Mutant KRAS drives glycolytic flux in lung cancer, potentially impacting aberrant protein glycosylation. Recent evidence suggests aberrant KRAS drives flux of glucose into the hexosamine biosynthetic pathway (HBP). HBP is required for various glycosylation processes, such as protein N- or O-glycosylation and glycolipid synthesis. However, its function during tumorigenesis is poorly understood. One contributor and proposed target of KRAS driven cancers is a developmentally conserved epithelial plasticity program called epithelial-mesenchymal transition (EMT). Here we show in novel autochthonous mouse models that EMT accelerates KrasG12D lung tumorigenesis by upregulating expression of key enzymes of the HBP pathway. We demonstrate that HBP is required for suppressing KrasG12D-induced senescence, and targeting HBP significantly delays KrasG12D lung tumorigenesis. To explore the mechanism, we investigated protein glycosylation downstream of HBP and found elevated levels of O-linked β-N-acetylglucosamine (O-GlcNAcylation) post-translational modification on intracellular proteins. O-GlcNAcylation suppressed KrasG12D oncogene-induced senescence (OIS) and accelerates lung tumorigenesis. Conversely, loss of O-GlcNAcylation delays lung tumorigenesis. O-GlcNAcylation of proteins SNAI1 and c-Myc correlates with the EMT-HBP axis and accelerated lung tumorigenesis. Our results demonstrate that O-GlcNAcylation is sufficient and required to accelerate KrasG12D lung tumorigenesis in vivo, which is reinforced by epithelial plasticity programs.
Kekoa Taparra, Hailun Wang, Reem Malek, Audrey Lafargue, Mustafa A. Barbhuiya, Xing Wang, Brian W. Simons, Matthew Ballew, Katriana Nugent, Jennifer Groves, Russell D. Williams, Takumi Shiraishi, James Verdone, Gokben Yildirir, Roger Henry, Bin Zhang, John Wong, Ken Kang-Hsin Wang, Barry D. Nelkin, Kenneth J. Pienta, Dean Felsher, Natasha E. Zachara, Phuoc T. Tran
First generation immune checkpoint inhibitors including anti-CTLA-4 and anti-PD-1 antibodies have led to major clinical progress, yet resistance frequently leads to treatment failure. Thus, new targets acting on T cells are needed. CD33-related Siglecs are pattern recognition immune receptors binding to a range of sialoglycan ligands, which appear to function as self-associated molecular patterns (SAMPs) that suppress autoimmune responses. Siglecs are expressed at very low levels on normal T cells, and these receptors were not yet considered as interesting targets on T cells for cancer immunotherapy. Here, we show an upregulation of Siglecs including Siglec-9 on tumor-infiltrating T cells from non-small cell lung (NSCLC), colorectal and ovarian cancer patients. Siglec-9 expressing T cells co-expressed several inhibitory receptors including PD-1. Targeting of the sialoglycan-SAMP/Siglec pathway in vitro and in vivo resulted in increased anti-cancer immunity. T cell expression of Siglec-9 in NSCLC patients correlated with a reduced survival, and Siglec-9 polymorphisms showed associations with the risk of developing lung and colorectal cancer. Our data identify the sialoglycan-SAMP/Siglec pathway as new potential target to improve T cell activation for immunotherapy.
Michal A. Stanczak, Shoib S. Siddiqui, Marcel P. Trefny, Daniela S. Thommen, Kayluz Frias Boligan, Stephan von Gunten, Alexandar Tzankov, Lothar Tietze, Didier Lardinois, Viola Heinzelmann-Schwarz, Michael S. von Bergwelt-Baildon, Wu Zhang, Heinz-Josef Lenz, Younghan Han, Christopher I. Amos, Mohammedyaseen Syedbasha, Adrian Egli, Frank Stenner, Daniel E. Speiser, Ajit Varki, Alfred Zippelius, Heinz Läubli
While T cells are important for the pathogenesis of systemic lupus erythematosus (SLE) and lupus nephritis, little is known about how T cells function after infiltrating the kidney. The current paradigm suggests that kidney infiltrating T cells (KITs) are activated effector cells contributing to tissue damage and ultimately organ failure. Herein, we demonstrate that the majority of CD4+ and CD8+ KITs in three murine lupus models are not effector cells, as hypothesized, but rather expressed multiple inhibitory receptors and proved highly dysfunctional with reduced cytokine production and proliferative capacity. Mechanistically this was linked directly to metabolic and specifically mitochondrial dysfunction. This was driven by the expression of an “exhausted” transcriptional signature. Our data thus reveal that the tissue parenchyma has the capability to suppress T cell responses and limit damage to self. These findings open novel avenues for the treatment of autoimmunity based on selectively exploiting the exhausted phenotype of tissue-infiltrating T cells.
