An in-depth understanding of immune escape mechanisms in cancer are likely to lead to innovative advances in immunotherapeutic strategies. However, much remains unknown regarding these mechanisms and how they impact immunotherapy resistance. Using several pre-clinical tumor models as well as clinical specimens, we report a newly identified mechanism whereby CD8+ T cell activation in response to PD-1 blockade induced a PD-L1-NLRP3 inflammasome signaling cascade that ultimately led to the recruitment of granulocytic myeloid-derived suppressor cells (PMN-MDSCs) into tumor tissues, thereby dampening the resulting anti-tumor immune response. The genetic and pharmacologic inhibition of NLRP3 suppressed PMN-MDSC tumor infiltration and significantly augmented the efficacy of anti-PD-1 antibody immunotherapy. This pathway therefore represents a tumor-intrinsic adaptive resistance mechanism to anti-PD-1 checkpoint inhibitor immunotherapy and is a promising target for future translational research.
Balamayooran Theivanthiran, Kathy S. Evans, Nicholas C. DeVito, Michael P. Plebanek, Michael Sturdivant, Lucas P. Wachsmuth, April K.S. Salama, Yubin Kang, David Hsu, Justin M. Balko, Douglas B. Johnson, Mark Starr, Andrew B. Nixon, Alisha Holtzhausen, Brent A. Hanks
Background: Neurofibroma/schwannoma hybrid nerve sheath tumors (N/S HNSTs) are neoplasms associated with larger nerves that occur sporadically and in the context of schwannomatosis or neurofibromatosis type 2 or 1. Clinical management of N/S HNST is challenging, especially for large tumors, and established systemic treatments are lacking. Methods: We used next-generation sequencing and array-based DNA methylation profiling to determine the clinically actionable genomic and epigenomic landscapes of N/S HNST. Results: Whole-exome sequencing within a precision oncology program identified an activating mutation (p.Asp769Tyr) in the catalytic domain of the ERBB2 receptor tyrosine kinase in a patient with schwannomatosis-associated N/S HNST, and targeted treatment with the small-molecule ERBB inhibitor lapatinib led to prolonged clinical benefit and a lasting radiographic and metabolic response. Analysis of a multicenter validation cohort revealed recurrent ERBB2 mutations (p.Leu755Ser, p.Asp769Tyr, p.Val777Leu) in N/S HNSTs occurring in patients who met diagnostic criteria for sporadic schwannomatosis (3 of 7 patients), but not in N/S HNSTs arising in the context of neurofibromatosis (6 patients) or outside a tumor syndrome (1 patient), and showed that ERBB2-mutant N/S HNSTs cluster in a distinct subgroup of peripheral nerve sheath tumors based on genome-wide DNA methylation patterns. Conclusion: These findings uncover a key biological feature of N/S HNST that may have important diagnostic and therapeutic implications. Funding: This work was supported by grant H021 from DKFZ-HIPO. MWR and PNH have received fellowships from UCT Frankfurt, and MWR has received funding from the Frankfurt Research Funding Clinician Scientist Program.
Michael W. Ronellenfitsch, Patrick N. Harter, Martina Kirchner, Christoph Heining, Barbara Hutter, Laura Gieldon, Jens Schittenhelm, Martin U. Schuhmann, Marcos Tatagiba, Gerhard Marquardt, Marlies Wagner, Volker Endris, Christian H. Brandts, Victor-Felix Mautner, Evelin Schröck, Wilko Weichert, Benedikt Brors, Andreas von Deimling, Michel Mittelbronn, Joachim P. Steinbach, David E. Reuss, Hanno Glimm, Albrecht Stenzinger, Stefan Fröhling
Hepatocellular carcinoma (HCC) is clearly age-related and represents one of the deadliest cancer types worldwide. Due to globally increasing risk factors including metabolic disorders, the incidence rates of HCC are still rising. However, the molecular hallmarks of HCC remain poorly understood. Neuropeptide Y (NPY) and NPY-receptors represent a highly conserved, stress-activated system which is involved in diverse cancer-related hallmarks including aging and metabolic alterations, but its impact on liver cancer had been unclear. Here, we observed increased NPY5-receptor (Y5R) expression in HCC which correlated with tumor growth and survival. Furthermore, we found that its ligand NPY was secreted by peri-tumorous hepatocytes. Hepatocyte-derived NPY promoted HCC progression by Y5R-activation. Transforming growth factor beta 1 (TGFβ1) was identified as a regulator of NPY in hepatocytes and induced Y5R in invasive cancer cells. Moreover, NPY-conversion by dipeptidylpeptidase 4 (DPP4) augmented Y5R-activation and function in liver cancer. The TGFβ-NPY-Y5R-axis and DPP4 represent attractive therapeutic targets for controlling liver cancer progression.
