Issue published September 8, 2022
- Volume 7, Issue 17
- Previous Issue | Next Issue
Janardhan et al. report that pathological MAPK activation disrupts the lymphatic basement membrane, causing lymphangiectasia, chylous pleural effusion, and mortality. The cover image is an H&E-stained pathological human tissue section that shows lymphangiectasia.
On the cover: Pathological activation of MAPK causes lymphangiectasia treatable with ravoxertinib
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Research Articles
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Abstract
Osteosarcoma (OS) is a lethal disease with few known targeted therapies. Here, we show that decreased ATRX expression is associated with more aggressive tumor cell phenotypes, including increased growth, migration, invasion, and metastasis. These phenotypic changes correspond with activation of NF-κB signaling, extracellular matrix remodeling, increased integrin αvβ3 expression, and ETS family transcription factor binding. Here, we characterize these changes in vitro, in vivo, and in a data set of human OS patients. This increased aggression substantially sensitizes ATRX-deficient OS cells to integrin signaling inhibition. Thus, ATRX plays an important tumor-suppression role in OS, and loss of function of this gene may underlie new therapeutic vulnerabilities. The relationship between ATRX expression and integrin binding, NF-κB activation, and ETS family transcription factor binding has not been described in previous studies and may impact the pathophysiology of other diseases with ATRX loss, including other cancers and the ATR-X α thalassemia intellectual disability syndrome.
Authors
Suzanne Bartholf DeWitt, Sarah Hoskinson Plumlee, Hailey E. Brighton, Dharshan Sivaraj, E.J. Martz, Maryam Zand, Vardhman Kumar, Maya U. Sheth, Warren Floyd, Jacob V. Spruance, Nathan Hawkey, Shyni Varghese, Jianhua Ruan, David G. Kirsch, Jason A. Somarelli, Ben Alman, William C. Eward
×Abstract
Acquired mutations in the ligand-binding domain (LBD) of the gene encoding estrogen receptor α (ESR1) are common mechanisms of endocrine therapy resistance in patients with metastatic ER+ breast cancer. The ESR1 Y537S mutation, in particular, is associated with development of resistance to most endocrine therapies used to treat breast cancer. Employing a high-throughput screen of nearly 1,200 Federal Drug Administration–approved (FDA-approved) drugs, we show that OTX015, a bromodomain and extraterminal domain (BET) inhibitor, is one of the top suppressors of ESR1 mutant cell growth. OTX015 was more efficacious than fulvestrant, a selective ER degrader, in inhibiting ESR1 mutant xenograft growth. When combined with abemaciclib, a CDK4/6 inhibitor, OTX015 induced more potent tumor regression than current standard-of-care treatment of abemaciclib + fulvestrant. OTX015 has preferential activity against Y537S mutant breast cancer cells and blocks their clonal selection in competition studies with WT cells. Thus, BET inhibition has the potential to both prevent and overcome ESR1 mutant–induced endocrine therapy resistance in breast cancer.
Authors
Sm N. Udden, Qian Wang, Sunil Kumar, Venkat S. Malladi, Shwu-Yuan Wu, Shuguang Wei, Bruce A. Posner, Sophie Geboers, Noelle S. Williams, Yulun Liu, Jayesh K. Sharma, Ram S. Mani, Srinivas Malladi, Karla Parra, Mia Hofstad, Ganesh V. Raj, Jose M. Larios, Reshma Jagsi, Max S. Wicha, Ben Ho Park, Gaorav P. Gupta, Arul M. Chinnaiyan, Cheng-Ming Chiang, Prasanna G. Alluri
×Abstract
B lymphocytes have long been recognized for their critical contributions to adaptive immunity, providing defense against pathogens through cognate antigen presentation to T cells and Ab production. More recently appreciated is that B cells are also integral in securing self-tolerance; this has led to interest in their therapeutic application to downregulate unwanted immune responses, such as transplant rejection. In this study, we found that PMA- and ionomycin-activated mouse B cells acquire regulatory properties following stimulation through TLR4/TLR9 receptors (Bregs-TLR). Bregs-TLR efficiently inhibited T cell proliferation in vitro and prevented allograft rejection. Unlike most reported Breg activities, the inhibition of alloimmune responses by Bregs-TLR relied on the expression of TGF-β and not IL-10. In vivo, Bregs-TLR interrupted donor-specific T cell expansion and induced Tregs in a TGF-β–dependent manner. RNA-Seq analyses corroborated the involvement of TGF-β pathways in Breg-TLR function, identified potential gene pathways implicated in preventing graft rejection, and suggested targets to foster Breg regulation.