Jeremy S. Tilstra, Lyndsay Avery, Ashley V. Menk, Rachael A. Gordon, Shuchi Smita, Lawrence P. Kane, Maria Chikina, Greg M. Delgoffe, Mark J. Shlomchik
Regulatory T-cells (Treg) are critical for maintaining immune homeostasis. However, current Treg immunotherapies do not optimally treat inflammatory diseases in patients. Understanding the cellular processes that control Treg function may allow for the augmentation of therapeutic efficacy. In contrast to activated conventional T-cells, where protein kinase C-θ (PKC-θ) localizes to the contact-point between T-cells and antigen-presenting cells, in human and mouse Treg, PKC-θ localizes to the opposite end of the cell in the distal pole complex (DPC). Here, using a phosphoproteomic screen, we identified the intermediate filament vimentin as a PKC-θ phospho-target and show that vimentin forms a DPC superstructure on which PKC-θ accumulates. Treatment of mouse Treg with either a clinically relevant PKC-θ inhibitor or vimentin siRNA disrupted vimentin and enhanced Treg metabolic and suppressive activity. Moreover, vimentin-disrupted mouse Treg were significantly better than controls in suppressing alloreactive T-cell priming in graft-versus-host disease, and graft-versus-host disease lethality, using a complete MHC mismatch mouse model of acute graft-versus-host disease (C57BL/6 donor in to BALB/c host). Interestingly, vimentin disruption augmented suppressor function of PKC-θ-deficient mouse Treg. This suggests that enhanced Treg activity after PKC-θ inhibition is secondary to effects on vimentin, not just PKC-θ kinase activity inhibition. Our data demonstrated that vimentin is a key metabolic and functional controller of Treg activity, and provide proof-of-principle that disrupting vimentin is a feasible, translationally relevant method to enhance Treg potency.
Cameron McDonald-Hyman, James T. Muller, Michael Loschi, Govindarajan Thangavelu, Asim Saha, Sudha Kumari, Dawn K. Reichenbach, Michelle J. Smith, Guoan Zhang, Brent H. Koehn, Jiqiang Lin, Jason S. Mitchell, Brian T. Fife, Angela Panoskaltsis-Mortari, Colby J. Feser, Andrew Kemal Kirchmeier, Mark J. Osborn, Keli L. Hippen, Ameeta Kelekar, Jonathan S. Serody, Laurence A. Turka, David H. Munn, Hongbo Chi, Thomas A. Neubert, Michael L. Dustin, Bruce R. Blazar
Graft-versus-host disease (GVHD) in the gastrointestinal (GI) tract remains the major cause of morbidity and non-relapse mortality after bone marrow transplantation (BMT). The Paneth cell protein, regenerating islet-derived 3-alpha (REG3α), is a biomarker specific for GI GVHD. REG3α serum levels rose in the systematic circulation as GVHD progressively destroyed Paneth cells and reduced GI epithelial barrier function. Paradoxically, GVHD suppressed intestinal REG3γ (the mouse homologue of human REG3α), and the absence of REG3γ in BMT recipients intensified GVHD but did not change the composition of the microbiome. IL-22 administration restored REG3γ production and prevented apoptosis of both intestinal stem cells (ISCs) and Paneth cells, but this protection was completely abrogated in Reg3g−/− mice. In vitro, addition of REG3α reduced the apoptosis of colonic cell lines. Strategies that increase intestinal REG3α/γ to promote crypt regeneration may offer a novel, non-immunosuppressive approach for GVHD and perhaps for other diseases involving the ISC niche such as inflammatory bowel disease.
