Cell biology
Abstract
Extracellular cold-inducible RNA-binding protein (eCIRP) is a key mediator of severity and mortality in sepsis. We found that stimulation of mouse bone marrow–derived neutrophils (BMDNs) with eCIRP generated a distinct neutrophil subpopulation, characterized by cell surface markers of both antigen-presenting cells and aged neutrophils as well as expression of IL-12, which we named antigen-presenting aged neutrophils (APANs). The frequency of APANs was significantly increased in the blood, spleen, and lungs of WT mice subjected to cecal ligation and puncture–induced sepsis but not in CIRP–/– mice. Patients with sepsis had a significant increase in circulating APAN counts compared with healthy individuals. Compared with non–APAN-transfered mice, APAN-transferred septic mice had increased serum levels of injury and inflammatory markers, exacerbated acute lung injury (ALI), and worsened survival. APANs and CD4+ T cells colocalized in the spleen, suggesting an immune interaction between these cells. APANs cocultured with CD4+ T cells significantly induced the release of IFN-γ via IL-12. BMDNs stimulated with eCIRP and IFN-γ underwent hyper-NETosis. Stimulating human peripheral blood neutrophils with eCIRP also induced APANs, and stimulating human neutrophils with eCIRP and IFN-γ caused hyper-NETosis. Thus, eCIRP released during sepsis induced APANs to aggravate ALI and worsen the survival of septic animals via CD4+ T cell activation, Th1 polarization, and IFN-γ–mediated hyper-NETosis.
Authors
Hui Jin, Monowar Aziz, Atsushi Murao, Molly Kobritz, Andrew J. Shih, Robert P. Adelson, Max Brenner, Ping Wang
Abstract
RAS mutations are among the most prevalent oncogenic drivers in cancers. RAS proteins propagate signals only when associated with cellular membranes as a consequence of lipid modifications that impact their trafficking. Here, we discovered that RAB27B, a RAB family small GTPase, controlled NRAS palmitoylation and trafficking to the plasma membrane, a localization required for activation. Our proteomic studies revealed RAB27B upregulation in CBL- or JAK2-mutated myeloid malignancies, and its expression correlated with poor prognosis in acute myeloid leukemias (AMLs). RAB27B depletion inhibited the growth of CBL-deficient or NRAS-mutant cell lines. Strikingly, Rab27b deficiency in mice abrogated mutant but not WT NRAS–mediated progenitor cell growth, ERK signaling, and NRAS palmitoylation. Further, Rab27b deficiency significantly reduced myelomonocytic leukemia development in vivo. Mechanistically, RAB27B interacted with ZDHHC9, a palmitoyl acyltransferase that modifies NRAS. By regulating palmitoylation, RAB27B controlled c-RAF/MEK/ERK signaling and affected leukemia development. Importantly, RAB27B depletion in primary human AMLs inhibited oncogenic NRAS signaling and leukemic growth. We further revealed a significant correlation between RAB27B expression and sensitivity to MEK inhibitors in AMLs. Thus, our studies presented a link between RAB proteins and fundamental aspects of RAS posttranslational modification and trafficking, highlighting future therapeutic strategies for RAS-driven cancers.
Authors
Jian-Gang Ren, Bowen Xing, Kaosheng Lv, Rachel A. O’Keefe, Mengfang Wu, Ruoxing Wang, Kaylyn M. Bauer, Arevik Ghazaryan, George M. Burslem, Jing Zhang, Ryan M. O’Connell, Vinodh Pillai, Elizabeth O. Hexner, Mark R. Philips, Wei Tong
Abstract
Neutrophil (PMN) mobilization to sites of insult is critical for host defense and requires transendothelial migration (TEM). TEM involves several well-studied sequential adhesive interactions with vascular endothelial cells (ECs); however, what initiates or terminates this process is not well-understood. Here we describe what we believe to be a new mechanism where vessel associated macrophages (VAMs) through localized interactions primed EC responses to form ICAM-1 “hot spots”, to support PMN TEM. Using real-time intravital microscopy (IVM) on lipopolysaccharide (LPS)-inflamed intestines in CX3CR1-EGFP macrophage-reporter mice, complemented by whole-mount tissue imaging and flow cytometry, we found that macrophage vessel association is critical for the initiation of PMN-EC adhesive interactions, PMN TEM and subsequent accumulation in the intestinal mucosa. Anti-colony stimulating factor 1 receptor (CSF1R) antibody-mediated macrophage depletion in the lamina propria and at the vessel wall resulted in elimination of ICAM-1 hot spots impeding PMN-EC interactions and TEM. Mechanistically, the use of human clinical specimens, TNFα knockout macrophage chimeras, TNFα/TNF receptor (TNFR) neutralization and multi-cellular macrophage-EC-PMN cocultures revealed that macrophage-derived TNFα and EC TNFR2 axis mediated this regulatory mechanism and was required for PMN TEM. As such, our findings identified clinically relevant mechanism by which macrophages regulate PMN trafficking in inflamed mucosa.