Authors
Kang Mi Lee, Qiang Fu, Guoli Huai, Kevin Deng, Ji Lei, Lisa Kojima, Divyansh Agarwal, Peter van Galen, Shoko Kimura, Naoki Tanimine, Laura Washburn, Heidi Yeh, Ali Naji, Charles G. Rickert, Christian LeGuern, James F. Markmann
×Abstract
Lymphangiectasia, an anomalous dilation of lymphatic vessels first described in the 17th century, is frequently associated with chylous effusion, respiratory failure, and high mortality in young patients, yet the underlying molecular pathogenesis and effective treatments remain elusive. Here, we identify an unexpected causal link between MAPK activation and defective development of the lymphatic basement membrane that drives lymphangiectasia. Human pathological tissue samples from patients diagnosed with lymphangiectasia revealed sustained MAPK activation within lymphatic endothelial cells. Endothelial KRASG12D–mediated sustained MAPK activation in newborn mice caused severe pulmonary and intercostal lymphangiectasia, accumulation of chyle in the pleural space, and complete lethality. Pathological activation of MAPK in murine vasculature inhibited the Nfatc1-dependent genetic program required for laminin interactions, collagen crosslinking, and anchoring fibril formation, driving defective development of the lymphatic basement membrane. Treatment with ravoxertinib, a pharmacological inhibitor of MAPK, reverses nuclear-to-cytoplasmic localization of Nfatc1, basement membrane development defects, lymphangiectasia, and chyle accumulation, ultimately improving survival of endothelial KRAS mutant neonatal mice. These results reveal defective lymphatic basement membrane assembly and composition as major causes of thoracic lymphangiectasia and provide a potential treatment.
Authors
Harish P. Janardhan, Karen Dresser, Lloyd Hutchinson, Chinmay M. Trivedi
×Abstract
The sarcoplasmic reticulum (SR) plays an important role in calcium homeostasis. SR calcium mishandling is described in pathological conditions, such as myopathies. Here, we investigated whether the nuclear receptor subfamily 1 group D member (NR1D1, also called REV-ERBα) regulates skeletal muscle SR calcium homeostasis. Our data demonstrate that NR1D1 deficiency in mice impaired sarco/endoplasmic reticulum calcium ATPase–dependent (SERCA-dependent) SR calcium uptake. NR1D1 acts on calcium homeostasis by repressing the SERCA inhibitor myoregulin through direct binding to its promoter. Restoration of myoregulin counteracted the effects of NR1D1 overexpression on SR calcium content. Interestingly, myoblasts from patients with Duchenne muscular dystrophy displayed lower NR1D1 expression, whereas pharmacological NR1D1 activation ameliorated SR calcium homeostasis and improved muscle structure and function in dystrophic mdx/Utr+/– mice. Our findings demonstrate that NR1D1 regulates muscle SR calcium homeostasis, pointing to its therapeutic potential for mitigating myopathy.
Authors
Alexis Boulinguiez, Christian Duhem, Alicia Mayeuf-Louchart, Benoit Pourcet, Yasmine Sebti, Kateryna Kondratska, Valérie Montel, Stéphane Delhaye, Quentin Thorel, Justine Beauchamp, Aurore Hebras, Marion Gimenez, Marie Couvelaere, Mathilde Zecchin, Lise Ferri, Natalia Prevarskaya, Anne Forand, Christel Gentil, Jessica Ohana, France Piétri-Rouxel, Bruno Bastide, Bart Staels, Helene Duez, Steve Lancel
×Abstract
Despite intensive therapy, children with high-risk neuroblastoma are at risk of treatment failure. We applied a multiomic system approach to evaluate metabolic vulnerabilities in human neuroblastoma. We combined metabolomics, CRISPR screening, and transcriptomic data across more than 700 solid tumor cell lines and identified dihydroorotate dehydrogenase (DHODH), a critical enzyme in pyrimidine synthesis, as a potential treatment target. Of note, DHODH inhibition is currently under clinical investigation in patients with hematologic malignancies. In neuroblastoma, DHODH expression was identified as an independent risk factor for aggressive disease, and high DHODH levels correlated to worse overall and event-free survival. A subset of tumors with the highest DHODH expression was associated with a dismal prognosis, with a 5-year survival of less than 10%. In xenograft and transgenic neuroblastoma mouse models treated with the DHODH inhibitor brequinar, tumor growth was dramatically reduced, and survival was extended. Furthermore, brequinar treatment was shown to reduce the expression of MYC targets in 3 neuroblastoma models in vivo. A combination of brequinar and temozolomide was curative in the majority of transgenic TH-MYCN neuroblastoma mice, indicating a highly active clinical combination therapy. Overall, DHODH inhibition combined with temozolomide has therapeutic potential in neuroblastoma, and we propose this combination for clinical testing.