Peter Dietrich, Laura Wormser, Valerie Fritz, Tatjana Seitz, Monica De Maria, Alexandra Schambony, Andreas E. Kremer, Claudia Günther, Timo Itzel, Wolfgang E. Thasler, Andreas Teufel, Jonel Trebicka, Arndt Hartmann, Markus F. Neurath, Stephan von Hörsten, Anja Bosserhoff, Claus Hellerbrand
Treating neuropathic pain is challenging and novel non-opioid based medicines are needed. Using unbiased receptomics, transcriptomic analyses, immunofluorescence and in situ hybridization, we found the expression of the orphan GPCR (oGPCR) Gpr160 and GPR160 increased in the rodent dorsal horn of the spinal cord (DH-SC) following traumatic nerve injury. Genetic and immunopharmacological approaches demonstrated that GPR160 inhibition in the spinal cord prevented and reversed neuropathic pain in male and female rodents without altering normal pain response. GPR160 inhibition in the spinal cord attenuated sensory processing in the thalamus, a key relay in the sensory discriminative pathways of pain. We also identified cocaine- and amphetamine-regulated transcript peptide (CARTp) as a GPR160 ligand. Inhibiting endogenous CARTp signaling in spinal cord attenuated neuropathic pain, whereas exogenous intrathecal (i.th.) CARTp evoked painful hypersensitivity through GPR160-dependent ERK and cAMP response element-binding protein (CREB). Our findings de-orphanize GPR160, identify it as a determinant of neuropathic pain and potential therapeutic target, and provide insights to its signaling pathways. CARTp is involved in many diseases including depression, reward and addiction, de-orphanization of GPR160 is a major step forward understanding the role of CARTp signaling in health and disease.
Gina LC Yosten, Caron M. Harada, Christopher J. Haddock, Luigino Antonio Giancotti, Grant R. Kolar, Ryan Patel, Chun Guo, Zhoumou Chen, Jinsong Zhang, Timothy M. Doyle, Anthony H. Dickenson, Willis K. Samson, Daniela Salvemini
Lamin A is a component of the inner nuclear membrane that, together with epigenetic factors, organizes the genome in higher order structures required for transcriptional control. Mutations in the Lamin A/C gene cause several diseases, belonging to the class of laminopathies, including muscular dystrophies. Nevertheless, molecular mechanisms involved in the pathogenesis of Lamin A-dependent dystrophies are still largely unknown. Polycomb group of proteins (PcG) are epigenetic repressors and Lamin A interactors, primarily involved in the maintenance of cell identity. Using a murine model of Emery-Dreifuss Muscular Dystrophy (EDMD), we showed here that Lamin A loss deregulated PcG positioning in muscle satellite stem cells leading to de-repression of non-muscle specific genes and p16INK4a, a senescence driver encoded in the Cdkn2a locus. This aberrant transcriptional programme caused impairment in self-renewal, loss of cell identity and premature exhaustion of quiescent satellite cell pool. Genetic ablation of Cdkn2a locus restored muscle stem cell properties in Lamin A/C null dystrophic mice. Our findings established a direct link between Lamin A and PcG epigenetic silencing and indicated that Lamin A-dependent muscular dystrophy can be ascribed to intrinsic epigenetic dysfunctions of muscle stem cells.
