Therapeutics
Abstract
Intravenous administration of a high affinity carbon monoxide (CO)-binding molecule, recombinant neuroglobin, can improve survival in CO poisoning mouse models. The current study aims to understand how biochemical variables of the scavenger determine the CO removal from the RBCs by evaluating three readily available hemoproteins, 2,3-diphosphoglycerate stripped human hemoglobin (StHb), N-ethylmaleimide modified hemoglobin (NEMHb), and equine myoglobin (Mb). These molecules efficiently sequester CO from hemoglobin in erythrocytes in vitro. A kinetic model was developed to predict the CO binding efficacy for hemoproteins, based on their measured in vitro oxygen and CO binding affinities, suggesting that the therapeutic efficacy of hemoproteins for CO poisoning relates to a high M value, which is the binding affinity for CO relative to oxygen (KA,CO/KA,O2). In a lethal CO poisoning mouse model, StHb, NEMHb, and Mb improved survival by 100%, 100%, and 60%, respectively, compared with saline controls, and were well tolerated in 48-hour toxicology assessments. In conclusion, both StHb and NEMHb have high CO binding affinities and M values and scavenge CO efficiently in vitro and in vivo, highlighting their therapeutic potential for point-of-care antidotal therapy of CO poisoning.
Authors
Qinzi Xu, Jason J. Rose, Xiukai Chen, Ling Wang, Anthony W. DeMartino, Matthew R. Dent, Sagarika Tiwari, Kaitlin Bocian, Xueyin N. Huang, Qin Tong, Charles F. McTiernan, Lanping Guo, Elmira Alipour, Trevor C. Jones, Kamil Burak Ucer, Daniel B. Kim-Shapiro, Jesus Tejero, Mark T. Gladwin
Abstract
Therapy with radiation plus cisplatin kills HPV+ oropharyngeal squamous cell carcinomas (OPSCCs) by increasing reactive oxygen species beyond cellular antioxidant capacity. To explore why these standard treatments fail for some patients, we evaluated whether the variation in HPV oncoprotein levels among HPV+ OPSCCs affects mitochondrial metabolism, a source of antioxidant capacity. In cell line and patient-derived xenograft models, levels of HPV full-length E6 (fl-E6) inversely correlated with oxidative phosphorylation, antioxidant capacity, and therapy resistance, and fl-E6 was the only HPV oncoprotein to display such correlations. Ectopically expressing fl-E6 in models with low baseline levels reduced mitochondrial mass, depleted antioxidant capacity, and sensitized to therapy. In this setting, fl-E6 repressed the peroxisome proliferator–activated receptor gamma co-activator 1α/estrogen-related receptor α (PGC-1α/ERRα) pathway for mitochondrial biogenesis by reducing p53-dependent PGC-1α transcription. Concordant observations were made in 3 clinical cohorts, where expression of mitochondrial components was higher in tumors of patients with reduced survival. These tumors contained the lowest fl-E6 levels, the highest p53 target gene expression, and an activated PGC-1α/ERRα pathway. Our findings demonstrate that E6 can potentiate treatment responses by depleting mitochondrial antioxidant capacity and provide evidence for low E6 negatively affecting patient survival. E6’s interaction with the PGC-1α/ERRα axis has implications for predicting and targeting treatment resistance in OPSCC.
Authors
Malay K. Sannigrahi, Pavithra Rajagopalan, Ling Lai, Xinyi Liu, Varun Sahu, Hiroshi Nakagawa, Jalal B. Jalaly, Robert M. Brody, Iain M. Morgan, Bradford E. Windle, Xiaowei Wang, Phyllis A. Gimotty, Daniel P. Kelly, Elizabeth A. White, Devraj Basu
Abstract
Pancreatitis, the inflammatory disorder of the pancreas, has no specific therapy. Genetic, biochemical and animal model studies revealed that trypsin plays a central role in the onset and progression of pancreatitis. Here, we performed biochemical and preclinical mouse experiments to offer proof of concept that orally administered dabigatran etexilate can inhibit pancreatic trypsins and shows therapeutic efficacy in trypsin-dependent pancreatitis. We found that dabigatran competitively inhibited all human and mouse trypsin isoforms (Ki range 10-79 nM) and dabigatran plasma concentrations in mice given oral dabigatran etexilate well exceeded the Ki of trypsin inhibition. In the T7K24R trypsinogen mutant mouse model, a single oral gavage of dabigatran etexilate was effective against cerulein-induced progressive pancreatitis with a high degree of histological normalization. In contrast, spontaneous pancreatitis in T7D23A mice, which carry a more aggressive trypsinogen mutation, was not ameliorated by dabigatran etexilate, given either as daily gavages or by mixing it with solid chow. Taken together, our observations confirmed that benzamidine derivatives such as dabigatran are potent trypsin inhibitors and show therapeutic activity against trypsin-dependent pancreatitis in T7K24R mice. Lack of efficacy in T7D23A mice is likely related to the more severe pathology and insufficient drug concentrations in the pancreas.
