Research
Abstract
Asymmetric cell division (ACD) enables the maintenance of a stem cell population while simultaneously generating differentiated progeny. Cancer stem cells (CSCs) undergo multiple modes of cell division during tumor expansion and in response to therapy, yet the functional consequences of these division modes remain to be determined. Using a fluorescent reporter for cell surface receptor distribution during mitosis, we found that ACD generated a daughter cell with enhanced therapeutic resistance and increased co-enrichment of epidermal growth factor receptor (EGFR) and neurotrophin receptor (p75NTR) from a glioblastoma CSC. Stimulation of both receptors antagonized differentiation induction and promoted self-renewal capacity. p75NTR knockdown enhanced the therapeutic efficacy of EGFR inhibition, indicating that co-inheritance of p75NTR and EGFR promotes resistance to EGFR inhibition through a redundant mechanism. These data demonstrate that ACD produces progeny with co-enriched growth factor receptors, which contributes to the generation of a more therapeutically resistant CSC population.
Authors
Masahiro Hitomi, Anastasia P. Chumakova, Daniel J. Silver, Arnon M. Knudsen, W. Dean Pontius, Stephanie Murphy, Neha S. Anand, Bjarne Winther Kristensen, Justin Lathia
Abstract
Paucity of the Glucose Transporter1 (Glut1) protein resulting from haploinsufficiency of the SLC2A1 gene arrests cerebral angiogenesis and disrupts brain function to cause Glucose Transporter1 Deficiency Syndrome (Glut1 DS). Restoring Glut1 to Glut1 DS model mice prevents disease but the precise cellular sites of action of the transporter, its temporal requirements and the mechanism(s) linking scarcity of the protein to brain cell dysfunction remain poorly understood. Here we show that Glut1 functions in a cell-autonomous manner in the cerebral microvasculature to affect endothelial tip cells and thus brain angiogenesis. Moreover, brain endothelial cell-specific Glut1 depletion not only triggers a severe neuro-inflammatory response in the Glut1 DS brain but also reduces levels of brain-derived neurotrophic factor (BDNF) and causes overt disease. Reduced BDNF correlated with fewer neurons in the Glut1 DS brain. Controlled depletion of the protein demonstrated that brain pathology and disease severity was greatest when Glut1 scarcity was induced neonatally, during brain angiogenesis. Reducing Glut1 at later stages had mild or little effect. Our results suggest that targeting brain endothelial cells during early development is important to ensure proper brain angiogenesis, prevent neuro-inflammation, maintain BDNF levels and preserve neuron numbers. This requirement will be essential for any disease-modifying therapeutic strategy for Glut1 DS.
Authors
Maoxue Tang, Sarah H. Park, Sabrina Petri, Hang Yu, Carlos B. Rueda, E. Dale Abel, Carla Y. Kim, Elizabeth M. C. Hillman, Fanghua Li, Yeojin Lee, Lei Ding, Smitha Jagadish, Wayne N. Frankel, Darryl C. De Vivo, Umrao R. Monani
Abstract
Aberrant activation of NLRP3 inflammasome has been implicated in a variety of human inflammatory diseases, however currently no pharmacological NLRP3 inhibitor has been approved in clinic. In this study, we showed that echinatin, the ingredient of the traditional herbal medicine licorice, effectively suppresses the activation of NLRP3 inflammasome in vitro and in vivo. Further investigation revealed that echinatin exerts its inhibitory effect on NLRP3 inflammasome by binding to heat-shock protein 90 (HSP90), inhibiting its ATPase activity, and disrupting the association between the cochaperone SGT1 and HSP90-NLRP3. Importantly, in vivo experiments demonstrated that administration of echinatin obviously inhibits NLRP3 inflammasome activation and ameliorates LPS-induced septic shock and DSS-induced colitis in mice. Moreover, echinatin exerted favorable pharmacological effects on liver inflammation and fibrosis in mouse model of non-alcoholic steatohepatitis (NASH). Collectively, our study identified echinatin as a novel inhibitor of NLRP3 inflammasome and may be developed as a potentially therapeutic approach for the treatment of NLRP3-driven diseases.
