The MALT1 paracaspase plays an essential role in Activated B-cell like Diffuse Large B cell Lymphoma (ABC DLBCL) downstream of B cell and Toll-like receptor pathway genes mutated in these tumors. Although MALT1 is considered to be a compelling therapeutic target, development of tractable and specific MALT1 protease inhibitors has thus far been elusive. Herein, we developed a target engagement assay that provides a quantitative readout for specific MALT1 inhibitory effects in living cells. This enabled a structure-guided medicinal chemistry effort culminating in the discovery of pharmacologically tractable irreversible substrate-mimetic compounds that bind the MALT1 active site. We confirmed MALT1 targeting with compound #3 is effective at suppressing ABC DLBCL cells in vitro and in vivo. We show that reduction in serum IL10 levels exquisitely correlates with drug PK and degree of MALT1 inhibition in vitro and in vivo and could constitute a useful pharmacodynamic biomarker to evaluate these compounds in clinical trials. Compound #3 revealed insights into the biology of MALT1 in ABC DLBCL, such as driving JAK-STAT signaling and suppressing type I interferon (IFN) response and MHC class II expression, suggesting that MALT1 inhibition could prime lymphomas for immune recognition by cytotoxic immune cells.
Lorena Fontán, Qi Qiao, John M. Hatcher, Gabriella Casalena, Ilkay Us, Matt Teater, Matthew Durant, Guangyan Du, Min Xia, Natalia Bilchuk, Spandan Chennamadhavuni, Giuseppe Palladino, Giorgio Inghirami, Ulrike Philippar, Hao Wu, David A. Scott, Nathanael S. Gray, Ari Melnick
HIV post-treatment controllers (PTCs) represent a natural model of sustained HIV remission, but they are rare and little is known about their viral reservoir. We obtained 1450 proviral sequences after near-full-length amplification for 10 PTCs and 16 post-treatment non-controllers (NCs). Before treatment interruption, the median intact and total reservoir size in PTCs was 7-fold lower than in NCs, but the proportion of intact, defective and total clonally-expanded viral genomes was not significantly different between the two groups. Quantification of total, but not intact, proviral genome copies predicted sustained HIV remission as 81% of NCs, but none of the PTCs, had a total proviral genome >4 copies per million PBMCs. The results highlight the restricted intact and defective HIV reservoir in PTCs and suggest that total proviral genome burden could act as the first biomarker for identifying PTCs. Defective, but not intact, proviral copy numbers correlated with levels of cell-associated HIV RNA, activated NK cell percentages and both HIV-specific CD4+ and CD8+ responses. These results support the concept that defective HIV genomes lead to viral antigen production and interact with both the innate and adaptive immune systems.
Radwa Sharaf, Guinevere Q. Lee, Xiaoming Sun, Behzad Etemad, Layla M. Aboukhater, Zixin Hu, Zabrina L. Brumme, Evgenia Aga, Ronald J. Bosch, Ying Wen, Golnaz Namazi, Ce Gao, Edward P. Acosta, Rajesh T. Gandhi, Jeffrey M. Jacobson, Daniel Skiest, David M. Margolis, Ronald Mitsuyasu, Paul Volberding, Elizabeth Connick, Daniel R. Kuritzkes, Michael M. Lederman, Xu G. Yu, Mathias Lichterfeld, Jonathan Z. Li
Nucleophosmin (NPM1) is amongst the most frequently mutated genes in acute myeloid leukemia (AML). It is not known, however, how the resulting oncoprotein mutant-NPM1 is leukemogenic. To reveal the cellular machinery in which NPM1 participates in myeloid cells, we analyzed the endogenous NPM1 protein-interactome by mass-spectrometry, and discovered abundant amounts of the master transcription factor driver of monocyte lineage-differentiation PU.1 (SPI1). Mutant-NPM1, which aberrantly accumulates in cytoplasm, dislocated PU.1 into cytoplasm with it. CEBPA and RUNX1, the master transcription factors that collaborate with PU.1 to activate granulo-monocytic lineage-fates, remained nuclear, but without PU.1, their coregulator interactions were toggled from coactivators to corepressors, repressing instead of activating greater than 500 granulocyte and monocyte terminal-differentiation genes. An inhibitor of nuclear export, selinexor, by locking mutant-NPM1/PU.1 in the nucleus, activated terminal monocytic fates. Direct depletion of the corepressor DNA methyltransferase 1 (DNMT1) from the CEBPA/RUNX1 protein interactome using the clinical drug decitabine activated terminal granulocytic fates. Together, these non-cytotoxic treatments extended survival by greater than 160 days versus vehicle in a patient-derived xenotransplant model of NPM1/FLT3-mutated AML. In sum, mutant-NPM1 represses monocyte and granulocyte terminal-differentiation by disrupting PU.1/CEBPA/RUNX1 collaboration, a transforming action that can be reversed by pharmacodynamically-directed dosing of clinical small molecules.