Authors
Thale Kristin Olsen, Cecilia Dyberg, Bethel Tesfai Embaie, Adele Alchahin, Jelena Milosevic, Jane Ding, Jörg Otte, Conny Tümmler, Ida Hed Myrberg, Ellen M. Westerhout, Jan Koster, Rogier Versteeg, Han-Fei Ding, Per Kogner, John Inge Johnsen, David B. Sykes, Ninib Baryawno
×Abstract
Huntington’s disease (HD) is a late-onset neurological disorder for which therapeutics are not available. Its key pathological mechanism involves the proteolysis of polyglutamine-expanded (polyQ-expanded) mutant huntingtin (mHTT), which generates N-terminal fragments containing polyQ, a key contributor to HD pathogenesis. Interestingly, a naturally occurring spliced form of HTT mRNA with truncated exon 12 encodes an HTT (HTTΔ12) with a deletion near the caspase-6 cleavage site. In this study, we used a multidisciplinary approach to characterize the therapeutic potential of targeting HTT exon 12. We show that HTTΔ12 was resistant to caspase-6 cleavage in both cell-free and tissue lysate assays. However, HTTΔ12 retained overall biochemical and structural properties similar to those of wt-HTT. We generated mice in which HTT exon 12 was truncated and found that the canonical exon 12 was dispensable for the main physiological functions of HTT, including embryonic development and intracellular trafficking. Finally, we pharmacologically induced HTTΔ12 using the antisense oligonucleotide (ASO) QRX-704. QRX-704 showed predictable pharmacology and efficient biodistribution. In addition, it was stable for several months and inhibited pathogenic proteolysis. Furthermore, QRX-704 treatments resulted in a reduction of HTT aggregation and an increase in dendritic spine count. Thus, ASO-induced HTT exon 12 splice switching from HTT may provide an alternative therapeutic strategy for HD.
Authors
Hyeongju Kim, Sophie Lenoir, Angela Helfricht, Taeyang Jung, Zhana K. Karneva, Yejin Lee, Wouter Beumer, Geert B. van der Horst, Herma Anthonijsz, Levi C.M. Buil, Frits van der Ham, Gerard J. Platenburg, Pasi Purhonen, Hans Hebert, Sandrine Humbert, Frédéric Saudou, Pontus Klein, Ji-Joon Song
×Abstract
Development of resistance to chemo- and immunotherapies often occurs following treatment of melanoma brain metastasis (MBM). The brain microenvironment (BME), particularly astrocytes, cooperate toward MBM progression by upregulating secreted factors, among which we found that monocyte chemoattractant protein-1 (MCP-1) and its receptors, CCR2 and CCR4, were overexpressed in MBM compared with primary lesions. Among other sources of MCP-1 in the brain, we show that melanoma cells altered astrocyte secretome and evoked MCP-1 expression and secretion, which in turn induced CCR2 expression in melanoma cells, enhancing in vitro tumorigenic properties, such as proliferation, migration, and invasion of melanoma cells. In vivo pharmacological blockade of MCP-1 or molecular knockout of CCR2/CCR4 increased the infiltration of cytotoxic CD8+ T cells and attenuated the immunosuppressive phenotype of the BME as shown by decreased infiltration of Tregs and tumor-associated macrophages/microglia in several models of intracranially injected MBM. These in vivo strategies led to decreased MBM outgrowth and prolonged the overall survival of the mice. Our findings highlight the therapeutic potential of inhibiting interactions between BME and melanoma cells for the treatment of this disease.
Authors
Sabina Pozzi, Anna Scomparin, Dikla Ben-Shushan, Eilam Yeini, Paula Ofek, Alessio D. Nahmad, Shelly Soffer, Ariel Ionescu, Antonella Ruggiero, Adi Barzel, Henry Brem, Thomas M. Hyde, Iris Barshack, Sanju Sinha, Eytan Ruppin, Tomer Weiss, Asaf Madi, Eran Perlson, Inna Slutsky, Helena F. Florindo, Ronit Satchi-Fainaro
×Abstract
STING gain-of-function mutations cause STING-associated vasculopathy with onset in infancy (SAVI) in humans, a disease characterized by spontaneous lung inflammation and fibrosis. Mice with STING gain-of-function mutations (SAVI mice) develop αβ T cell–dependent lung disease and also lack lymph nodes. Although SAVI has been regarded as a type I interferonopathy, the relative contributions of the three interferon receptors are incompletely understood. Here, we show that STING gain of function led to upregulation of IFN-γ–induced chemokines in the lungs of SAVI mice and that deletion of the type II IFN receptor (IFNGR1), but not the type I IFN receptor (IFNAR1) or type III IFN receptor (IFNλR1), ameliorated lung disease and restored lymph node development in SAVI mice. Furthermore, deletion of IFNGR1, but not IFNAR1 or IFNλR1, corrected the ratio of effector to Tregs in SAVI mice and in mixed bone marrow chimeric mice. Finally, cultured SAVI mouse macrophages were hyperresponsive to IFN-γ, but not IFN-β, in terms of Cxcl9 upregulation and cell activation. These results demonstrate that IFNGR1 plays a major role in autoinflammation and immune dysregulation mediated by STING gain of function.
