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.
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
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.
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
STAT2 is a transcription factor activated by type I and III interferons. We report 23 patients with loss of function variants causing autosomal recessive (AR), complete STAT2 deficiency. Both cells transfected with mutant STAT2 alleles and the patients’ cells display impaired expression of interferon stimulated genes and impaired control of in-vitro viral infections. Clinical manifestations from early childhood onward include severe adverse reaction to live attenuated viral vaccines (LAV, 12/17 patients) and severe viral infections (10/23 patients), particularly critical influenza pneumonia (6 patients), critical COVID-19 pneumonia (1 patient), and herpes simplex encephalitis (1 patient). The patients display various types of hyperinflammation, often triggered by viral infection or after LAV administration, which probably attests to unresolved viral infection in the absence of STAT2-dependent type I and III IFN immunity (7 patients). Transcriptomic analysis reveals that circulating monocytes, neutrophils, and CD8 memory T cells contribute to this inflammation. Eight patients died (35%, 2 months-7 years): one of HSV-1 encephalitis, one of fulminant hepatitis, and six of heart failure during a febrile illness with no identified etiology. 15 patients remain alive (5-40 years). AR complete STAT2 deficiency underlies severe viral diseases, with half of the patients surviving into teenage years or adulthood.
Giorgia Bucciol, Leen Moens, Masato Ogishi, Darawan Rinchai, Daniela Matuozzo, Mana Momenilandi, Nacim Kerrouche, Catherine M. Cale, Elsa R. Treffeisen, Mohammad Al Salamah, Bandar K. Al-Saud, Alain Lachaux, Remi Duclaux-Loras, Marie Meignien, Aziz Bousfiha, Ibtihal Benhsaien, Anna Shcherbina, Anna Roppelt, Florian Gothe, Nadhira Houhou-Fidouh, Scott J. Hackett, Lisa M. Bartnikas, Michelle C. Maciag, Mohammed F. Alosaimi, Janet Chou, Reem W. Mohammed, Bishara J. Freij, Emmanuelle Jouanguy, Shen-Ying Zhang, Stephanie Boisson-Dupuis, Vivien Béziat, Qian Zhang, Christopher J.A. Duncan, Sophie Hambleton, Jean-Laurent Casanova, Isabelle Meyts
Spastic paraplegia 50 (SPG50) is an ultrarare childhood-onset neurological disorder caused by biallelic loss-of-function variants in the AP4M1 gene. SPG50 is characterized by progressive spastic paraplegia, global developmental delay and subsequent intellectual disability, secondary microcephaly, and epilepsy. Preclinical studies evaluated an adeno-associated virus (AAV)/AP4M1 gene therapy for SPG50. In vitro studies demonstrated that transduction of patient-derived fibroblasts with AAV2/AP4M1 resulted in phenotypic rescue. To evaluate efficacy in vivo, Ap4m1 knockout mice were intrathecally (IT) injected with 5E11, 2.5E11, or 1.25E11 vg doses of AAV9/AP4M1 at postnatal day p7-10 (pre-manifesting cohorts) or p90 (early manifesting cohorts). Age- and dose-dependent effects were observed, with early intervention and higher doses achieving the best therapeutic benefits. In parallel, three toxicology studies in wild-type mice, rats, and non-human primates (NHPs) demonstrated that AAV9/AP4M1 had an acceptable safety profile up to a target human dose of 1E15 vg. Of note, similar degrees of minimal to mild dorsal root ganglia (DRG) toxicity were observed in both rats and NHPs, supporting the use of rats to monitor DRG toxicity in future IT AAV studies. These preclinical results identify an acceptably safe and efficacious dose of IT-administered AAV9/AP4M1, supporting an investigational gene transfer clinical trial to treat SPG50.
Xin Chen, Thomas Dong, Yuhui Hu, Raffaella De Pace, Rafael Mattera, Kathrin Eberhardt, Marvin Ziegler, Terry Pirovolakis, Mustafa Sahin, Juan S. Bonifacino, Darius Ebrahimi-Fakhari, Steven J. Gray
Patients with small cell lung cancer (SCLC) generally have a poor prognosis and a median overall survival of only about 13 months, indicating the urgent need for novel therapies. Delta-like protein 3 (DLL3) has been identified as a tumor-specific cell surface marker on neuroendocrine cancers including SCLC. In this study, we developed a chimeric antigen receptor (CAR) against DLL3 that displays antitumor efficacy in xenograft and murine SCLC models. CAR T cell expression of the proinflammatory cytokine interleukin-18 (IL-18) greatly enhanced the potency of DLL3-targeting CAR T cell therapy. In a murine metastatic SCLC model, IL-18 production increased the activation of both CAR T cells and endogenous tumor-infiltrating lymphocytes. We also observed an increased infiltration, repolarization and activation of antigen-presenting cells. Lastly, human IL-18-secreting anti-DLL3 CAR T cells showed an increased memory phenotype, less exhaustion and induced durable responses in multiple SCLC models, an effect that could be further enhanced with anti-PD-1 blockade. Together, these results define DLL3-targeting CAR T cells that produce IL-18 as a promising novel strategy against DLL3-expressing solid tumors.