Xiaorong Gu, Quteba Ebrahem, Reda Z. Mahfouz, Metis Hasipek, Francis Enane, Tomas Radivoyevitch, Nicolas Rapin, Bartlomiej Przychodzen, Zhenbo Hu, Ramesh Balusu, Claudiu V. Cotta, David Wald, Christian Argueta, Yosef Landesman, Maria Paola Martelli, Brunangelo Falini, Hetty Carraway, Bo T. Porse, Jaroslaw P. Maciejewski, Babal K. Jha, Yogen Saunthararajah
Germinal centers (GCs) are major sites of clonal B cell expansion and generation of long-lived, high-affinity antibody responses to pathogens. Signaling through toll-like receptors(TLRs) on B cells promotes many aspects of GC B cell responses, including affinity-maturation, class-switching and differentiation into long-lived memory and plasma cells. A major challenge for effective vaccination is identifying strategies to specifically promote GC B cell responses. Here we have identified a mechanism of regulation of GC B cell TLR signaling, mediated by αv integrins and non-canonical autophagy. Using B cell-specific αv-knockout mice, we show that loss of αv-mediated TLR regulation increased GC B cell expansion, somatic-hypermutation, class-switching, and generation of long-lived plasma cells after immunization with virus-like particles(VLPs) or antigens associated with TLR ligand adjuvants. Furthermore, targeting αv-mediated regulation increased the magnitude and breadth of antibody responses to influenza virus vaccination. These data therefore identify a mechanism of regulation of GC B cells, which can be targeted to enhance antibody responses to vaccination.
Fiona Raso, Sara Sagadiev, Samuel Du, Emily Gage, Tanvi Arkatkar, Genita Metzler, Lynda M. Stuart, Mark T. Orr, David Rawlings, Shaun Jackson, Adam Lacy-Hulbert, Mridu Acharya
Genome-wide association studies have repeatedly mapped susceptibility loci for emphysema to genes that modify hedgehog signaling, but the functional relevance of hedgehog signaling to this morbid disease remains unclear. In the current study, we identified a broad population of mesenchymal cells in the adult murine lung receptive to hedgehog signaling, characterized by higher activation of hedgehog surrounding the proximal airway relative to the distal alveoli. Single cell RNA-sequencing showed that the hedgehog-receptive mesenchyme is composed of mostly fibroblasts with distinct proximal and distal subsets with discrete identities. Ectopic hedgehog activation in the distal fibroblasts promoted expression of proximal fibroblast markers, and promoted loss of distal alveoli and airspace enlargement of over twenty percent compared to controls. We found that hedgehog suppressed mesenchymal-derived mitogens enriched in distal fibroblasts that regulate alveolar stem cell regeneration and airspace size. Finally, single cell analysis of the human lung mesenchyme showed that segregated proximal-distal identity with preferential hedgehog activation in the proximal fibroblasts is conserved between mice and humans. In conclusion, we showed that differential hedgehog activation segregates mesenchymal identities of distinct fibroblast subsets, and disruption of fibroblast identity can alter the alveolar stem cell niche leading to emphysematous changes in the murine lung.