Authors
W. Alexander Stinson, Cathrine A. Miner, Fang R. Zhao, Annena Jane Lundgren, Subhajit Poddar, Jonathan J. Miner
×Abstract
Cardiovascular diseases, especially atherosclerosis and its complications, are a leading cause of death. Inhibition of the noncanonical IκB kinases TANK-binding kinase 1 and IKKε with amlexanox restores insulin sensitivity and glucose homeostasis in diabetic mice and human patients. Here we report that amlexanox improves diet-induced hypertriglyceridemia and hypercholesterolemia in Western diet–fed (WD-fed) Ldlr–/– mice and protects against atherogenesis. Amlexanox ameliorated dyslipidemia, inflammation, and vascular dysfunction through synergistic actions that involve upregulation of bile acid synthesis to increase cholesterol excretion. Transcriptomic profiling demonstrated an elevated expression of key bile acid synthesis genes. Furthermore, we found that amlexanox attenuated monocytosis, eosinophilia, and vascular dysfunction during WD-induced atherosclerosis. These findings demonstrate the potential of amlexanox as a therapy for hypercholesterolemia and atherosclerosis.
Authors
Peng Zhao, Xiaoli Sun, Zhongji Liao, Hong Yu, Dan Li, Zeyang Shen, Christopher K. Glass, Joseph L. Witztum, Alan R. Saltiel
×Abstract
Checkpoint inhibitors (CPIs) targeting programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) and cytotoxic T lymphocyte antigen 4 (CTLA-4) have revolutionized cancer treatment but can trigger autoimmune complications, including CPI-induced diabetes mellitus (CPI-DM), which occurs preferentially with PD-1 blockade. We found evidence of pancreatic inflammation in patients with CPI-DM with shrinkage of pancreases, increased pancreatic enzymes, and in a case from a patient who died with CPI-DM, peri-islet lymphocytic infiltration. In the NOD mouse model, anti–PD-L1 but not anti–CTLA-4 induced diabetes rapidly. RNA sequencing revealed that cytolytic IFN-γ+CD8+ T cells infiltrated islets with anti–PD-L1. Changes in β cells were predominantly driven by IFN-γ and TNF-α and included induction of a potentially novel β cell population with transcriptional changes suggesting dedifferentiation. IFN-γ increased checkpoint ligand expression and activated apoptosis pathways in human β cells in vitro. Treatment with anti–IFN-γ and anti–TNF-α prevented CPI-DM in anti–PD-L1–treated NOD mice. CPIs targeting the PD-1/PD-L1 pathway resulted in transcriptional changes in β cells and immune infiltrates that may lead to the development of diabetes. Inhibition of inflammatory cytokines can prevent CPI-DM, suggesting a strategy for clinical application to prevent this complication.
Authors
Ana Luisa Perdigoto, Songyan Deng, Katherine C. Du, Manik Kuchroo, Daniel B. Burkhardt, Alexander Tong, Gary Israel, Marie E. Robert, Stuart P. Weisberg, Nancy Kirkiles-Smith, Angeliki M. Stamatouli, Harriet M. Kluger, Zoe Quandt, Arabella Young, Mei-Ling Yang, Mark J. Mamula, Jordan S. Pober, Mark S. Anderson, Smita Krishnaswamy, Kevan C. Herold
×Abstract
Lysosomal acid lipase (LAL) is a key enzyme in the metabolic pathway of neutral lipids. In the blood of LAL-deficient (Lal–/–) mice, increased CD11c+ cells were accompanied by upregulated programmed cell death ligand 1 (PD-L1) expression. Single-cell RNA sequencing of Lal–/– CD11c+ cells identified 2 distinctive clusters with a major metabolic shift toward glucose utilization and reactive oxygen species overproduction. Pharmacologically blocking pyruvate dehydrogenase in glycolysis not only reduced CD11c+ cells and their PD-L1 expression but also reversed their capabilities of T cell suppression and tumor growth stimulation. Colony-stimulating factor 1 receptor (CSF1R) played an essential role in controlling Lal–/– CD11c+ cell homeostasis and function and PD-L1 expression. Pharmacological inhibition of LAL activity increased CD11c, PD-L1, and CSF1R levels in both normal murine myeloid cells and human blood cells. Tumor-bearing mice and human patients with non–small cell lung cancer also showed CD11c+ cell expansion with PD-L1 and CSF1R upregulation and immunosuppression. There were positive correlations among CD11c, PD-L1, and CSF1R expression and negative correlations with LAL expression in patients with lung cancer or melanoma using The Cancer Genome Atlas database and patient samples. Therefore, CD11c+ cells switched their functions to immune suppression and tumor growth stimulation through CSF1R/PD-L1 upregulation and metabolic reprogramming.