Authors
Zsófia G. Pesei, Zsanett Jancsó, Alexandra Demcsák, Balázs Csaba Németh, Sandor Vajda, Miklós Sahin-Tóth
Abstract
Metastatic pancreatic cancer (PDAC) has a poor clinical outcome with a 5-year survival rate below 3%. Recent transcriptome profiling of PDAC biopsies has identified 2 clinically distinct subtypes — the “basal-like” (BL) subtype with poor prognosis and therapy resistance compared with the less aggressive and drug-susceptible “classical” (CLA) subtype. However, the mechanistic events and environmental factors that promote the BL subtype identity are not very clear. Using preclinical models, patient-derived xenografts, and FACS-sorted PDAC patient biopsies, we report here that the axon guidance receptor, roundabout guidance receptor 3 (ROBO3), promotes the BL metastatic program via a potentially unique AXL/IL-6/phosphorylated STAT3 (p-STAT3) regulatory axis. RNA-Seq identified a ROBO3-mediated BL-specific gene program, while tyrosine kinase profiling revealed AXL as the key mediator of the p-STAT3 activation. CRISPR/dCas9-based ROBO3 silencing disrupted the AXL/p-STAT3 signaling axis, thereby halting metastasis and enhancing therapy sensitivity. Transcriptome analysis of resected patient tumors revealed that AXLhi neoplastic cells associated with the inflammatory stromal program. Combining AXL inhibitor and chemotherapy substantially restored a CLA phenotypic state and reduced disease aggressiveness. Thus, we conclude that a ROBO3-driven hierarchical network determines the inflammatory and prometastatic programs in a specific PDAC subtype.
Authors
Niklas Krebs, Lukas Klein, Florian Wegwitz, Elisa Espinet, Hans Carlo Maurer, Mengyu Tu, Frederike Penz, Stefan Küffer, Xingbo Xu, Hanibal Bohnenberger, Silke Cameron, Marius Brunner, Albrecht Neesse, Uday Kishore, Elisabeth Hessmann, Andreas Trumpp, Philipp Ströbel, Rolf A. Brekken, Volker Ellenrieder, Shiv K. Singh
Abstract
Development of resistance to chemo- and immuno- therapies often occurs following treatment of melanoma brain metastasis (MBM). In this scenario, astrocytes cooperate towards MBM progression by upregulating secreted-factors, amongst which we found that monocyte chemoattractant protein-1 (MCP-1) and its receptors, CCR2 and CCR4, are overexpressed in activated astrocytes and in brain metastatic melanoma cells compared to primary lesions. We show that melanoma cells alter astrocytes-secretome and evoke MCP-1 expression and secretion, which in turn enhance vascular hyperpermeability and proliferation, migration, and invasion of CCR2-expressing melanoma cells, while inhibiting MCP-1 rescued this phenotype. Pharmacological or molecular inhibition of MCP-1/CCR2 in MBM mouse model activates an anti-tumor immune-mediated response as revealed by the enhanced infiltration of cytotoxic CD8+ T cells, attenuated immunosuppressive phenotype of tumor-associated macrophages, and reduced infiltration of regulatory T cells, leading to inhibition of MBM progression and prolonged survival. In addition, blocking this key target in MBM, improved the therapeutic response of anti-PD-1 immunotherapy, regardless of the tumor mutational load. These results show that the MCP-1/CCR2 axis polarizes the brain microenvironment towards an anti-inflammatory/pro-tumorigenic phenotype, highlighting the therapeutic relevance of this pathway as a potential immune checkpoint in MBM.