Chaoqun Wang, Nabora S. Reyes de Mochel, Stephanie A. Christenson, Monica Cassandras, Rebecca Moon, Alexis N. Brumwell, Lauren E. Byrnes, Alfred Li, Yasuyuki Yokosaki, Peiying Shan, Julie B. Sneddon, David Jablons, Patty J. Lee, Michael A. Matthay, Harold A. Chapman, Tien Peng
Cyclin D1 is an oncogene frequently overexpressed in human cancers that plays a dual function as cell cycle and transcriptional regulator, although the latter is widely unexplored. Here, we investigated the transcriptional role of cyclin D1 in lymphoid tumor cells with cyclin D1 oncogenic overexpression. Cyclin D1 showed widespread binding to the promoters of most actively transcribed genes and the promoter occupancy positively correlated with the transcriptional output of targeted genes. Despite this association, the overexpression of cyclin D1 in lymphoid cells led to a global transcriptional downmodulation that was proportional to cyclin D1 levels. This cyclin D1 dependent global transcriptional downregulation was associated with a reduced nascent transcription and an accumulation of promoter-proximal paused RNA Polymerase II (Pol II) that colocalized with cyclin D1. Concordantly, cyclin D1 overexpresion promoted an increment of the Poll II pausing index. This transcriptional impairment seems to be mediated by the interaction of cyclin D1 with the transcription machinery. In addition, cyclin D1 overexpression sensitized cells to transcription inhibitors revealing a synthetic lethality interaction that it was also observed in primary MCL cases. This global transcriptional dysregulation expands the oncogenic cyclin D1 functions and places the transcriptional machinery as a potential therapeutic target in cyclin D1 overexpressing tumors.
Robert Albero, Anna Enjuanes, Santiago Demajo, Giancarlo Castellano, Magda Pinyol, Noelia García, Cristina Capdevila, Guillem Clot, Helena Suárez-Cisneros, Mariko Shimada, Kennosuke Karube, Mónica López-Guerra, Dolors Colomer, Sílvia Beà, José Ignacio Martin-Subero, Elías Campo, Pedro Jares
Induction of TLR2 activation depends on its association with adapter protein MyD88. We have found that levels of TLR2 and MyD88 are elevated in the hippocampus and cortex of Alzheimer’s disease (AD) patients and 5XFAD mouse model of AD. Since there is no specific inhibitor of TLR2, to target induced TLR2 from therapeutic angle, we engineered a peptide corresponding to the TLR2-interacting domain of MyD88 (TIDM) that binds to the BB loop of only TLR2, but not other TLRs. Interestingly, wild type (wt) TIDM peptide inhibited microglial activation induced by fibrillar Aβ1-42 and lipoteichoic acid, but not 1-methyl-4-phenylpyridinium, double-stranded RNA, bacterial lipopolysaccharide, flagellin, and CpG DNA. After intranasal administration, wtTIDM peptide reached the hippocampus, reduced hippocampal glial activation, lowered Aβ burden, attenuated neuronal apoptosis, and improved memory and learning in 5XFAD mice. However, wtTIDM peptide was not effective in 5XFAD mice lacking TLR2. In addition to 5XFAD mice, wtTIDM peptide also suppressed the disease process in mice with experimental allergic encephalomyelitis and collagen-induced arthritis. Therefore, selective targeting of activated status of one component of the innate immune system by wtTIDM peptide may be beneficial in AD as well as other disorders in which TLR2-MyD88 signaling plays a role in disease pathogenesis.
Suresh B. Rangasamy, Malabendu Jana, Avik Roy, Grant T. Corbett, Madhuchhanda Kundu, Sujyoti Chandra, Susanta Mondal, Sridevi Dasarathi, Elliott J. Mufson, Rama K. Mishra, Chi-Hao Luan, David A. Bennett, Kalipada Pahan
Anaplastic thyroid carcinomas (ATC) have a high prevalence of BRAF and TP53 mutations. A trial of vemurafenib in non-melanoma BRAFV600E-mutant cancers showed significant, although short-lived, responses in ATCs, indicating that these virulent tumors remain addicted to BRAF despite their high mutation burden. To explore the mechanisms mediating acquired resistance to BRAF blockade we generated mice with thyroid-specific deletion of p53 and dox-dependent expression of BRAFV600E, 50% of which developed ATCs after dox treatment. Upon dox withdrawal there was complete regression in all mice, although recurrences were later detected in 85% of animals. The relapsed tumors had elevated MAPK transcriptional output, and retained responses to the MEK/RAF inhibitor CH5126766 in vivo and in vitro. Whole exome sequencing identified recurrent focal amplifications of chromosome 6, with a minimal region of overlap that included Met. Met-amplified recurrences overexpressed the receptor as well as its ligand Hgf. Growth, signaling and viability of Met-amplified tumor cells were suppressed in vitro and in vivo by the Met kinase inhibitors PF-04217903 and crizotinib, whereas primary ATCs and Met-diploid relapses were resistant. Hence, recurrences are the rule after BRAF suppression in murine ATCs, most commonly due to activation of HGF/MET signaling, which generates exquisite dependency to MET kinase inhibitors.