Andrea Bianchi, Chiara Mozzetta, Gloria Pegoli, Federica Lucini, Sara Valsoni, Valentina Rosti, Cristiano Petrini, Alice Cortesi, Francesco Gregoretti, Laura Antonelli, Gennaro Oliva, Marco De Bardi, Roberto Rizzi, Beatrice Bodega, Diego Pasini, Francesco Ferrari, Claudia Bearzi, Chiara Lanzuolo
Elevated pressure in the pancreatic gland is the central cause of pancreatitis following abdominal trauma, surgery, endoscopic retrograde cholangiopancreatography (ERCP), and gallstones. In the pancreas excessive intracellular calcium causes mitochondrial dysfunction, premature zymogen activation, and necrosis ultimately leading to pancreatitis. Although stimulation of the mechanically activated, calcium-permeable ion channel, Piezo1, in the pancreatic acinar cell is the initial step in pressure-induced pancreatitis, activation of Piezo1 produces only transient elevation in intracellular calcium that is insufficient to cause pancreatitis. Therefore, how pressure produces a prolonged calcium elevation necessary to induce pancreatitis is unknown. We demonstrate that Piezo1 activation in pancreatic acinar cells caused a prolonged elevation in intracellular calcium levels, mitochondrial depolarization, intracellular trypsin activation, and cell death. Notably, these effects were dependent on the degree and duration of force applied to the cell. Low or transient force were insufficient to activate these pathological changes whereas higher and prolonged application of force triggered sustained elevation in intracellular calcium leading to enzyme activation and cell death. All of these pathological events were rescued in acinar cells treated with a Piezo1 antagonist and in acinar cells from mice with genetic deletion of Piezo1. We discovered that Piezo1 stimulation triggered TRPV4 channel opening which was responsible for the sustained elevation in intracellular calcium that caused intracellular organelle dysfunction. Moreover, TRPV4 gene knockout mice were protected from Piezo1 agonist- and pressure-induced pancreatitis. These studies unveil a calcium signaling pathway in which Piezo1-induced TRPV4 channel opening causes pancreatitis.
Sandip M. Swain, Joelle M.J. Romac, Rafiq A. Shahid, Stephen J. Pandol, Wolfgang Liedtke, Steven R. Vigna, Rodger A. Liddle
Hypoxia-inducible factor (HIF) is strikingly upregulated in many types of cancer and there is great interest in applying inhibitors of HIF as anti-cancer therapeutics. The most advanced of these are small molecules that target the HIF-2 isoform through binding the PAS-B domain of HIF-2α. These molecules are undergoing clinical trials with promising results in renal and other cancers where HIF-2 is considered to be driving growth. Nevertheless, a central question remains as to whether such inhibitors impact on physiological responses to hypoxia at relevant doses. Here we show that pharmacological HIF-2α inhibition with PT2385, at doses similar to those reported to inhibit tumour growth, rapidly impaired ventilatory responses to hypoxia, abrogating both ventilatory acclimatisation and carotid body cell proliferative responses to sustained hypoxia. Mice carrying a HIF-2α PAS-B S305M mutation that disrupts PT2385 binding, but not dimerisation with HIF-1β, did not respond to PT2385 indicating that these effects are on target. Furthermore, the finding of a hypomorphic ventilatory phenotype in untreated HIF-2α S305M mutant mice suggests a function for the HIF-2α PAS-B domain beyond heterodimerisation with HIF-1β. Although PT2385 was well-tolerated, the findings indicate the need for caution in patients who are dependent on hypoxic ventilatory drive.
Xiaotong Cheng, Maria Prange-Barczynska, James W. Fielding, Minghao Zhang, Alana L. Burrell, Joanna D.C.C. Lima, Luise Eckardt, Isobel L.A. Argles, Christopher W. Pugh, Keith J. Buckler, Peter A. Robbins, Emma J. Hodson, Richard K. Bruick, Lucy M. Collinson, Fraydoon Rastinejad, Tammie Bishop, Peter J. Ratcliffe
Fibroblasts are key-effector cells in tissue remodeling. They remain persistently activated in fibrotic diseases, resulting in progressive deposition of extracellular matrix. Although fibroblast activation maybe initiated by external factors, prolonged activation can induce an “autonomous”, self-maintaining pro-fibrotic phenotype in fibroblasts. Accumulating evidence suggests that epigenetic alterations play a central role to establish this persistently activated pathologic phenotype of fibroblasts. We demonstrated that in fibrotic skin of patients with systemic sclerosis (SSc), a prototypical idiopathic fibrotic disease, transforming growth factor-β (TGFβ) induced the expression of DNA-methyltransferase 3A (DNMT3A) and DNMT1 in fibroblasts in a SMAD-dependent manner to silence the expression of suppressor of cytokine signaling 3 (SOCS3) by promoter hypermethylation. Downregulation of SOCS3 facilitated activation of signal transducers and activators of transcription 3 (STAT3) to promote fibroblast-to–myofibroblast transition, collagen release and fibrosis in vitro and in vivo. Re-establishment of the epigenetic control of STAT3 signaling by genetic or pharmacological inactivation of DNMT3A reversed the activated phenotype of SSc fibroblasts in tissue culture, inhibited TGFβ-dependent fibroblast activation and ameliorated experimental fibrosis in murine models. These findings identify a novel pathway of epigenetic imprinting of fibroblasts in fibrotic disease with translational implications for the development of new targeted therapies in fibrotic diseases.
