Therapeutics
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
Abstract
Disrupted liver regeneration following hepatectomy represents an “undruggable” clinical challenge associated with poor patient outcomes. Yes-associated protein (YAP), a transcriptional co-activator which is repressed by the Hippo pathway, is instrumental in liver regeneration. We have previously described an alternative, Hippo-independent mechanism of YAP activation mediated by tyrosine-protein phosphatase non-receptor type 11 (SHP2) inhibition. Herein, we examined the effects of YAP activation with a selective SHP1/SHP2 inhibitor, NSC-87877, on liver regeneration in murine partial hepatectomy models. In our studies, NSC-87877 led to accelerated hepatocyte proliferation, improved liver regeneration, and decreased markers of injury following partial hepatectomy. The effects of NSC-87877 were lost in mice with hepatocyte-specific Yap/Taz deletion, which demonstrated dependence on these molecules for the enhanced regenerative response. Furthermore, administration of NSC-87877 to murine models of non-alcoholic steatohepatitis was associated with improved survival and decreased markers of injury post-hepatectomy. Evaluation of transcriptomic changes in the context of NSC-87877 administration revealed reduction in fibrotic signaling and augmentation of cell cycle signaling. Cytoprotective changes included downregulation of Nr4a1, an apoptosis inducer. Collectively, the data suggest that SHP2 inhibition induces a pro-proliferative and cytoprotective enhancement of liver regeneration dependent on YAP.
Authors
Ryan D. Watkins, EeeLN H. Buckarma, Jennifer L. Tomlinson, Chantal E. McCabe, Jennifer A. Yonkus, Nathan W. Werneburg, Rachel L. Bayer, Patrick P. Starlinger, Keith D. Robertson, Chen Wang, Gregory J. Gores, Rory L. Smoot
Abstract
The ribosomal protein S6 kinase 1 (S6K1) is a relevant effector downstream the mammalian target of rapamycin complex 1 (mTORC1), best known for its role in the control of lipid homeostasis. Consistent with this, mice lacking the S6k1 gene have a defect in their ability to induce the commitment of fat precursor cells to the adipogenic lineage, which contributes to a significant reduction of fat mass. Here, we assess the therapeutic blockage of S6K1 in diet-induced obese mice challenged with LY2584702 tosylate, a specific oral S6K1 inhibitor initially developed for the treatment of solid tumours. We show that diminished S6K1 activity hampers fat mass expansion and ameliorates dyslipidaemia and hepatic steatosis, while modifying transcriptome-wide gene expression programs relevant for adipose and liver function. Accordingly, impaired mTORC1 signalling in fat (decreased) and liver (increased) co-segregated with defective epithelial-mesenchymal transition, being prominent the decreased expression of Cd36 (coding for a fatty acid translocase) and Lgals1 (Galectin 1) in both tissues. All these factors combined align with reduced adipocyte size and improved lipidomic signatures in the liver, while hepatic steatosis and hypertriglyceridemia were improved in treatments lasting either 3 months or 6 weeks.
Authors
Aina Lluch, Sonia R. Veiga, Jèssica Latorre, José M. Moreno-Navarrete, Núria Bonifaci, Van Dien Nguyen, You Zhou, Marcus Horing, Gerhard Liebisch, Vesa M. Olkkonen, David Llobet-Navas, George Thomas, Ruth Rodriguez-Barrueco, José M. Fernández-Real, Sara C. Kozma, Francisco J. Ortega
Abstract
Natural killer (NK) cell exhaustion is caused by chronic exposure to activating stimuli during viral infection, tumorigenesis, and prolonged cytokine treatment. Evidence suggests that exhaustion may play a role in disease progression, however relative to T cell exhaustion, the mechanisms underlying NK cell exhaustion and methods of reversing it are poorly understood. Here, we describe a novel in vitro model of exhaustion that employs plate-bound agonists of the NK cell activating receptors NKp46 and NKG2D to induce canonical exhaustion phenotypes. In this model, prolonged activation results in downregulation of activating receptors, upregulation of checkpoint markers, decreased cytokine production and cytotoxicity in vitro, defects in glycolytic metabolism, and decreased persistence, function, and tumor control in vivo. Furthermore, we discover a beneficial effect of NK cell inhibitory receptor signaling during exhaustion. By simultaneously engaging the inhibitory receptor NKG2A during activation in our model, cytokine production and cytotoxicity defects can be mitigated, suggesting that balancing positive and negative signals integrated by effector NK cells can be beneficial for anti-tumor immunity. Together, these data uncover some of the mechanisms underlying NK cell exhaustion in humans and establish our novel in vitro model as a valuable tool for studying the processes regulating exhaustion.
Authors
Jacob A. Myers, Dawn Schirm, Laura Bendzick, Rachel Hopps, Carly Selleck, Peter Hinderlie, Martin Felices, Jeffrey S. Miller
Abstract
Epileptic seizures are common sequelae of stroke, acute brain injury, and chronic neurodegenerative diseases, including Alzheimer’s disease (AD), and cannot be effectively controlled in approximately 40% of patients, necessitating the development of novel therapeutic agents. Activation of the A1 receptor (A1R) by endogenous adenosine is an intrinsic mechanism to self-terminate seizures and protect neurons from excitotoxicity. However, targeting A1R for neurological disorders has been hindered by side effects associated with its broad expression outside the nervous system. Here we aim to target the neural-specific A1R/neurabin/regulator of G protein signaling 4 (A1R/neurabin/RGS4) complex that dictates A1R signaling strength and response outcome in the brain. We developed a peptide that blocks the A1R-neurabin interaction to enhance A1R activity. Intracerebroventricular or i.n. administration of this peptide shows marked protection against kainate-induced seizures and neuronal death. Furthermore, in an AD mouse model with spontaneous seizures, nasal delivery of this blocking peptide reduces epileptic spike frequency. Significantly, the anticonvulsant and neuroprotective effects of this peptide are achieved through enhanced A1R function in response to endogenous adenosine in the brain, thus, avoiding side effects associated with A1R activation in peripheral tissues and organs. Our study informs potentially new anti-seizure therapy applicable to epilepsy and other neurological illness with comorbid seizures.
Authors
Shalini Saggu, Yunjia Chen, Liping Chen, Diana Pizarro, Sandipan Pati, Wen Jing Law, Lori McMahon, Kai Jiao, Qin Wang
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