Jeffrey A. Knauf, Kathleen A. Luckett, Kuen-Yuan Chen, Francesca Voza, Nicholas D. Socci, Ronald Ghossein, James A. Fagin
Bi-allelic loss-of-function mutations of the NCF4 gene, encoding the p40phox subunit of the phagocyte NADPH oxidase, have been described in only one patient. We report 24 p40phox-deficient patients from 12 additional families in eight countries. These patients display eight different in-frame or out-of-frame mutations of NCF4, homozygous in 11 families and compound heterozygous in another. When overexpressed in NB4 neutrophil-like cells and EBV-transformed B cells in vitro, the mutant alleles were found to be loss-of-function, with the exception of the p.R58C and c.120_134del alleles, which were hypomorphic. Particle-induced NADPH oxidase activity was subnormal in the patients’ neutrophils, whereas PMA-induced DHR oxidation, which is widely used as a diagnostic test for CGD, was normal in some of the patients. Moreover, the NADPH oxidase activity of EBV-transformed B cells was also subnormal, whereas that of mononuclear phagocytes was normal. Finally, the killing of Candida albicans and Aspergillus fumigatus hyphae by neutrophils was conserved in these patients. The patients described here suffer from hyperinflammation and peripheral infections, but they do not display any of the invasive bacterial and fungal infections seen in CGD. In conclusion, inherited p40phox deficiency underlies a distinctive condition, resembling a mild, atypical form of CGD
Annemarie van de Geer, Alejandro Nieto-Patlán, Douglas B. Kuhns, Anton T.J. Tool, Andrés A. Arias, Matthieu Bouaziz, Martin de Boer, José Luis Franco, Roel P. Gazendam, John L. van Hamme, Michel van Houdt, Karin van Leeuwen, Paul J.H. Verkuijlen, Timo K. van den Berg, Juan F. Alzate, Carlos A. Arango-Franco, Vritika Batura, Andrea R. Bernasconi, Barbara Boardman, Claire Booth, Siobhan O. Burns, Felipe Cabarcas, Nadine Cerf Bensussan, Fabienne Charbit-Henrion, Anniek Corveleyn, Caroline Deswarte, María Esnaola Azcoiti, Dirk Foell, John I. Gallin, Carlos Garcés, Margarida Guedes, Claas H. Hinze, Steven M. Holland, Stephen M. Hughes, Patricio Ibañez, Harry L. Malech, Isabelle Meyts, Marcela Moncada-Velez, Kunihiko Moriya, Esmeralda Neves, Matias Oleastro, Laura Perez, Vimel Rattina, Carmen Oleaga-Quintas, Neil Warner, Aleixo M. Muise, Jeanet Serafin López, Eunice Trindade, Julia Vasconselos, Severine Vermeire, Helmut Wittkowski, Austen Worth, Laurent Abel, Mary C. Dinauer, Peter D. Arkwright, Dirk Roos, Jean-Laurent Casanova, Taco W. Kuijpers, Jacinta Bustamante
Red blood cells (RBCs) influence rheology, release ADP, ATP and nitric oxide suggesting a role for RBCs in hemostasis and thrombosis. Here we provide evidence for a significant contribution of RBCs to thrombus formation. Anemic mice showed enhanced occlusion times upon injury of the carotid artery. A small population of RBCs was located to platelet thrombi and enhanced platelet activation by a direct cell contact via the FasL-FasR (CD95) pathway known to induce apoptosis. Activation of platelets in the presence of RBCs led to platelet FasL exposure that activated FasR on RBCs responsible for externalization of phosphatidylserine (PS) on the RBC membrane. Inhibition or genetic deletion of either FasL or FasR resulted in reduced PS exposure of RBCs and platelets, decreased thrombin generation and reduced thrombus formation in vitro and protection against arterial thrombosis in vivo. Direct cell contacts of platelets and RBCs via FasL-FasR were shown after ligation of the inferior vena cava (IVC) and in surgical specimens of patients after thrombectomy. In a flow restriction model of the IVC, reduced thrombus formation was observed in FasL–/– mice. Taken together, our data reveal a significant contribution of RBCs to thrombosis by the FasL-FasR pathway.