Authors
Ting Zhao, Sheng Liu, Xinchun Ding, Erica M. Johnson, Nasser H. Hanna, Kanhaiya Singh, Chandan K. Sen, Jun Wan, Hong Du, Cong Yan
×Abstract
Increased red cell distribution width (RDW), which measures erythrocyte mean corpuscular volume (MCV) variability (anisocytosis), has been linked to early mortality in many diseases and in older adults through unknown mechanisms. Hypoxic stress has been proposed as a potential mechanism. However, experimental models to investigate the link between increased RDW and reduced survival are lacking. Here, we show that lifelong hypobaric hypoxia (~10% O2) increased erythrocyte numbers, hemoglobin, and RDW, while reducing longevity in male mice. Compound heterozygous knockout (hKO) mutations in succinate dehydrogenase (Sdh; mitochondrial complex II) genes Sdhb, Sdhc, and Sdhd reduced Sdh subunit protein levels, reduced RDW, and increased healthy life span compared with WT mice in chronic hypoxia. RDW-SD, a direct measure of MCV variability, and the SD of MCV showed the most statistically significant reductions in Sdh hKO mice. Tissue metabolomic profiling of 147 common metabolites showed the largest increase in succinate with elevated succinate/fumarate and succinate/oxoglutarate (2-ketoglutarate) ratios in Sdh hKO mice. These results demonstrate that mitochondrial complex II level is an underlying determinant of both RDW and healthy life span in hypoxia and suggest that therapeutic targeting of Sdh might reduce high RDW–associated clinical mortality in hypoxic diseases.
Authors
Bora E. Baysal, Abdulrahman A. Alahmari, Tori C. Rodrick, Debra Tabaczynski, Leslie Curtin, Mukund Seshadri, Drew R. Jones, Sandra Sexton
×Abstract
Junctional adhesion molecule-A (JAM-A) is expressed in several cell types, including epithelial and endothelial cells, as well as some leukocytes. In intestinal epithelial cells (IEC), JAM-A localizes to cell junctions and plays a role in regulating barrier function. In vitro studies with model cell lines have shown that JAM-A contributes to IEC migration; however, in vivo studies investigating the role of JAM-A in cell migration–dependent processes such as mucosal wound repair have not been performed. In this study, we developed an inducible intestinal epithelial–specific JAM-A–knockdown mouse model (Jam-aERΔIEC). While acute induction of IEC-specific loss of JAM-A did not result in spontaneous colitis, such mice had significantly impaired mucosal healing after chemically induced colitis and after biopsy colonic wounding. In vitro primary cultures of JAM-A–deficient IEC demonstrated impaired migration in wound healing assays. Mechanistic studies revealed that JAM-A stabilizes formation of protein signaling complexes containing Rap1A/Talin/β1 integrin at focal adhesions of migrating IECs. Loss of JAM-A in primary IEC led to decreased Rap1A activity and protein levels of Talin and β1 integrin, and it led to a reduction in focal adhesion structures. These findings suggest that epithelial JAM-A plays a critical role in controlling mucosal repair in vivo through dynamic regulation of focal adhesions.
Authors
Shuling Fan, Kevin Boerner, Chithra K. Muraleedharan, Asma Nusrat, Miguel Quiros, Charles A. Parkos
×Abstract
Gut microbiota dysbiosis is associated with inflammatory bowel diseases and with cardiometabolic, neurological, and autoimmune diseases. Gut microbiota composition has a direct effect on the immune system, and vice versa, and it has a particular effect on Treg homeostasis. Low-dose IL-2 (IL-2LD) stimulates Tregs and is a promising treatment for autoimmune and inflammatory diseases. We aimed to evaluate the impact of IL-2LD on gut microbiota and correlatively on the immune system. We used 16S ribosomal RNA profiling and metagenomics to characterize gut microbiota of mice and humans treated or not with IL-2LD. We performed fecal microbiota transplantation (FMT) from IL-2LD–treated to naive recipient mice and evaluated its effects in models of gut inflammation and diabetes. IL-2LD markedly affected gut microbiota composition in mice and humans. Transfer of an IL-2–tuned microbiota by FMT protected C57BL/6J mice from dextran sulfate sodium–induced colitis and prevented diabetes in NOD mice. Metagenomic analyses highlighted a role for several species affected by IL-2LD and for microbial pathways involved in the biosynthesis of amino acids, short-chain fatty acids, and L-arginine. Our results demonstrate that IL-2LD induced changes in gut microbiota that are involved in the immunoregulatory effects of IL-2LD and suggest a crosstalk between Tregs and gut microbiota. These results provide potentially novel insight for understanding the mode of action of Treg-directed therapies.
Authors
Nicolas Tchitchek, Otriv Nguekap Tchoumba, Gabriel Pires, Sarah Dandou, Julien Campagne, Guillaume Churlaud, Gwladys Fourcade, Thomas W. Hoffmann, Francesco Strozzi, Camille Gaal, Christophe Bonny, Emmanuelle Le Chatelier, Stanislav Dusko Erlich, Harry Sokol, David Klatzmann
×Abstract
It is known that tumor-reactive T cells are initially activated in the draining lymph node, but it is not well known whether and how tumor-infiltrating lymphocytes (TILs) are reactivated in the tumor microenvironment (TME). We hypothesize that defective T cell receptor (TCR) signaling and cosignals in the TME limit T cell reactivation. To address this, we designed a mesenchymal stromal cell–based delivery of local membrane-bound anti-CD3 and/or cosignals to explore their contribution to reactivate T cells inside the TME. Combined anti-CD3 and CD40L rather than CD80 led to superior antitumor efficacy compared with either alone. Mechanistically, TCR activation of preexisting CD8+ T cells synergized with CD40L activation of DCs inside the TME for optimum tumor control. Exogenous TCR signals could better reactivate TILs that then exited to attack distal tumors. This study supplies further evidence that TCR signaling for T cell reactivation in the TME is defective but can be rescued by proper exogenous signals.