Authors
Xingsheng Ren, Laura D. Manzanares, Enzo B. Piccolo, Jessica M. Urbanczyk, David P. Sullivan, Lenore K. Yalom, Triet M. Bui, Connor Lantz, Hinda Najem, Parambir S. Dulai, Amy B. Heimberger, Edward B. Thorp, Ronen Sumagin
Abstract
Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the gradual loss of midbrain dopaminergic neurons in association with aggregation of α-synuclein. Oxidative damage has been widely implicated in this disease, though the mechanisms involved remain elusive. Here, we demonstrated that preferential accumulation of peroxidized phospholipids and loss of the antioxidant enzyme glutathione peroxidase 4 (GPX4) were responsible for vulnerability of midbrain dopaminergic neurons and progressive motor dysfunctions in a mouse model of PD. We also established a mechanism wherein iron-induced dopamine oxidation modified GPX4, thereby rendering it amenable to degradation via the ubiquitin-proteasome pathway. In conclusion, this study unraveled what we believe to be a novel pathway for dopaminergic neuron degeneration during PD pathogenesis, driven by dopamine-induced loss of antioxidant GPX4 activity.
Authors
Jie Sun, Xiao-Min Lin, Dan-Hua Lu, Meng Wang, Kun Li, Sheng-Rong Li, Zheng-Qiu Li, Cheng-Jun Zhu, Zhi-Min Zhang, Chang-Yu Yan, Ming-Hai Pan, Hai-Biao Gong, Jing-Cheng Feng, Yun-Feng Cao, Feng Huang, Wan-Yang Sun, Hiroshi Kurihara, Yi-Fang Li, Wen-Jun Duan, Gen-Long Jiao, Li Zhang, Rong-Rong He
Abstract
Targeted therapies such as venetoclax (Bcl-2 inhibitor) have revolutionized the treatment of chronic lymphocytic leukemia (CLL). We previously reported that persister CLL cells in treated patients overexpress multiple anti-apoptotic proteins and display resistance to pro-apoptotic agents. Here, we demonstrated that multidrug resistant CLL cells in vivo exhibit apoptosis restriction at a premitochondrial level due to insufficient activation of the Bax and Bak proteins. Co-immunoprecipitation analyses with selective BH-domain antagonists revealed that the pleotropic pro-apoptotic protein (Bim) is prevented from activating Bax/Bak by “switching” interactions to other upregulated anti-apoptotic proteins (Mcl-1/Bcl-xL/Bcl-2). Hence, treatments that bypass Bax/Bak restriction are required to deplete these resistant cells in patients. Protein Phosphatase 2A (PP2A) contributes to oncogenesis and treatment resistance. We observed that a small molecule activator of PP2A (SMAP) induced cytotoxicity in multiple cancer cell lines and CLL samples, including multidrug resistant leukemia/lymphoma cells. The SMAP (DT-061) activated apoptosis in multidrug resistant CLL cells through induction of mitochondrial permeability transition pores (mPTP), independent of Bax/Bak. DT-061 inhibited the growth of wild type and Bax/Bak double knockout multidrug resistant CLL cells in a xenograft mouse model. Collectively, we discovered multidrug resistant CLL cells in patients, and validated a pharmacologically tractable pathway to deplete this reservoir.