Christoph Klatt, Irena Krüger, Saskia Zey, Kim-Jürgen Krott, Martina Spelleken, Nina Sarah Gowert, Alexander Oberhuber, Lena Pfaff, Wiebke Lückstädt, Kerstin Jurk, Martin Schaller, Hadi Al-Hasani, Jürgen Schrader, Steffen Massberg, Konstantin Stark, Hubert Schelzig, Malte Kelm, Margitta Elvers
DNA damaging chemotherapy and radiation therapy are integrated into the treatment paradigm of the majority of cancer patients. Recently, immunotherapy that targets the immunosuppressive interaction between Programmed Death 1 (PD-1) and its ligand PD-L1 has been approved for malignancies including non-small lung cancer (NSCLC), melanoma, and head and neck squamous cell carcinoma (HNSCC). ATR is a DNA damage signaling kinase activated at damaged replication forks and ATR kinase inhibitors potentiate the cytotoxicity of DNA damaging chemotherapies. We show here that the ATR kinase inhibitor AZD6738 combines with conformal radiation therapy to attenuate radiation-induced CD8+ T cell exhaustion and potentiate CD8+ T cell activity in mouse models of Kras-mutant cancer. Mechanistically, AZD6738 blocks radiation-induced PD-L1 upregulation on tumor cells and dramatically decreases the number of tumor-infiltrating T regulatory (Treg) cells. Remarkably, AZD6738 combines with conformal radiation therapy to generate immunologic memory in complete responder mice. Our work raises the exciting possibility that a single pharmacologic agent may enhance the cytotoxic effects of radiation while concurrently potentiating radiation-induced antitumor immune responses.
Frank P. Vendetti, Pooja Karukonda, David A. Clump, Troy Teo, Ronald Lalonde, Katriana Nugent, Matthew Ballew, Brian F. Kiesel, Jan H. Beumer, Saumendra N. Sarkar, Thomas P. Conrads, Mark J. O'Connor, Robert L. Ferris, Phuoc T. Tran, Greg M. Delgoffe, Christopher J. Bakkenist
Cancer cell dependence on activated oncogenes is targeted therapeutically, but acquired resistance is virtually unavoidable. Here we show that the treatment of addicted melanoma cells with BRAF-inhibitors, and of breast cancer cells with HER2-targeted drugs, led to an adaptive rise in Neuropilin-1 (NRP1) expression, which is crucial for the onset of acquired resistance to therapy. Moreover, NRP1 levels dictated the efficacy of MET oncogene-inhibitors in addicted stomach and lung carcinoma cells. Mechanistically, NRP1 induced a JNK-dependent signaling cascade leading to the upregulation of alternative effector kinases, EGFR or IGF1R, which in turn sustained cancer cell growth and mediated acquired resistance to BRAF, HER2, or MET inhibitors. Notably, the combination with NRP1-interfering molecules improved the efficacy of oncogene-targeted drugs, and prevented, or even reversed, the onset of resistance in cancer cells and tumor models. Our study provides the rationale for targeting the NRP1-dependent upregulation of tyrosine kinases, responsible for loss of responsiveness to oncogene-targeted therapies.