Authors
Casey Moore, Joonbeom Bae, Longchao Liu, Huiyu Li, Yang-Xin Fu, Jian Qiao
×Abstract
BACKGROUND Systemic sclerosis (SSc) is an autoimmune, connective tissue disease characterized by vasculopathy and fibrosis of the skin and internal organs.METHODS We randomized 15 participants with early diffuse cutaneous SSc to tofacitinib 5 mg twice a day or matching placebo in a phase I/II double-blind, placebo-controlled trial. The primary outcome measure was safety and tolerability at or before week 24. To understand the changes in gene expression associated with tofacitinib treatment in each skin cell population, we compared single-cell gene expression in punch skin biopsies obtained at baseline and 6 weeks following the initiation of treatment.RESULTS Tofacitinib was well tolerated; no participants experienced grade 3 or higher adverse events before or at week 24. Trends in efficacy outcome measures favored tofacitnib. Baseline gene expression in fibroblast and keratinocyte subpopulations indicated IFN-activated gene expression. Tofacitinib inhibited IFN-regulated gene expression in SFRP2/DPP4 fibroblasts (progenitors of myofibroblasts) and in MYOC/CCL19, representing adventitial fibroblasts (P < 0.05), as well as in the basal and keratinized layers of the epidermis. Gene expression in macrophages and DCs indicated inhibition of STAT3 by tofacitinib (P < 0.05). No clinically meaningful inhibition of T cells and endothelial cells in the skin tissue was observed.CONCLUSION These results indicate that mesenchymal and epithelial cells of a target organ in SSc, not the infiltrating lymphocytes, may be the primary focus for therapeutic effects of a Janus kinase inhibitor.TRIAL REGISTRATION ClinicalTrials.gov NCT03274076.FUNDING Pfizer, NIH/National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) R01 AR070470, NIH/NIAMS K24 AR063120, Taubman Medical Research Institute and NIH P30 AR075043, and NIH/NIAMS K01 AR072129.
Authors
Dinesh Khanna, Cristina Padilla, Lam C. Tsoi, Vivek Nagaraja, Puja P. Khanna, Tracy Tabib, J. Michelle Kahlenberg, Amber Young, Suiyuan Huang, Johann E. Gudjonsson, David A. Fox, Robert Lafyatis
×Abstract
Plasmacytoid dendritic cells (pDCs) perform dual proinflammatory and immunosuppressive roles. We recently reported the potential of pDC therapy for treatment of intractable acute liver failure. However, establishment of efficient methods to deliver pDCs to the liver is essential for future clinical therapeutic applications. The present study demonstrates a higher abundance of liver and peripheral blood pDCs in mice lacking C-C motif chemokine receptor 9 (CCR9), a pDC gut-homing receptor, than in WT mice. Adoptive transfer of Ccr9–/– pDCs resulted in a higher efficiency of migration to the liver than WT pDCs did, while WT pDCs migrated efficiently to the original target organ, the small intestine. Further, Ccr9–/– pDCs consistently migrated efficiently to livers with concanavalin A–induced inflammation, and exerted a more effective immunosuppressive effect, resulting in better protection against acute liver inflammation than that demonstrated by WT pDCs. These findings highlight the therapeutic potential of the manipulation of the CCR9 axis as an approach to improve migration of immunosuppressive pDCs to the liver in order to exploit their beneficial effects in acute liver disease.
Authors
Yuzo Koda, Nobuhiro Nakamoto, Po-Sung Chu, Toshiaki Teratani, Akihisa Ueno, Takeru Amiya, Nobuhito Taniki, Sayako Chiba, Kentaro Miyamoto, Michiie Sakamoto, Takanori Kanai
×Abstract
Allogeneic hematopoietic stem cell transplantation (allo-HSCT) efficacy is complicated by graft-versus-host disease (GVHD), a leading cause of morbidity and mortality. Regulatory T cells (Tregs) have shown efficacy in preventing GVHD. However, high Treg doses are often required, necessitating substantial ex vivo or in vivo expansion that may diminish suppressor function. To enhance in vivo suppressor function, murine Tregs were transduced to express an anti–human CD19 chimeric antigen receptor (hCAR19) and infused into lethally irradiated, hCD19-transgenic recipients for allo-HSCT. Compared with recipients receiving control transduced Tregs, those receiving hCAR19 Tregs had a marked decrease in acute GVHD lethality. Recipient hCD19 B cells and murine hCD19 TBL12-luciferase (TBL12luc) lymphoma cells were both cleared by allogeneic hCAR19 Tregs, which was indicative of graft-versus-tumor (GVT) maintenance and potentiation. Mechanistically, hCAR19 Tregs killed syngeneic hCD19+ but not hCD19– murine TBL12luc cells in vitro in a perforin-dependent, granzyme B–independent manner. Importantly, cyclophosphamide-treated, hCD19-transgenic mice given hCAR19 cytotoxic T lymphocytes without allo-HSCT experienced rapid lethality due to systemic toxicity that has been associated with proinflammatory cytokine release; in contrast, hCAR19 Treg suppressor function enabled avoidance of this severe complication. In conclusion, hCAR19 Tregs are a potentially novel and effective strategy to suppress GVHD without loss of GVT responses.