Clara Dees, Sebastian Pötter, Yun Zhang, Christina Bergmann, Xiang Zhou, Markus Luber, Thomas Wohlfahrt, Emmanuel Karouzakis, Andreas Ramming, Kolja Gelse, Akihiko Yoshimura, Rudolf Jaenisch, Oliver Distler, Georg Schett, Jörg H.W. Distler
Colitis caused by C. difficile infection is an increasing cause of human morbidity and mortality, especially after antibiotic use in healthcare settings. The natural immunity of newborn infants and protective host immune mediators against C. difficile infection are not fully understood, with data suggesting that inflammation can be either protective or pathogenic. Here we show an essential role for IL-17A produced by γδ T cells in host defense against C. difficile infection. Fecal extracts of children with C. difficile infection showed increased IL-17A and T cell receptor γ-chain expression, and IL-17 production by intestinal γδ T cells was efficiently induced after infection in mice. C. difficile induced tissue inflammation and mortality were each significantly increased in mice deficient in IL-17A or γδ T cells. neonatal mice, with naturally expanded ROR-γ+ γδ T cells poised for IL-17 production were resistant to C. difficile infection, whereas eliminating γδ T cells or IL-17A each efficiently overturned neonatal resistance against infection. These results reveal an expanded role for IL-17 producing γδ T cells in neonatal host defense against infection and provide a mechanistic explanation for the clinically observed resistance of infants to C. difficile colitis.
Yee-Shiuan Chen, Iuan-Bor Chen, Giang Pham, Tzu-Yu Shao, Hansraj Bangar, Sing Sing Way, David B. Haslam
Background. Preclinical experiments have shown that donor blood cells, modified in vitro by an alkylating agent (MIC, modified immune cells), induced long-term specific immunosuppression against the allogeneic donor. Methods. In this phase-I trial, patients received either 1.5x106 MIC per kg b.w. on day -2 (N=3, group A), or 1.5x108 MIC per kg b.w. on day -2 (N=3, group B) or day -7 (N=4, group C) before living donor kidney transplantation in addition to post-transplant immunosuppression. Primary outcome measure was the frequency of adverse events (AE) until day 30 (study phase) with follow-up to day 360. Results. MIC infusions were extremely well tolerated. During the study phase, a total of 69 AE occurred in 10 treated patients which were unlikely/not related to MIC infusion. No donor-specific human leukocyte antigen antibodies or rejection episodes were noted even though the patients received up to 1.3x1010 of donor mononuclear cells prior to transplantation. Group C patients with low immunosuppression during follow-up showed no in vitro reactivity against stimulatory donor blood cells on day 360 while reactivity against third party cells was preserved. Frequencies of CD19+CD24highCD38high transitional B lymphocytes (Breg) increased from a median of 6% before MIC infusion to 20% on day 180, which was 19- and 68-fold higher, respectively, than in two independent cohorts of transplanted controls. The majority of Breg produced immunosuppressive cytokine IL-10. MIC-treated patients showed the Immune Tolerance Network operational tolerance signature. Conclusion. MIC administration was safe and could be a future tool for the targeted induction of tolerogenic Breg.
Christian Morath, Anita Schmitt, Christian Kleist, Volker Daniel, Gerhard Opelz, Caner Süsal, Eman H. Ibrahim, Florian Kälble, Claudius Speer, Christian Nusshag, Luiza Pego da Silva, Claudia Sommerer, Lei Wang, Ming Ni, Angela Hückelhoven-Krauss, David Czock, Uta Merle, Arianeb Mehrabi, Anja Sander, Matthes Hackbusch, Christoph Eckert, Rüdiger Waldherr, Paul Schnitzler, Carsten Müller-Tidow, Jörg D. Hoheisel, Shakhawan A. Mustafa, Mohamed S.S. Alhamdani, Andrea S Bauer, Jochen Reiser, Martin Zeier, Michael Schmitt, Matthias Schaier, Peter Terness
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