Sabrina Rizzolio, Gabriella Cagnoni, Chiara Battistini, Stefano Bonelli, Claudio Isella, Jo A. Van Ginderachter, René Bernards, Federica Di Nicolantonio, Silvia Giordano, Luca Tamagnone
BACKGROUND. The circadian clock is a fundamental and pervasive biological program that coordinates 24-hour rhythms in physiology, metabolism and behaviour, and it is essential to health. Whereas time-of-day adapted therapy is increasingly reported to be highly successful, it needs to be personalized since internal circadian time is different for each individual. In addition, internal time is not a stable trait, but is influenced by many factors including genetic predisposition, age, gender, environmental light levels and season. An easy and convenient diagnostic tool is currently missing. METHODS. To establish a validated test, we followed a three-stage biomarker development strategy: (i) using circadian transcriptomics of blood monocytes from 12 individuals in a constant routine protocol combined with machine learning approaches, we identified biomarkers for internal time; (ii) these biomarkers were migrated to a clinically relevant gene expression-profiling platform (NanoString), and (iii) externally validated using an independent study with 28 early or late chronotypes. RESULTS. We developed a highly accurate and simple assay (BodyTime) to estimate the internal circadian time in humans from a single blood sample. Our assay needs only a small set of blood-based transcript biomarkers and is as accurate as the current gold standard dim light melatonin onset method at smaller monetary, time and sample number cost. CONCLUSION. The BodyTime assay provides a new diagnostic tool for personalization of healthcare according to the patient’s circadian clock. FUNDING. This study was supported by the Bundesministerium für Bildung und Forschung, Germany (FKZ: 13N13160 and 13N13162) and Intellux GmbH, Germany.
Nicole Wittenbrink, Bharath Ananthasubramaniam, Mirjam Münch, Barbara Koller, Bert Maier, Charlotte Weschke, Frederik Bes, Jan de Zeeuw, Claudia Nowozin, Amely Wahnschaffe, Sophia Wisniewski, Mandy Zaleska, Osnat Bartok, Reut Ashwal-Fluss, Hedwig Lammert, Hanspeter Herzel, Michael Hummel, Sebastian Kadener, Dieter Kunz, Achim Kramer
Dormant or slow-cycling tumour cells can form a residual chemoresistant reservoir responsible for relapse in patients, years after curative surgery and adjuvant therapy. We have adapted the pulse-chase expression of H2BeGFP for labelling and isolating slow-cycling cancer cells (SCCC). SCCC showed cancer-initiation potential and enhanced chemoresistance. Cells at this slow-cycling status presented a distinctive non-genetic and cell-autonomous gene expression profile shared across different tumour types. We identified TET2 epigenetic enzyme as key factor controlling SCCC numbers, survival and tumour recurrence. 5-Hydroxymethylcytosine (5hmC), generated by TET2 enzymatic activity, labelled SCCC genome in carcinomas and was a predictive biomarker of relapse and survival in cancer patients. We have shown the enhanced chemoresistance of SCCC, revealed 5hmC as a biomarker for their clinical identification, and TET2 as a potential drug-target for SCCC elimination that could extend patients’ survival.
Isabel Puig, Stephan P. Tenbaum, Irene Chicote, Oriol Arqués, Jordi Martínez-Quintanilla, Estefania Cuesta-Borrás, Lorena Ramírez, Pilar Gonzalo, Atenea Soto, Susana Aguilar, Cristina Eguizabal, Ginevra Caratù, Aleix Prat, Guillem Argilés, Stefania Landolfi, Oriol Casanovas, Violeta Serra, Alberto Villanueva, Alicia G. Arroyo, Luigi Terracciano, Paolo Nuciforo, Joan Seoane, Juan A. Recio, Ana Vivancos, Rodrigo Dienstmann, Josep Tabernero, Héctor G. Palmer
In the mid-1990s, whole-cell (wP) pertussis vaccines were associated with local and systemic adverse events, which prompted their replacement with acellular (aP) vaccines in many high-income countries. In the past decade rates of pertussis disease have increased in children receiving only acellular pertussis vaccines. We compared the immune responses to acellular pertussis boosters in children who received their initial doses with either wP or aP vaccines using activation-induced marker (AIM) assays. Specifically, we examined pertussis-specific memory CD4+ T cell responses ex vivo, highlighting a Type 2/Th2 versus Type 1/Th1 and Th17 differential polarization as a function of childhood vaccination. Remarkably, after a contemporary aP booster, cells from donors originally primed with aP were 1) associated with increased IL-4, IL-5, IL-13, IL-9 and TGF-β and decreased IFNγ and IL-17 production; 2) defective in their ex vivo capacity to expand memory cells; and 3) less capable to proliferate in vitro. These differences appeared to be T cell-specific, since equivalent increases of antibody titers and plasmablasts after aP boost were seen in both groups. In conclusion, our data suggest that long lasting effects and differential polarization and proliferation exists between adults originally vaccinated with aP versus wP despite repeated acellular boosters.
