biology of GPCR-activated YAP/TAZ.

Authors

Daisuke Yasuda, Daiki Kobayashi, Noriyuki Akahoshi, Takayo Ohto-Nakanishi, Kazuaki Yoshioka, Yoh Takuwa, Seiya Mizuno, Satoru Takahashi, Satoshi Ishii

The alveolar immune cell landscape is dysregulated in checkpoint inhibitor pneumonitis

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Abstract

Background: Checkpoint inhibitor pneumonitis (CIP) is a highly morbid complication of immune checkpoint immunotherapy (ICI), one which precludes the continuation of ICI. Yet, the mechanistic underpinnings of CIP are unknown. Methods: To better understand the mechanism of lung injury in CIP, we prospectively collected bronchoalveolar lavage (BAL) samples in ICI-treated patients with (n=12) and without CIP (n=6), prior to initiation of first-line therapy for CIP (high dose corticosteroids. We analyzed BAL immune cell populations using a combination of traditional multicolor flow cytometry gating, unsupervised clustering analysis and BAL supernatant cytokine measurements. Results: We found increased BAL lymphocytosis, predominantly CD4+ T cells, in CIP. Specifically, we observed increased numbers of BAL central memory T-cells (Tcm), evidence of Type I polarization, and decreased expression of CTLA-4 and PD-1 in BAL Tregs, suggesting both activation of pro-inflammatory subsets and an attenuated suppressive phenotype. CIP BAL myeloid immune populations displayed enhanced expression of IL-1β and decreased expression of counter-regulatory IL-1RA. We observed increased levels of T cell chemoattractants in the BAL supernatant, consistent with our pro-inflammatory, lymphocytic cellular landscape. Conclusion: We observe several immune cell subpopulations that are dysregulated in CIP, which may represent possible targets that could lead to therapeutics for this morbid immune related adverse event.

Authors

Karthik Suresh, Jarushka Naidoo, Qiong Zhong, Ye Xiong, Jennifer Mammen, Marcia Villegas de Flores, Laura Cappelli, Aanika Balaji, Tsvi Palmer, Patrick M. Forde, Valsamo Anagnostou, David S. Ettinger, Kristen A. Marrone, Ronan J. Kelly, Christine L. Hann, Benjamin Levy, Josephine L. Feliciano, Cheng-Ting Lin, David Feller-Kopman, Andrew D. Lerner, Hans Lee, Majid Shafiq, Lonny Yarmus, Evan J. Lipson, Mark Soloski, Julie R. Brahmer, Sonye K. Dannoff, Franco D'Alessio

Tumor suppressor TET2 promotes cancer immunity and immunotherapy efficacy

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Abstract

Loss-of-function mutations in genes encoding TET DNA dioxygenase occur frequently in hematopoietic malignancy, but rarely in solid tumors which instead commonly have reduced activity. The impact of decreased TET activity in solid tumors is not known. Here we show that TET2 mediates interferon γ (IFNγ)-JAK-STAT signaling pathway to control chemokine and PD-L1 expression, lymphocyte infiltration and cancer immunity. IFNγ stimulated STAT1 to bind TET2 and recruit TET2 to hydroxymethylate chemokine and PD-L1 genes. Reduced TET activity was associated with decreased TH1-type chemokines and tumor-infiltrating lymphocytes (TILs) and the progression of human colon cancer. Deletion of Tet2 in murine melanoma and colon tumor cells reduced chemokine expression and TILs, enabling tumors to evade anti-tumor immunity and to resist anti-PD-L1 therapy. Conversely, stimulating TET activity by systematic injection of its co-factor, ascorbate/vitamin C, increased chemokine and TILs, leading to enhanced anti-tumor immunity and anti-PD-L1 efficacy and extended lifespan of tumor-bearing mice. These results suggest an IFNγ-JAK-STAT-TET signaling pathway that mediates tumor response to anti-PD-L1/PD-1 therapy and is frequently disrupted in solid tumors. Our findings also suggest TET activity as a biomarker for predicting the efficacy and patient response to anti-PD-1/PD-L1 therapy, and stimulating TET activity as an adjuvant immunotherapy of solid tumors.

Authors

Yan-ping Xu, Lei Lv, Ying Liu, Matthew D. Smith, Wen-Cai Li, Xian-ming Tan, Meng Cheng, Zhijun Li, Michael Bovino, Jeffrey Aubé, Yue Xiong