Authors
Guang Xu, Shubin Fu, Xiaoyan Zhan, Zhilei Wang, Ping Zhang, Wei Shi, Nan Qin, Yuanyuan Chen, Chunyu Wang, Ming Niu, Yuming Guo, Jia-bo Wang, Zhaofang Bai, Xiaohe Xiao
Abstract
Tumor antigen-specific CD4 T cells accumulate at tumor sites evoking their involvement in antitumor effector functions in situ. Contrarily to CD8 cytotoxic T-lymphocyte exhaustion, that of CD4 T cells remains poorly appreciated. Here, using phenotypic, transcriptomic and functional approaches, we characterized CD4 T-cell exhaustion in head and neck, cervical and ovarian cancer patients. We identified a CD4 tumor-infiltrating lymphocyte (TIL) population, defined by high PD-1 and CD39 expression, which contained high proportions of cytokine-producing cells, although the quantity of cytokines produced by these cells was low evoking an exhausted state. Terminal exhaustion of CD4 TILs was instated regardless of TIM-3 expression suggesting divergence with CD8 T-cell exhaustion. ScRNA-Seq and further phenotypic analyses uncovered, however, similarities with the CD8 T-cell exhaustion program. In particular, PD-1hiCD39+ CD4 TILs expressed the exhaustion transcription factor TOX and the chemokine CXCL13 and were tumor antigen-specific. In vitro, PD-1 blockade enhanced CD4 TIL activation, as evidenced by increased CD154 expression and cytokine secretion, leading to improved dendritic cell maturation and consequently to higher tumor-specific CD8 T-cell proliferation. Our data identify CD4 TIL exhaustion as a player of responsiveness to immune checkpoint blockade.
Authors
Camille-Charlotte Balança, Anna Salvioni, Clara-Maria Scarlata, Marie Michelas, Carlos Martinez-Gomez, Carlos Gomez-Roca, Victor Sarradin, Marie Tosolini, Carine Valle, Frédéric Pont, Gwénaël Ferron, Laurence Gladieff, Sébastien Vergez, Agnès Dupret-Bories, Eliane Mery, Philippe Rochaix, Jean-Jacques Fournié, Jean-Pierre Delord, Christel Devaud, Alejandra Martinez, Maha Ayyoub
Abstract
CIS43 is a potent neutralizing human monoclonal antibody (mAb) that targets a highly conserved ‘junctional’ epitope in the Plasmodium falciparum (Pf) circumsporozoite protein (PfCSP). Enhancing the durability of CIS43 in vivo will be important for clinical translation. Here, two approaches were used to improve the durability of CIS43 in vivo while maintaining potent neutralization. First, the Fc domain was modified with the “LS” mutations (CIS43LS) to increase CIS43 binding affinity for the neonatal Fc receptor (FcRn). CIS43LS and CIS43 showed comparable in vivo protective efficacy. CIS43LS had nine to thirteen-fold increased binding affinity for human (6.2 nM vs. 54.2 nM) and rhesus (25.1 nM vs. 325.8 nM) FcRn at endosomal pH 6.0 compared to CIS43. Importantly, the half-life of CIS43LS in macaques increased from 22 days to 39 days compared to CIS43. The second approach for sustaining antibody levels of CIS43 in vivo is through adeno-associated virus (AAV) expression. Mice administered once with AAV expressing CIS43 had sustained antibody levels of ~ 300 μg/mL and mediated protection against sequential malaria challenges up to 36 weeks. Based on these data, CIS43LS has advanced to Phase I clinical trials and AAV delivery provides a potential next generation approach.