Ricardo da Silva Antunes, Mariana Babor, Chelsea Carpenter, Natalie Khalil, Mario Cortese, Alexander J Mentzer, Grégory Seumois, Christopher D. Petro, Lisa A. Purcell, Pandurangan Vijayanand, Shane Crotty, Bali Pulendran, Bjorn Peters, Alessandro Sette
Epithelial cell dysfunction is postulated as an important component in the pathogenesis of Idiopathic Pulmonary Fibrosis (IPF). Mutations in the Surfactant Protein C [SP-C] gene [SFTPC], an alveolar type 2 (AT2) cell restricted protein, have been found in sporadic and familial IPF. To causally link these events, we developed a knock-in mouse model capable of regulated expression of an IPF-associated Isoleucine to Threonine substitution at codon 73 [I73T] in Sftpc (SP-CI73T). Tamoxifen treated SP-CI73T cohorts developed rapid increases in SftpcI73T mRNA and misprocessed proSP-CI73T protein accompanied by increased early mortality (days 7-14). This acute phase was marked by diffuse parenchymal lung injury, tissue infiltration by monocytes, polycellular alveolitis, and elevations in bronchoalveolar lavage and AT2 mRNA contents of select inflammatory cytokines. Resolution of alveolitis (2-4 weeks), commensurate with a rise in TGFB1, was followed by aberrant remodeling marked by collagen deposition, AT2 cell hyperplasia, a-SMA positive cells, and restrictive lung physiology. The translational relevance of the model was supported by detection of multiple IPF biomarkers previously reported in human cohorts. These data provide proof of principle that mutant SP-C expression in vivo causes spontaneous lung fibrosis strengthening the role of AT2 dysfunction as a key upstream driver of IPF pathogenesis.
Shin-Ichi Nureki, Yaniv Tomer, Alessandro Venosa, Jeremy Katzen, Scott J. Russo, Sarita Jamil, Matthew Barrett, Vivian Nguyen, Meghan Kopp, Surafel Mulugeta, Michael F. Beers
Control of cellular metabolism is critical for efficient cell function, although little is known about the interplay between cell subset-specific metabolites in situ, especially in the tumor setting. Here, we determine how a macrophage-specific metabolite, itaconic acid, can regulate tumor progression in the peritoneum. We show peritoneal tumors (B16 melanoma or ID8 ovarian carcinoma) elicited a fatty acid oxidation-mediated increase in oxidative phosphorylation (OXPHOS) and glycolysis in peritoneal tissue-resident macrophages (pResMφ). Unbiased metabolomics identified itaconic acid, the product of Irg1-mediated catabolism of mitochondrial cis-aconitate, among the most highly upregulated metabolites in pResMφ of tumor-bearing mice. Administration of lentivirally-encoded Irg1 shRNA significantly reduced peritoneal tumors. This resulted in reductions in OXPHOS and OXPHOS-driven production of reactive oxygen species (ROS) in pResMφ and ROS-mediated MAP kinase activation in tumor cells. Our findings demonstrate that tumors profoundly alter pResMφ metabolism, leading to the production of itaconic acid, which potentiates tumor growth. Monocytes isolated from ovarian carcinoma patient ascites fluid expressed significantly elevated levels of Irg1. Therefore, Irg1 in pResMφ represents a potential therapeutic target for peritoneal tumors.
Jonathan M. Weiss, Luke C. Davies, Megan Karwan, Lilia Ileva, Michelle K. Ozaki, Robert Y.S. Cheng, Lisa A. Ridnour, Christina M. Annunziata, David A. Wink, Daniel W. McVicar
Rearrangements involving the neurotrophic receptor kinase genes (NTRK1, NTRK2, and NTRK3; hereafter referred to as TRK) produce oncogenic fusions in a wide variety of cancers in adults and children. Although TRK fusions occur in <1% of all solid tumors, inhibition of TRK results in profound therapeutic responses resulting in breakthrough FDA-approval of the TRK inhibitor larotrectinib for adult and pediatric solid tumor patients regardless of histology. In contrast to solid tumors, the frequency of TRK fusions and clinical effects of targeting TRK in hematologic malignancies is unknown. Here, through an evaluation for TRK fusions across > 7,000 patients with hematologic malignancies, we identified TRK fusions in acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), histiocytosis, multiple myeloma and dendritic cell neoplasms. Although TRK fusions occurred in only 0.1% of patients (8 out of 7,311 patients), they conferred responsiveness to TRK inhibition in vitro and in vivo in a patient-derived xenograft and a corresponding AML patient with ETV6-NTRK2 fusion. These data identify that despite their individual rarity, collectively TRK fusions are present in a wide variety of hematologic malignancies and predict clinically significant therapeutic responses to TRK inhibition.