Authors
Kallesh D. Jayappa, Brian Tran, Vicki L. Gordon, Christopher G. Morris, Shekhar Saha, Caroline C. Farrington, Caitlin M. O’Connor, Kaitin P. Zawacki, Krista M. Isaac, Mark Kester, Timothy P. Bender, Michael E. Williams, Craig A. Portell, Michael J. Weber, Goutham Narla
Abstract
How cancer cells evade the therapeutic effects of immune checkpoint blockade is largely unknown. Here, we report that fibrinogen-like protein 1 (FGL1), a newly identified immune checkpoint ligand, was modified by acetylation at Lys 98 in hepatocellular carcinoma (HCC), which targeted it for proteasomal degradation. Sirtuin 2 (SIRT2) deacetylated and stabilized FGL1, thus promoting immune evasion. Notably, the SIRT2 inhibitor 2-Cyano-3-[5-(2,5-dichlorophenyl)-2-furanyl]-N-5-quinolinyl-2-propenamide (AGK2) enhanced acetylation of FGL1 and reduced FGL1 protein levels in vitro. The combination of AGK2 and programmed death ligand 1 (PD-L1) blockade effectively suppressed tumor growth and improved overall survival of mice. Furthermore, aspirin, an old drug, could directly acetylate FGL1 at Lys 98 and promote its degradation in vitro. Aspirin enhanced the immunotherapeutic efficacy, induced tumor regression, and extended the lifespan of tumor-bearing mice. Furthermore, the SIRT2/FGL1 axis was expressed in HCC specimens. Collectively, these findings unveil an acetylation-mediated regulation of FGL1, identify a potential target for HCC immunotherapy, and provide therapeutic strategies for the clinical treatment of HCC.
Authors
Mingen Lin, Jing He, Xinchao Zhang, Xue Sun, Wenjing Dong, Ruonan Zhang, Yanping Xu, Lei Lv
Abstract
Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy that harbors mutations in homologous recombination (HR) repair proteins in 20-25% of cases. Defects in HR impart to tumor cells a specific vulnerability to poly-ADP ribose polymerase inhibitors and platinum-containing chemotherapy. However, not all patients who receive these therapies respond, and many who initially respond ultimately develop resistance. Inactivation of the HR pathway is associated with the overexpression of polymerase theta (Polθ, or POLQ). This key enzyme regulates the microhomology-mediated end-joining (MMEJ) pathway of double-strand break (DSB) repair. Using human and murine HR-deficient PDAC models, we find that POLQ knockdown is synthetically lethal with mutations in HR genes (BRCA1 and BRCA2) and the DNA damage repair gene ATM. Further, POLQ knockdown enhances cytosolic micronuclei formation and activates cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling, leading to enhanced infiltration of activated CD8+ T cells in BRCA2-deficient PDAC tumors in vivo. Overall, POLQ, a key mediator in the MMEJ pathway, is critical for DSB repair in BRCA2-deficient PDAC. Its inhibition represents a synthetic lethal approach to block tumor growth while simultaneously stimulating an immune response.
Authors
Grace Oh, Annie Wang, Lidong Wang, Jiufeng Li, Gregor Werba, Daniel Weissinger, Ende Zhao, Surajit Dhara, Rosmel E. Hernandez, Amanda Ackermann, Sarina Porcella, Despoina Kalfakakou, Igor Dolgalev, Emily A. Kawaler, Talia Golan, Theodore H. Welling, Agnel Sfeir, Diane M. Simeone
Abstract
Neural differentiation, synaptic transmission, and action potential propagation depend on membrane sphingolipids, whose metabolism is tightly regulated. Mutations in the ceramide transporter CERT (CERT1), which is involved in sphingolipid biosynthesis, are associated with intellectual disability, but the pathogenic mechanism remains obscure. Here, we characterize 31 individuals with de novo missense variants in CERT1. Several variants fall into a previously uncharacterized dimeric helical domain that enables CERT homeostatic inactivation, without which sphingolipid production goes unchecked. The clinical severity reflects the degree to which CERT autoregulation is disrupted, and inhibiting CERT pharmacologically corrects morphological and motor abnormalities in a Drosophila model of the disease, which we call CerTra syndrome. These findings uncover a central role for CERT autoregulation in the control of the sphingolipid biosynthetic flux, provide unexpected insight into the structural organisation of CERT, and suggest a possible therapeutic approach for CerTra syndrome patients.