Janneke E. Jaspers, Jonathan F. Khan, William D. Godfrey, Andrea V. Lopez, Metamia Ciampricotti, Charles M. Rudin, Renier J. Brentjens
Endothelial cells (ECs) are constitutively an anticoagulant surface but switch to support coagulation following pathogenic stimuli. This switch promotes thrombotic cardiovascular disease. To generate thrombin at physiologic rates, coagulation proteins assemble on a membrane containing anionic phospholipid, most notably phosphatidylserine (PS). PS can be rapidly externalized to the outer cell membrane leaflet by phospholipid “scramblases”, such as TMEM16F. TMEM16F-dependent PS externalization is well-characterized in platelets. In contrast, how ECs externalize phospholipids to support coagulation is not understood. We employed a focused genetic screen to evaluate the contribution of transmembrane phospholipid transport on EC procoagulant activity. We identified two TMEM16 family members, TMEM16F, and its closest paralog, TMEM16E, which were both required to support coagulation on ECs via PS externalization. Applying an intravital laser-injury model of thrombosis, we observed, unexpectedly, that PS externalization was concentrated at the vessel wall, not on platelets. TMEM16E-null mice demonstrated reduced vessel-wall dependent fibrin formation. The TMEM16 inhibitor benzbromarone prevented PS externalization and EC procoagulant activity and protected mice from thrombosis without increasing bleeding following tail transection. These findings indicate the activated endothelial surface is a source of procoagulant phospholipid contributing to thrombus formation. TMEM16 phospholipid scramblases may be a therapeutic target for thrombotic cardiovascular disease.
Alec A. Schmaier, Papa F. Anderson, Siyu M. Chen, Emale El-Darzi, Ivan Aivasovsky, Milan P. Kaushik, Kelsey D. Sack, H. Criss Hartzell, Samir M. Parikh, Robert Flaumenhaft, Sol Schulman
Mucosal infections pose a significant global health burden. Antigen-specific tissue resident T cells are critical to maintaining barrier immunity. Previous studies in the context of systemic infection suggest that memory CD8 T cells may also provide innate-like protection against antigenically unrelated pathogens independent of TCR engagement. Whether "bystander T cell activation" is also an important defense mechanism in the mucosa is poorly understood. Here, we investigated if innate-like memory CD8 T cells could protect against a model mucosal virus infection, herpes simplex virus 2 (HSV-2). We found that immunization with an irrelevant antigen delayed disease progression from lethal HSV-2 challenge, suggesting that memory CD8 T cells may mediate protection despite the lack of antigen-specificity. Upon HSV-2 infection, we observed an early infiltration, rather than substantial local proliferation, of antigen-non-specific CD8 T cells, which became bystander-activated only within the infected mucosal tissue. Critically, we show that bystander-activated CD8 T cells are sufficient to reduce early viral burden after HSV-2 infection. Finally, local cytokine cues within the tissue microenvironment after infection were sufficient for bystander activation of mucosal tissue memory CD8 T cells from mice and humans. Altogether, our findings suggest that local bystander-activation of CD8 memory T cells contribute a fast and effective innate-like response to infection in mucosal tissue.