Authors
Sabina Pozzi, Anna Scomparin, Dikla Ben-Shushan, Eilam Yeini, Paula Ofek, Alessio D. Nahmad, Shelly Soffer, Ariel Ionescu, Antonella Ruggiero, Adi Barzel, Henry Brem, Thomas M. Hyde, Iris Barshack, Sanju Sinha, Eytan Ruppin, Tomer Weiss, Asaf Madi, Eran Perlson, Inna Slutsky, Helena F. Florindo, Ronit Satchi-Fainaro
Abstract
Despite intensive therapy, children with high-risk neuroblastoma are at risk of treatment failure. We applied a multi-omic system approach to evaluate metabolic vulnerabilities in human neuroblastoma. We combined metabolomics, CRISPR screening and transcriptomic data across >700 solid tumor cell lines and identified dihydroorotate dehydrogenase (DHODH), a critical enzyme in pyrimidine synthesis, as a potential treatment target. Of note, DHODH inhibition is currently under clinical investigation in patients with hematologic malignancies. In neuroblastoma, DHODH expression was identified as an independent risk factor for aggressive disease, and high DHODH levels correlated to worse overall and event-free survival. A subset of tumors with the highest DHODH expression was associated with a dismal prognosis, with a 5-year survival of <10%. In xenograft and transgenic neuroblastoma mouse models treated with the DHODH inhibitor brequinar, tumor growth was dramatically reduced, and survival was extended. Furthermore, brequinar treatment was shown to reduce the expression of MYC targets in three different neuroblastoma models in vivo. A combination of brequinar and temozolomide was curative in the majority of transgenic TH-MYCN neuroblastoma mice, indicating a highly active clinical combination therapy. Overall, DHODH inhibition combined with temozolomide has therapeutic potential in neuroblastoma and we propose this combination for clinical testing.
Authors
Thale Kristin Olsen, Cecilia Dyberg, Bethel Tesfai Embaie, Adele M. Alchahin, Jelena Milosevic, Jane Ding, Jörg Otte, Conny Tümmler, Ida Hed Myrberg, Ellen M. Westerhout, Jan Koster, Rogier Versteeg, Han-Fei Ding, Per Kogner, John Inge Johnsen, David B. Sykes, Ninib Baryawno
Abstract
Gut microbiota (GM) dysbiosis is associated with inflammatory bowel diseases and also with cardiometabolic, neurologic, and autoimmune diseases. GM composition has a direct effect on the immune system, and vice versa, and particularly on regulatory T cell (Treg) homeostasis. Low-dose interleukin-2 (IL-2LD) stimulates Tregs and is a promising treatment for autoimmune and inflammatory diseases. We aimed to evaluate the impacts of IL-2LD on GM, and correlatively on the immune system. We used 16S ribosomal RNA profiling and metagenomics to characterize GM of mice and humans treated or not with IL-2LD. We performed faecal microbiota transplantation (FMT) from IL-2LD-treated to naïve recipient mice and evaluated its effects in models of gut inflammation and diabetes. IL-2LD markedly affects GM composition in mice and humans. Transfer of an IL-2-tuned microbiota by FMT protected C57BL/6J mice from dextran sulphate sodium-induced colitis and prevented diabetes in NOD mice. Metagenomic analyses highlighted a role for several species impacted by IL-2LD and for microbial pathways involved in the biosynthesis of amino acids, short-chain fatty acids, and L-arginine. Our results demonstrate that IL-2LD induces changes in GM that are involved in the immunoregulatory effects of IL-2LD and suggest a cross-talk between Tregs and GM. These results provide novel insights for understanding the mode of action of Treg-directed therapies.