Authors
Neville Kielau Kisalu, Lais Da Silva Pereira, Keenan J. Ernste, Yevel Flores-Garcia, Azza H. Idris, Mangaiarkarasi Asokan, Marlon Dillon, Scott MacDonald, Wei Shi, Xuejun Chen, Amarendra Pegu, Arne Schön, Fidel Zavala, Alejandro B. Balazs, Joseph R. Francica, Robert A. Seder
Abstract
Subjects with obesity frequently have elevated serum vasopressin levels, noted by the stable analog, copeptin. Vasopressin acts primarily to reabsorb water via urinary concentration. However, fat is also a source of metabolic water, raising the possibility that vasopressin might have a role in fat accumulation. Fructose has also been reported to stimulate vasopressin. Here we tested the hypothesis that fructose induced metabolic syndrome is mediated by vasopressin. Orally administered fructose, glucose or high fructose corn syrup increased vasopressin (copeptin) concentrations and was mediated by fructokinase, an enzyme specific for fructose metabolism. Suppressing vasopressin with hydration both prevented and ameliorated fructose-induced metabolic syndrome. The vasopressin effects were mediated by the Vasopressin 1b receptor, as Vasopressin 1b receptor knockout mice were completely protected while V1a knockout paradoxically showed worse metabolic syndrome. The mechanism is likely mediated in part by de novo expression of V1b in the liver that amplifies fructokinase expression in response to fructose. Thus, our studies document a new role for vasopressin in water conservation via the accumulation of fat as a source of metabolic water. Clinically, it also suggests that increased water intake may be a beneficial way to both prevent or treat metabolic syndrome.
Authors
Ana Andres-Hernando, Thomas J. Jensen, Masanari Kuwabara, David J. Orlicky, Christina Cicerchi, Nanxing Li, Carlos A. Roncal-Jimenez, Gabriela E. Garcia, Takuji Ishimoto, Paul S. Maclean, Petter Bjornstad, Laura Gabriela Sanchez-Lozada, Mehmet Kanbay, Takahiko Nakagawa, Richard Johnson, Miguel Lanaspa
Abstract
The G/T transversion, rs35705950, located approximately 3 kb upstream of the MUC5B start site, is the cardinal risk factor for idiopathic pulmonary fibrosis (IPF). Here, we investigate the function and chromatin structure of this -3 kb region and provide evidence that it functions as a classically defined enhancer subject to epigenetic programming. We use nascent transcript analysis to show that RNA polymerase II loads within 10 bp of the G/T transversion site, definitively establishing enhancer function for the region. By integrating Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) analysis of fresh and cultured human airway epithelial cells with nuclease sensitivity data, we demonstrate that this region is in accessible chromatin that affects the expression of MUC5B. Through applying paired single nucleus RNA- and ATAC-seq to frozen tissue from IPF lungs, we extend these findings directly to disease, with results indicating that epigenetic programming of the -3 kb enhancer in IPF occurs in both MUC5B-expressing and non-expressing lineages. In aggregate, our results indicate that the MUC5B-associated variant, rs35705950, resides within an enhancer that is subject to epigenetic remodeling and contributes to pathologic misexpression in IPF.
Authors
Fabienne Gally, Sarah K. Sasse, Jonathan Kurche, Margaret A. Gruca, Jonathan H. Cardwell, Tsukasa Okamoto, Hong Wei Chu, Xiaomeng Hou, Olivier Poirion, Justin Buchanan, Sebastian Preissl, Bing Ren, Sean P. Colgan, Robin D. Dowell, Ivana V. Yang, David A. Schwartz, Anthony N. Gerber
Abstract
Limited experimental evidence bridges nutrition and cancer immunosurveillance. Here, we show that ketogenic diet (KD) or its principal ketone body, 3-hydroxybutyrate (3HB), most specifically in an intermittent scheduling, induced T cell-dependent tumor growth retardation of aggressive tumor models. In conditions in which anti-PD-1, alone or in combination with anti-CTLA-4, failed to reduce tumor growth in mice receiving a standard diet, KD or oral supplementation of 3HB reestablished therapeutic responses. Supplementation of KD with sucrose (which breaks ketogenesis, abolishing 3HB production) or with a pharmacological antagonist of the 3HB receptor GPR109A abolished the antitumor effects. Mechanistically, 3HB prevented the ICB-linked upregulation of PD-L1 on myeloid cells while favoring the expansion of CXCR3+ T cells. KD induced compositional changes of the gut microbiota with distinct species such as Eisenbergiella massiliensis commonly emerging in mice and humans subjected to carbohydrate low diet interventions and highly correlating with serum concentrations of 3HB. Altogether, these results demonstrate that KD induces a 3HB-mediated antineoplastic effect that relies on T-cell mediated cancer immunosurveillance.