Tanvi Arkatkar, Veronica A. Davé, Irene Cruz Talavera, Jessica B. Graham, Jessica L. Swarts, Sean M. Hughes, Timothy A. Bell, Pablo Hock, Joe Farrington, Ginger D. Shaw, Anna C. Kirby, Michael Fialkow, Meei-Li Huang, Keith R. Jerome, Martin T. Ferris, Florian Hladik, Joshua T. Schiffer, Martin Prlic, Jennifer M. Lund
Sphingolipids function as membrane constituents and signaling molecules, with crucial roles in human diseases, from neurodevelopmental to cancer, best exemplified in the inborn errors of sphingolipid metabolism in lysosomes. The dihydroceramide desaturase DEGS1 acts in the last step of a sector of the sphingolipid pathway, de novo ceramide biosynthesis. Defects in DEGS1 cause the recently described hypomyelinating leukodystrophy-18 (HLD18, OMIM #618404). Here, we reveal that DEGS1 is a mitochondria-associated endoplasmic reticulum membrane (MAM)-resident enzyme, refining previous reports locating DEGS1 at the endoplasmic reticulum only. Using patient fibroblasts, multi-omics and enzymatic assays, we show that DEGS1 deficiency disrupts the main core functions of the MAM: i) mitochondrial dynamics, with a hyperfused mitochondrial network associated with decreased activation of dynamin-related protein 1; ii) cholesterol metabolism, with impaired sterol O-acyltransferase activity and decreased cholesteryl esters; iii) phospholipid metabolism, with increased phosphatidic acid and phosphatidylserine and decreased phosphatidylethanolamine; iv) biogenesis of lipid droplets, with increased size and numbers. Moreover, we detected increased mitochondrial superoxide species production in fibroblasts and mitochondrial respiration impairment in patient muscle biopsy tissues. Our findings shed light on the pathophysiology of HLD18 and broaden our understanding of the role of sphingolipid metabolism in MAMs function.
Laura Planas-Serra, Nathalie Launay, Leire Goicoechea, Bénédicte Heron, Cristina Jou, Natalia Juliá-Palacios, Montserrat Ruiz, Stéphane Fourcade, Carlos Casasnovas, Carolina De La Torre, Antoinette Gelot, Maria Marsal, Pablo Loza-Alvarez, Àngels García-Cazorla, Ali Fatemi, Isidre Ferrer, Manuel Portero-Otin, Estela Area-Gómez, Aurora Pujol
Cutaneous Skeletal Hypophosphatemia Syndrome (CSHS) is a mosaic RASopathy characterized by the association of dysplastic skeletal lesions, congenital skin nevi of epidermal and/or melanocytic origin, and fibroblast growth factor-23 (FGF23)-mediated hypophosphatemia. The primary physiological source of circulating FGF23 is bone cells. However, several reports have suggested skin lesions as the source of excess FGF23 in CSHS. Consequently, without convincing evidence of efficacy, many patients with CSHS have undergone painful removal of cutaneous lesions in an effort to normalize blood phosphate levels.This study aims to elucidate whether the source of FGF23 excess in CSHS is RAS mutation-bearing bone or skin lesions. Towards this end, we analyzed the expression and activity of Fgf23 in two mouse models expressing similar HRAS/Hras activating mutations in a mosaic-like fashion in either bone or epidermal tissue. We found that HRAS hyperactivity in bone, not skin, caused excess of bioactive intact FGF23, hypophosphatemia and osteomalacia. Our findings support RAS-mutated dysplastic bone as the primary source of physiologically active FGF23 excess in patients with CSHS. This evidence informs the care of patients with CSHS, arguing against the practice of nevi removal to decrease circulating, physiologically active FGF23.
Diana Ovejero, Zachary Michel, Christophe Cataisson, Amanda Saikali, Rebeca Galisteo, Stuart H. Yuspa, Michael T. Collins, Luis F. de Castro
Plasma IL-6 is elevated after myocardial infarction (MI) and is associated with increased morbidity and mortality. Which cardiac cell type preferentially contributes to IL-6 and how its production is regulated is largely unknown. Here, we studied the cellular source and purinergic regulation of IL-6 formation in a murine MI model. IL-6, measured in various cell types in post MI hearts by qPCR, RNAscope and at protein level, was preferentially formed by fibroblasts (CFs). scRNAseq in infarcted mouse and human hearts confirmed this finding. Adenosine stimulated fibroblast IL-6 formation via A2bR in a Gq-dependent manner. CFs highly expressed Adora2b, rapidly degraded extracellular ATP to AMP but lacked CD73. In mice and humans Adora2B was also mainly expressed by fibroblasts (scRNAseq). Global IL-6 formation was assessed in isolated hearts in mice lacking CD73 on T-cells (CD4CD73-/-) a condition known to be associated with adverse cardiac remodeling. The ischemia-induced release of IL-6 was strongly attenuated in CD4CD73-/- mice, suggesting adenosine-mediated modulation. Together this demonstrates that post-MI IL-6 is mainly derived from activated CFs and is controlled by T-cell derived adenosine. Purinergic metabolic cooperation between CFs and T-cells is a novel mechanism with therapeutic potential which modulates IL6 formation by the heart.
Christina Alter, Anne Sophie Henseler, Christoph Owenier, Julia Hesse, Zhaoping Ding, Tobias Lautwein, Jasmin Bahr, Sikander Hayat, Rafael Kramann, Eva Kostenis, Jürgen Scheller, Jürgen Schrader
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