Authors
Nicolas Tchitchek, Otriv Nguekap Tchoumba, Gabriel Pires, Sarah Dandou, Julien Campagne, Guillaume Churlaud, Gwladys Fourcade, Thomas W. Hoffmann, Francesco Strozzi, Camille Gaal, Christophe Bonny, Emmanuelle Le Chatelier, Stanislav Dusko Erlich, Harry Sokol, David Klatzmann
Abstract
Polyamine dysregulation plays key roles in a broad range of human diseases from cancer to neurodegeneration. Snyder-Robinson syndrome (SRS) is the first known genetic disorder of the polyamine pathway, caused by X-linked recessive loss-of-function mutations in spermine synthase. In the Drosophila SRS model, altered spermidine/spermine balance has been associated with increased generation of ROS and aldehydes, consistent with elevated spermidine catabolism. These toxic byproducts cause mitochondrial and lysosomal dysfunction, which are also observed in cells from SRS patients. No efficient therapy is available. We explored the biochemical mechanism and discovered acetyl-CoA reduction and altered protein acetylation as potentially novel pathomechanisms of SRS. We repurposed the FDA-approved drug phenylbutyrate (PBA) to treat SRS using an in vivo Drosophila model and patient fibroblast cell models. PBA treatment significantly restored the function of mitochondria and autolysosomes and extended life span in vivo in the Drosophila SRS model. Treating fibroblasts of patients with SRS with PBA ameliorated autolysosome dysfunction. We further explored the mechanism of drug action and found that PBA downregulates the first and rate-limiting spermidine catabolic enzyme spermidine/spermine N1-acetyltransferase 1 (SAT1), reduces the production of toxic metabolites, and inhibits the reduction of the substrate acetyl-CoA. Taken together, we revealed PBA as a potential modulator of SAT1 and acetyl-CoA levels and propose PBA as a therapy for SRS and potentially other polyamine dysregulation–related diseases.
Authors
Xianzun Tao, Yi Zhu, Zoraida Diaz-Perez, Seok-Ho Yu, Jackson R. Foley, Tracy Murray Stewart, Robert A. Casero Jr., Richard Steet, R. Grace Zhai
Abstract
Merkel cell carcinoma (MCC) is an aggressive neuroendocrine carcinoma of the skin with 2 etiologies. Merkel cell polyomavirus (MCPyV) integration is present in about 80% of all MCC. Virus-positive MCC (MCCP) tumors have few somatic mutations and usually express WT p53 (TP53). By contrast, virus-negative MCC (MCCN) tumors present with a high tumor mutational burden and predominantly UV mutational signature. MCCN tumors typically contain mutated TP53. MCCP tumors express 2 viral proteins: MCPyV small T antigen and a truncated form of large T antigen. MCPyV ST specifically activates expression of MDM2, an E3 ubiquitin ligase of p53, to inhibit p53-mediated tumor suppression. In this study, we assessed the efficacy of milademetan, a potent, selective, and orally available MDM2 inhibitor in several MCC models. Milademetan reduced cell viability of WT p53 MCC cell lines and triggered a rapid and sustained p53 response. Milademetan showed a dose-dependent inhibition of tumor growth in MKL-1 xenograft and patient-derived xenograft models. Here, along with preclinical data for the efficacy of milademetan in WT p53 MCC tumors, we report several in vitro and in vivo models useful for future MCC studies.
Authors
Varsha Ananthapadmanabhan, Thomas C. Frost, Kara M. Soroko, Aine Knott, Brianna J. Magliozzi, Prafulla C. Gokhale, Vijaya G. Tirunagaru, Robert C. Doebele, James A. DeCaprio
Abstract
Striated preferentially expressed protein kinase (SPEG), a myosin light chain kinase, is mutated in centronuclear myopathy (CNM) and/or dilated cardiomyopathy. No precise therapies are available against this disorder, and gene replacement therapy is not a feasible option due to the large size of SPEG. We evaluated the potential of dynamin-2 (DNM2) reduction as a potential therapeutic strategy as it has been shown to revert muscle phenotypes in mouse models of CNM caused by MTM1, DNM2, and BIN1 mutations. We determined that SPEGβ interacts with DNM2, and SPEG deficiency causes an increase in DNM2 levels. The DNM2 reduction strategy in Speg-KO mice was associated with an increase in life span, body weight, and motor performance. Additionally, it normalized the distribution of triadic proteins, triad ultrastructure, and triad number, and restored phosphatidylinositol-3-phosphate levels in SPEG-deficient skeletal muscles. While DNM2 reduction rescued the myopathy phenotype, it did not improve cardiac dysfunction, indicating a differential tissue-specific function. Combining DNM2 reduction with other strategies may be needed to target both the cardiac and skeletal defects associated with SPEG deficiency. DNM2 reduction should be explored as a therapeutic strategy against other genetic myopathies (and dystrophies) associated with a high level of DNM2.
Authors
Qifei Li, Jasmine Lin, Jeffrey J. Widrick, Shiyu Luo, Gu Li, Yuanfan Zhang, Jocelyn Laporte, Mark A. Perrella, Xiaoli Liu, Pankaj B. Agrawal
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