Authors
Gladys Ferrere, Maryam Tidjani Alou, Peng Liu, Anne-Gaëlle Goubet, Marine Fidelle, Oliver Kepp, Sylvère Durand, Valerio Iebba, Aurélie Fluckiger, Romain Daillère, Cassandra Thelemaque, Claudia Grajeda-Iglesias, Carolina Alves Costa Silva, Fanny Aprahamian, Deborah Lefevre, Liwei Zhao, Bernhard Ryffel, Emeline Colomba, Monica Arnedos, Damien Drubay, Conrad Rauber, Didier Raoult, Francesco Asnicar, Tim Spector, Nicola Segata, Lisa Derosa, Guido Kroemer, Laurence Zitvogel.
A roadmap from single-cell transcriptome to patient classification for the immune response to trauma
Abstract
Immune dysfunction is an important factor driving mortality and adverse outcomes after trauma but remains poorly understood, especially at cellular level. To deconvolute trauma-induced immune response, we applied single-cell RNA sequencing to circulating and bone marrow mononuclear cells in injured mice and circulating mononuclear cells in trauma patients. In mice, the greatest changes in gene expression were seen in monocytes across both compartments. After systemic injury, the gene expression pattern of monocytes markedly deviated from steady state with corresponding changes in critical transcription factors (TFs), which can be traced back to myeloid progenitors. These changes were largely recapitulated in human single-cell analysis. We generalized the major changes in human CD14+ monocytes into six signatures, which further defined two trauma patient subtypes (SG1 vs. SG2) identified in the whole blood leukocyte transcriptome in the initial 12h after injury. Compared with SG2, SG1 patients exhibited delayed recovery, more severe organ dysfunction and a higher incidence of infection and non-infectious complications. The two patient subtypes were also recapitulated in burn and sepsis patients, revealing a shared pattern of immune response across critical illness. Our data will be broadly useful to further explore the immune response to inflammatory diseases and critical illness.
Authors
Tianmeng Chen, Matthew J. Delano, Kong Chen, Jason L. Sperry, Rami A. Namas, Ashley J. Lamparello, Meihong Deng, Julia Conroy, Lyle L. Moldawer, Philip A. Efron, Patricia A. Loughran, Christopher W. Seymour, Derek C. Angus, Yoram Vodovotz, Wei Chen, Timothy R. Billiar
Abstract
Management of Gastrointestinal stromal tumor (GIST) has been revolutionized by the identification of activating mutations in KIT and PDGFRA and clinical application of receptor tyrosine kinase (RTK) inhibitors in advanced disease. Stratification of GIST into molecularly defined subsets provides insight into clinical behavior and response to approved targeted therapies. Although these RTK inhibitors are effective in most GIST, resistance remains a significant clinical problem. Development of effective treatment strategies for refractory GIST requires identification of novel targets to provide additional therapeutic options. Global kinome profiling has potential to identify critical signaling networks and reveal protein kinases essential in GIST. Using Multiplexed Inhibitor Beads and Mass Spectrometry, we explored the majority of the kinome in GIST specimens from the three most common molecular subtypes (KIT-mutant, PDGFRA-mutant, Succinate dehydrogenase (SDH)-deficient) to identify novel kinase targets. Kinome profiling with loss-of-function assays identified an important role for G2-M tyrosine kinase, Wee1, in GIST cell survival. In vitro and in vivo studies revealed significant efficacy of MK-1775 (Wee1 inhibitor) in combination with avapritinib in KIT and PDGFRA-mutant GIST cell lines, and notable efficacy of MK-1775 as a monotherapy in the PDGFRA-mutant line. These studies provide strong preclinical justification for the use of MK-1775 in GIST.
Authors
Shuai Ye, Dinara Sharipova, Marya Kozinova, Lillian R. Klug, Jimson W. D'Souza, Martin G. Belinsky, Katherine J. Johnson, Margret B. Einarson, Karthik Devarajan, Yan Zhou, Samuel Litwin, Michael C. Heinrich, Ronald P. DeMatteo, Margaret von Mehren, James S. Duncan, Lori Rink
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