Justin Taylor, Dean Pavlick, Akihide Yoshimi, Christina Marcelus, Stephen S. Chung, Jaclyn F. Hechtman, Ryma Benayed, Emiliano Cocco, Benjamin H. Durham, Lillian Bitner, Daichi Inoue, Young Rock Chung, Kerry Mullaney, Justin M. Watts, Eli L. Diamond, Lee A. Albacker, Tariq I. Mughal, Kevin Ebata, Brian B. Tuch, Nora Ku, Maurizio Scaltriti, Mikhail Roshal, Maria Arcila, Siraj Ali, David M. Hyman, Jae H. Park, Omar Abdel-Wahab
Lysine-63 (K63)–linked polyubiquitination of TRAF3 coordinates the engagement of pattern recognition receptors to recruited adaptor proteins and downstream activator TBK1 in pathways that induce type I interferon (IFN). Whether auto-ubiquitination or other E3 ligases mediate K63-linked TRAF3 polyubiquitination remains unclear. We demonstrated that mice deficient in E3 ligase gene Hectd3 remarkably increased host defense against infection by intracellular bacteria F. novicida, Mycobacterium, and Listeria by limiting bacterial dissemination. In the absence of HECTD3, type I IFN response was impaired during bacterial infection both in vivo and in vitro. HECTD3 regulated type I IFN production by mediating K63-linked polyubiquitination of TRAF3 at residue K138. The catalytic domain of HECTD3 regulated TRAF3 K63 polyubiquitination, which enabled TRAF3–TBK1 complex formation. Our study offers novel insights into mechanisms of TRAF3 modulation and provides potential therapeutic targets against infections by intracellular bacteria and inflammatory diseases.
Fubing Li, Yang Li, Huichun Liang, Tao Xu, Yanjie Kong, Maobo Huang, Ji Xiao, Xi Chen, Houjun Xia, Yingying Wu, Zhongmei Zhou, Xiaomin Guo, Chunmiao Hu, Chuanyu Yang, Xu Cheng, Ceshi Chen, Xiaopeng Qi
Myeloid-derived suppressor cells (MDSCs) densely accumulate into tumors and potently suppress anti-tumor immune responses promoting tumor development. Targeting MDSCs in tumor immunotherapy has been hampered by lack of understanding of the molecular pathways that govern MDSC differentiation and function. Herein, we identify autophagy as a crucial pathway for MDSC-mediated suppression of anti-tumor immunity. Specifically, MDSCs in melanoma patients and mouse melanoma exhibited increased levels of functional autophagy. Ablation of autophagy in myeloid cells, significantly delayed tumor growth and endowed anti-tumor immune responses. Notably, tumor-infiltrating autophagy-deficient monocytic MDSCs (M-MDSCs) demonstrated impaired suppressive activity in vitro and in vivo, while transcriptome analysis revealed significant differences in genes related to lysosomal function. Accordingly, autophagy-deficient M-MDSCs exhibited impaired lysosomal degradation thereby enhancing surface expression of MHC class II molecules, resulting in efficient activation of tumor-specific CD4+ T cells. Finally, targeting of the membrane-associated RING-CH1 (MARCH1) E3 ubiquitin ligase, that mediates the lysosomal degradation of MHC II, in M-MDSCs, attenuated their suppressive function, and resulted in significantly decreased tumor volume followed by development of a robust anti-tumor immunity. Collectively, these findings depict autophagy as a novel molecular target of MDSC-mediated suppression of anti-tumor immunity.
Themis Alissafi, Aikaterini Hatzioannou, Konstantinos Mintzas, Roza Maria Barouni, Aggelos Banos, Sundary Sormendi, Alexandros Polyzos, Maria Xilouri, Ben Wielockx, Helen Gogas, Panayotis Verginis