Authors
Sara Bolivar-Wagers, Michael L. Loschi, Sujeong Jin, Govindarajan Thangavelu, Jemma H. Larson, Cameron S. McDonald-Hyman, Ethan G. Aguilar, Asim Saha, Brent H. Koehn, Mehrdad Hefazi, Mark J. Osborn, Michael C. Jensen, John E. Wagner, Christopher A. Pennell, Bruce R. Blazar
×Abstract
In rodent models of type 2 diabetes (T2D), central administration of FGF1 normalizes elevated blood glucose levels in a manner that is sustained for weeks or months. Increased activity of NPY/AgRP neurons in the hypothalamic arcuate nucleus (ARC) is implicated in the pathogenesis of hyperglycemia in these animals, and the ARC is a key brain area for the antidiabetic action of FGF1. We therefore sought to determine whether FGF1 inhibits NPY/AgRP neurons and, if so, whether this inhibitory effect is sufficiently durable to offer a feasible explanation for sustained diabetes remission induced by central administration of FGF1. Here, we show that FGF1 inhibited ARC NPY/AgRP neuron activity, both after intracerebroventricular injection in vivo and when applied ex vivo in a slice preparation; we also showed that the underlying mechanism involved increased input from presynaptic GABAergic neurons. Following central administration, the inhibitory effect of FGF1 on NPY/AgRP neurons was also highly durable, lasting for at least 2 weeks. To our knowledge, no precedent for such a prolonged inhibitory effect exists. Future studies are warranted to determine whether NPY/AgRP neuron inhibition contributes to the sustained antidiabetic action elicited by intracerebroventricular FGF1 injection in rodent models of T2D.
Authors
Eunsang Hwang, Jarrad M. Scarlett, Arian F. Baquero, Camdin M. Bennett, Yanbin Dong, Dominic Chau, Jenny M. Brown, Aaron J. Mercer, Thomas H. Meek, Kevin L. Grove, Bao Anh N. Phan, Gregory J. Morton, Kevin W. Williams, Michael W. Schwartz
×Abstract
The immune factors associated with impaired SARS-CoV-2 vaccine response in elderly people are mostly unknown. We studied individuals older than 60 and younger than 60 years, who had been vaccinated with SARS-CoV-2 BNT162b2 mRNA, before and after the first and second dose. Aging was associated with a lower anti–RBD IgG levels and a decreased magnitude and polyfunctionality of SARS-CoV-2–specific T cell response. The dramatic decrease in thymic function in people > 60 years, which fueled alteration in T cell homeostasis, and their lower CD161+ T cell levels were associated with decreased T cell response 2 months after vaccination. Additionally, deficient DC homing, activation, and TLR-mediated function, along with a proinflammatory functional profile in monocytes, were observed in the > 60-year-old group, which was also related to lower specific T cell response after vaccination. These findings might be relevant for the improvement of the current vaccination strategies and for the development of new vaccine prototypes.
Authors
Joana Vitallé, Alberto Pérez-Gómez, Francisco José Ostos, Carmen Gasca-Capote, María Reyes Jiménez-León, Sara Bachiller, Inmaculada Rivas-Jeremías, Maria del Mar Silva-Sánchez, Anabel M. Ruiz-Mateos, María Ángeles Martín-Sánchez, Luis Fernando López-Cortes, Mohammed Rafii-El-Idrissi Benhnia, Ezequiel Ruiz-Mateos
×Abstract
The complex genomic landscape of prostate cancer evolves across disease states under therapeutic pressure directed toward inhibiting androgen receptor (AR) signaling. While significantly altered genes in prostate cancer have been extensively defined, there have been fewer systematic analyses of how structural variation shapes the genomic landscape of this disease across disease states. We uniformly characterized structural alterations across 531 localized and 143 metastatic prostate cancers profiled by whole genome sequencing, 125 metastatic samples of which were also profiled via whole transcriptome sequencing. We observed distinct significantly recurrent breakpoints in localized and metastatic castration-resistant prostate cancers (mCRPC), with pervasive alterations in noncoding regions flanking the AR, MYC, FOXA1, and LSAMP genes enriched in mCRPC and TMPRSS2-ERG rearrangements enriched in localized prostate cancer. We defined 9 subclasses of mCRPC based on signatures of structural variation, each associated with distinct genetic features and clinical outcomes. Our results comprehensively define patterns of structural variation in prostate cancer and identify clinically actionable subgroups based on whole genome profiling.