Authors
Charlotte Gehin, Museer A. Lone, Winston Lee, Laura Capolupo, Sylvia Ho, Adekemi M. Adeyemi, Erica H. Gerkes, Alexander P.A. Stegmann, Estrella López-Martín, Eva Bermejo-Sánchez, Beatriz Martínez-Delgado, Christiane Zweier, Cornelia Kraus, Bernt Popp, Vincent Strehlow, Daniel Gräfe, Ina Knerr, Eppie R. Jones, Stefano Zamuner, Luciano A. Abriata, Vidya Kunnathully, Brandon E. Moeller, Anthony Vocat, Samuel Rommelaere, Jean-Philippe Bocquete, Evelyne Ruchti, Greta Limoni, Marine Van Campenhoudt, Samuel Bourgeat, Petra Henklein, Christian Gilissen, Bregje W. van Bon, Rolph Pfundt, Marjolein H. Willemsen, Jolanda H. Schieving, Emanuela Leonardi, Fiorenza Soli, Alessandra Murgia, Hui Guo, Qiumeng Zhang, Kun Xia, Christina R. Fagerberg, Christoph P. Beier, Martin J. Larsen, Irene Valenzuela, Paula Fernández-Álvarez, Shiyi Xiong, Robert Śmigiel, Vanesa López-González, Lluís Armengol, Manuela Morleo, Angelo Selicorni, Annalaura Torella, Moira Blyth, Nicola S. Cooper, Valerie Wilson, Renske Oegema, Yvan Herenger, Aurore Garde, Ange-Line Bruel, Frederic Tran Mau-Them, Alexis B.R. Maddocks, Jennifer M. Bain, Musadiq A. Bhat, Gregory Costain, Peter Kannu, Ashish Marwaha, Neena L. Champaigne, Michael J. Friez, Ellen B. Richardson, Vykuntaraju K. Gowda, Varunvenkat M. Srinivasan, Yask Gupta, Tze Y. Lim, Simone Sanna-Cherchi, Bruno Lemaitre, Toshiyuki Yamaji, Kentaro Hanada, John E. Burke, Ana Marija Jakšić, Brian D. McCabe, Paolo De Los Rios, Thorsten Hornemann, Giovanni D'Angelo, Vincenzo A Gennarino
Abstract
CRISPR-Cas9 has been proposed as a treatment for genetically inherited skin disorders. Here we report that CRISPR transfection activates STING-dependent antiviral responses in keratinocytes, resulting in heightened endogenous interferon (IFN) responses through induction of IFN-κ leading to decreased plasmid stability secondary to induction of the cytidine deaminase APOBEC3G. Notably, CRISPR-generated KO keratinocytes had permanent suppression of IFN-κ and IFN-stimulated gene (ISG) expression, secondary to hypermethylation of the IFNK promoter region by the DNA methyltransferase DNMT3B. JAK inhibition via baricitinib prior to CRISPR transfection increased transfection efficiency, prevented IFNK promoter hypermethylation, and restored normal IFN-κ activity and ISG responses. This work shows that CRISPR-mediated gene correction alters antiviral responses in keratinocytes, has implications for future gene therapies of inherited skin diseases using CRISPR technology, and suggests pharmacologic JAK inhibition as a tool for facilitating and attenuating inadvertent selection effects in CRISPR-Cas9 therapeutic approaches.
Authors
Mrinal K. Sarkar, Ranjitha Uppala, Chang Zeng, Allison C. Billi, Lam C. Tsoi, Austin Kidder, Xianying Xing, Bethany E. Perez White, Shuai Shao, Olesya Plazyo, Sirisha Sirobhushanam, Enze Xing, Yanyun Jiang, Katherine A. Gallagher, John J. Voorhees, J. Michelle Kahlenberg, Johann E. Gudjonsson
Abstract
The origin of breast cancer, whether primary or recurrent, is unknown. Here, we show that invasive breast cancer cells exposed to hypoxia release small extracellular vesicles (sEV) that disrupt the differentiation of normal mammary epithelia, expand stem and luminal progenitor cells, and induce atypical ductal hyperplasia and intraepithelial neoplasia. This was accompanied by systemic immunosuppression with increased myeloid cell release of the “alarmin”, S100A9, and oncogenic traits of EMT, angiogenesis, and local and disseminated luminal cell invasion, in vivo. In the presence of a mammary gland driver oncogene (MMTV-PyMT), hypoxic sEV accelerated bilateral breast cancer onset and progression. Mechanistically, genetic or pharmacologic targeting of hypoxia-inducible factor-1α (HIF1α) packaged in hypoxic sEV, or homozygous deletion of S100A9 normalized mammary gland differentiation, restored T cell function and prevented atypical hyperplasia. The transcriptome of sEV-induced mammary gland lesions resembled luminal breast cancer, and detection of HIF1α in plasma circulating sEV from luminal breast cancer patients correlated with disease recurrence. Therefore, sEV-HIF1α signaling drives both local and systemic mechanisms of mammary gland transformation at high risk for evolution to multifocal breast cancer. This pathway may provide a readily accessible biomarker of luminal breast cancer progression.
Authors
Irene Bertolini, Michela Perego, Yulia Nefedova, Cindy Lin, Andrew T. Milcarek, Peter Vogel, Jagadish C. Ghosh, Andrew V. Kossenkov, Dario C. Altieri
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ISSN: 0021-9738 (print), 1558-8238 (online)