Dongchang Zhao, Yeung-Hyen Kim, Seihwan Jeong, Joel K. Greenson, Mohammed S. Chaudhry, Matthias Hoepting, Erik R. Anderson, Marcel R.M. van den Brink, Jonathan U. Peled, Antonio L.C. Gomes, Ann E. Slingerland, Michael J. Donovan, Andrew C. Harris, John E. Levine, Umut Özbek, Lora V. Hooper, Thaddeus S. Stappenbeck, Aaron M. Ver Heul, Ta-Chiang Liu, Pavan Reddy, James L.M. Ferrara
Previous findings showed that in mice, complete knockout of activity-dependent neuroprotective protein (ADNP) abolishes brain formation, while haploinsufficiency (Adnp+/–) causes cognitive impairments. We hypothesized that mutations in ADNP lead to a developmental/autistic syndrome in children. Indeed, recent phenotypic characterization of children harboring ADNP mutations (ADNP syndrome children) revealed global developmental delays and intellectual disabilities, including speech and motor dysfunctions. Mechanistically, ADNP includes a SIP motif embedded in the ADNP-derived snippet, drug candidate NAP (NAPVSIPQ also known as CP201), which binds to microtubule end binding protein 3, essential for dendritic spine formation. Here, we established a unique neuronal membrane tagged green fluorescent protein expressing Adnp+/– mouse line allowing in vivo synaptic pathology quantification. We discovered that Adnp deficiency reduced dendritic spine density and altered synaptic gene expression, both of which were partly ameliorated by NAP treatment. Adnp+/– mice further exhibited global developmental delays, vocalization impediments, gait/motor dysfunctions and social/object memory impairments, all partially reversed by daily NAP administration (systemic/nasal). In conclusion, we now connected ADNP-related synaptic pathology to developmental/behavioral outcomes, establishing NAP in vivo target engagement and identifying potential biomarkers. Together, these studies pave the path toward clinical development of NAP (CP201) in the ADNP syndrome.
Gal Hacohen-Kleiman, Shlomo Sragovich, Gidon Karmon, Andy Y. L. Gao, Iris Grigg, Metsada Pasmanik-Chor, Albert Le, Vlasta Korenková, R. Anne McKinney, Illana Gozes
B cells are increasingly recognised to play an important role in the ongoing control of hepatitis B virus (HBV). The development of antibodies against the viral surface antigen (HBsAg) constitutes the hallmark of resolution of acute infection and is a therapeutic goal for functional cure of chronic HBV (CHB). We characterised B cells directly ex vivo from the blood and liver of patients with CHB to investigate constraints on their antiviral potential. Unexpectedly, we found that HBsAg-specific B cells persisted in the blood and liver of many patients with CHB and were enriched for T-bet, a signature of antiviral potential in B cells. However purified, differentiated HBsAg-specific B cells from patients with CHB had defective antibody production, consistent with undetectable anti-HBs antibodies in vivo. HBsAg-specific and global B cells had an accumulation of CD21–CD27– atypical memory B cells (atMBC) with high expression of inhibitory receptors including PD-1. These atMBC demonstrated altered signalling, homing, differentiation into antibody-producing cells, survival and antiviral/pro-inflammatory cytokine production, that could be partially rescued by PD-1 blockade. Analysis of B cells within healthy and HBV-infected livers implicated the combination of this tolerogenic niche and HBV infection in driving PD-1hiatMBC and impairing B cell immunity.