Authors
Meng Zhou, Minjeong Ko, Anna C.H. Hoge, Kelsey Luu, Yuzhen Liu, Magdalena L. Russell, William W. Hannon, Zhenwei Zhang, Jian Carrot-Zhang, Rameen Beroukhim, Eliezer M. Van Allen, Atish D. Choudhury, Peter S. Nelson, Matthew L. Freedman, Mary-Ellen Taplin, Matthew Meyerson, Srinivas R. Viswanathan, Gavin Ha
×Abstract
Pneumocystis is the most common fungal pulmonary infection in children under the age of 5 years. In children with primary immunodeficiency, Pneumocystis often presents at 3–6 months of age, a time period that coincides with the nadir of maternal IgG and when IgM is the dominant Ig isotype. Because B cells are the dominant antigen-presenting cells for Pneumocystis, we hypothesized the presence of fungal-specific IgMs in humans and mice and that these IgM specificities would predict T cell antigens. We detected fungal-specific IgMs in human and mouse sera and utilized immunoprecipitation to determine whether any antigens were similar across donors. We then assessed T cell responses to these antigens and found anti-Pneumocystis IgM in WT mice, Aicda–/– mice, and in human cord blood. Immunoprecipitation of Pneumocystis murina with human cord blood identified shared antigens among these donors. Using class II MHC binding prediction, we designed peptides with these antigens and identified robust peptide-specific lung T cell responses after P. murina infection. After mice were immunized with 2 of the antigens, adoptive transfer of vaccine-elicited CD4+ T cells showed effector activity, suggesting that these antigens contain protective Pneumocystis epitopes. These data support the notion that germline-encoded IgM B cell receptors are critical in antigen presentation and T cell priming in early Pneumocystis infection.
Authors
Kristin Noell, Guixiang Dai, Dora Pungan, Anna Ebacher, Janet E. McCombs, Samuel J. Landry, Jay K. Kolls
×Abstract
Fibrolamellar hepatocellular carcinoma (FLC) is a rare and often lethal liver cancer with no proven effective systemic therapy. Inhibition of the antiapoptotic protein BCL-XL was found to synergize with a variety of systemic therapies in vitro using cells dissociated from patient-derived xenografts (PDX) of FLC or cells dissociated directly from surgical patient resections. As BCL-XL is physiologically expressed in platelets, prior efforts to leverage this vulnerability in other cancers have been hampered by severe thrombocytopenia. To overcome this toxicity, we treated FLC models with DT2216, a proteolysis targeting chimera (PROTAC) that directs BCL-XL for degradation via the von Hippel-Lindau (VHL) E3 ligase, which is minimally expressed in platelets. The combination of irinotecan and DT2216 in vitro on cells directly acquired from patients or in vivo using several xenografts derived from patients with FLC demonstrated remarkable synergy and at clinically achievable doses not associated with significant thrombocytopenia.
Authors
Bassem Shebl, Denise Ng, Gadi Lalazar, Carly Rosemore, Tova M. Finkelstein, Rachael D. Migler, Guangrong Zheng, Peiyi Zhang, Caroline S. Jiang, Adam Qureshi, Roger Vaughan, Mark Yarchoan, Ype P. de Jong, Charles M. Rice, Philip Coffino, Michael V. Ortiz, Daohong Zhou, Sanford M. Simon
×Abstract
Heterozygous mutations in FLT3ITD, TET2, and DNMT3A are associated with hematologic malignancies in humans. In patients, cooccurrence of mutations in FLT3ITD combined with TET2 (TF) or FLT3ITD combined with DNMT3A (DF) are frequent. However, in some rare complex acute myeloid leukemia (AML), all 3 mutations cooccur — i.e., FLT3ITD, TET2, and DNMT3A (TFD). Whether the presence of these mutations in combination result in quantitative or qualitative differences in disease manifestation has not been investigated. We generated mice expressing heterozygous Flt3ITD and concomitant for either heterozygous loss of Tet2 (TF) or Dnmt3a (DF) or both (TFD). TF and DF mice did not induce disease early on, in spite of similar changes in gene expression; during the same time frame, an aggressive form of transplantable leukemia was observed in TFD mice, which was mostly associated with quantitative but not qualitative differences in gene expression relative to TF or DF mice. The gene expression signature of TFD mice showed remarkable similarity to the human TFD gene signature at the single-cell RNA level. Importantly, TFD-driven AML responded to a combination of drugs that target Flt3ITD, inflammation, and methylation in a mouse model, as well as in a PDX model of AML bearing 3 mutations.
Authors
Baskar Ramdas, Palam Lakshmi Reddy, Raghuveer Singh Mali, Santhosh Kumar Pasupuleti, Ji Zhang, Mark R. Kelley, Sophie Paczesny, Chi Zhang, Reuben Kapur