Alice R. Burton, Laura J. Pallett, Laura E. McCoy, Kornelija Suveizdyte, Oliver E. Amin, Leo Swadling, Elena Alberts, Brian R. Davidson, Patrick T.F. Kennedy, Upkar S. Gill, Claudia Mauri, Paul A. Blair, Nadege Pelletier, Mala K. Maini
Chronic HBV (CHB) infection suppresses virus-specific T cells, but its impact on humoral immunity has been poorly analyzed. Here, we developed a dual staining method, which utilizes HBsAg labelled with fluorochromes as “baits”, for specific ex vivo detection of HBsAg-specific B cells and analysis of their quantity, function and phenotype. We studied healthy vaccinated subjects (n=18) and patients with resolved (n=21), acute (n=11) or chronic (n=96) HBV infection and observed that frequencies of circulating HBsAg-specific B cells are independent of the HBV infection status. In contrast, serum HBsAg presence affects function and phenotype of HBsAg-specific B cells that were unable to mature in vitro into antibody-secreting cells and displayed an increased expression of markers linked to hyperactivation (CD21low) and exhaustion (PD-1). Importantly, B cell alterations were not limited to HBsAg-specific B cells but affected the global B cell population. HBsAg-specific B cell maturation could be partially restored by a method involving the combination of IL-2, IL-21 and CD40L-expressing feeder cells, and further boosted by addition of anti-PD-1 antibodies.In conclusion, HBV infection has a marked impact on global and HBV-specific humoral immunity, yet HBsAg-specific B cells are amenable to a partial rescue by B cell maturing cytokines and PD-1 blockade.
Loghman Salimzadeh, Nina Le Bert, Charles-A. Dutertre, Upkar S. Gill, Evan W. Newell, Christian Frey, Magdeleine Hung, Nikolai Novikov, Simon Fletcher, Patrick T.F. Kennedy, Antonio Bertoletti
Zika virus (ZIKV) is a teratogenic mosquito-borne flavivirus which can be sexually transmitted from man to woman. High viral loads and prolonged viral shedding in semen suggest that ZIKV replicates within the human male genital tract, but its target organs are unknown. Using ex vivo infection of organotypic cultures, we demonstrated here that ZIKV replicates in human testicular tissue and infects a broad range of cell types, including germ cells, which we also identified as infected in the semen from ZIKV-infected donors. ZIKV had no major deleterious effect on the morphology and hormonal production of the human testis explants. Infection induced a broad antiviral response but no interferon up-regulation and minimal pro-inflammatory response in testis explants, with no cytopathic effect. Finally, we studied ZIKV infection in mouse testis, and compared it to human infection. This study provides key insights into how ZIKV may persist in semen and alter semen parameters, as well as a valuable tool for testing antiviral agents.
Giulia Matusali, Laurent Houzet, Anne-Pascale Satie, Dominique Mahé, Florence Aubry, Thérèse Couderc, Julie Frouard, Salomé Bourgeau, Karim Bensalah, Sylvain Lavoué, Guillaume Joguet, Louis Bujan, André Cabié, Gleide F. Avelar, Marc Lecuit, Anna Le Tortorec, Nathalie Dejucq-Rainsford
Prostate cancer is an androgen-dependent disease subject to interactions between the tumor epithelia and its microenvironment. Here, we found epigenetic changes in cancer-associated prostatic fibroblasts (CAF) initiated a cascade of stromal-epithelial interactions. This facilitated lethal prostate cancer growth and development of resistance to androgen signaling deprivation therapy (ADT). We identified that a Ras inhibitor, RASAL3, is epigenetically silenced in human prostatic CAF, leading to oncogenic Ras activity driving macropinocytosis-mediated glutamine synthesis. Interestingly, ADT further promoted RASAL3 epigenetic silencing and glutamine secretion by prostatic fibroblasts. In a orthotopic xenograft model, subsequent inhibition of macropinocytosis and glutamine transport resulted in antitumor effects. Stromal glutamine served as a source of energy through anaplerosis and as a mediator of neuroendocrine differentiation for prostate adenocarcinoma. Antagonizing the uptake of glutamine restored sensitivity to ADT in a castrate resistant xenograft model. In validating these findings, we found that prostate cancer patients on ADT with therapeutic resistance had elevated blood glutamine levels compared to those with therapeutically responsive disease (odds ratio = 7.451, P = 0.02). Identification of epigenetic regulation of RAS activity in prostatic CAF revealed RASAL3 as a sensor for metabolic and neuroendocrine reprogramming in prostate cancer patients failing ADT.
Rajeev Mishra, Subhash Haldar, Veronica Placencio, Anisha Madhav, Krizia Rohena-Rivera, Priyanka Agarwal, Frank Duong, Bryan Angara, Manisha Tripathi, Zhenqiu Liu, Roberta A. Gottlieb, Shawn Wagner, Edwin M. Posadas, Neil A. Bhowmick
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