Single cancer cell sequencing studies currently use randomly-selected cells, limiting correlations between genomic aberrations, morphology and spatial localization. We laser-captured microdissected single cells from morphologically-distinct areas of primary breast cancer and corresponding lymph node metastasis and performed whole-exome or deep-target sequencing of greater than 100 such cells. Two major subclones co-existed in different areas of the primary tumor, and the lymph node metastasis originated from a minor subclone in the invasive front of the primary tumor with additional copy number changes including 8q gain, but no additional point mutations in driver genes. Lack of metastasis-specific driver events lead us to assess whether other clonal and subclonal genomic aberrations pre-existing in primary tumors contribute to lymph node metastasis. Gene mutations and copy number variations analyzed in five breast cancer tissue sample sets revealed that copy number variations in several genomic regions, including areas within chromosome 1p, 8q, 9p, 12q and 20q, harboring several metastasis-associated genes, were consistently associated with lymph node metastasis. Moreover, clonal expansion was observed in an area of morphologically-normal breast epithelia, likely driven by a driver mutation and a subsequent amplification in chromosome 1q. Our study illuminates the molecular evolution of breast cancer and genomic aberrations contributing to metastases.
Li Bao, Zhaoyang Qian, Maria B. Lyng, Ling Wang, Yuan Yu, Ting Wang, Xiuqing Zhang, Huanming Yang, Nils Brünner, Jun Wang, Henrik J. Ditzel
Spinocerebellar ataxia type 1 (SCA1) is an adult-onset neurodegenerative disease caused by a polyglutamine expansion in the protein ATXN1, which is involved in transcriptional regulation. Although symptoms appear relatively late in life, primarily from cerebellar dysfunction, pathogenesis begins early, with brain-wide transcriptional changes detectable as early as a week after birth in SCA1 knock-in mice. Given the importance of this postnatal period for cerebellar development, we asked whether this region might be developmentally altered by mutant ATXN1. We found that expanded ATXN1 stimulates the proliferation of postnatal cerebellar stem cells in SCA1 mice. These hyper-proliferating stem cells tended to differentiate into GABAergic inhibitory interneurons rather than astrocytes; this significantly increased the GABAergic inhibitory interneuron synaptic connections, disrupting cerebellar Purkinje cell function in a non-cell autonomous manner. We confirmed the increased basket cell-Purkinje cell connectivity in human SCA1 patients. Mutant ATXN1 thus alters the neural circuitry of the developing cerebellum, setting the stage for the later vulnerability of Purkinje cells to SCA1. We propose that other late-onset degenerative diseases may also be rooted in subtle developmental derailments.
Chandrakanth Reddy Edamakanti, Jeehaeh Do, Alessandro Didonna, Marco Martina, Puneet Opal
Recent studies reveal that airway epithelial cells are critical pulmonary circadian pacemaker cells, mediating rhythmic inflammatory responses. Using mouse models, we now identify the rhythmic circadian repressor REV-ERB as essential to the mechanism coupling the pulmonary clock to innate immunity, involving both myeloid, and bronchial epithelial cells in temporal gating and determining amplitude of response to inhaled endotoxin. Dual mutation of REV-ERBα and its paralog REV-ERBβ in bronchial epithelia further augmented inflammatory responses and chemokine activation, but also initiated a basal inflammatory state, revealing a critical homeostatic role for REV-ERB proteins in the suppression of the endogenous pro-inflammatory mechanism in un-challenged cells. However, REV-ERBα plays the dominant role as deletion of REV-ERBβ alone had no impact on inflammatory responses. In turn, inflammatory challenges cause striking changes in stability and degradation of REV-ERBα protein, driven by SUMOylation and ubiquitination. We developed a novel selective oxazole-based inverse agonist of REV-ERB, which protects REV-ERBα protein from degradation and used this to reveal how pro-inflammatory cytokines trigger rapid degradation of REV-ERα in the elaboration of an inflammatory response. Thus, dynamic changes in stability of REV-ERα protein couple the core clock to innate immunity.
Marie Pariollaud, Julie Gibbs, Thomas Hopwood, Sheila Brown, Nicola Begley, Ryan Vonslow, Toryn Poolman, Baoqiang Guo, Ben Saer, D. Heulyn Jones, James P. Tellam, Stefano Bresciani, Nicholas C.O. Tomkinson, Justyna Wojno-Picon, Anthony W.J. Cooper, Dion A. Daniels, Ryan P. Trump, Daniel Grant, William Zuercher, Timothy M. Willson, Andrew S. MacDonald, Brian Bolognese, Patricia L. Podolin, Yolanda Sanchez, Andrew S.I. Loudon, David W. Ray
ONC201 is a first-in-class, orally active anti-tumor agent that upregulates cytotoxic TRAIL pathway signaling in cancer cells. ONC201 has demonstrated safety and preliminary efficacy in the first-in-human trial where patients were dosed every 3 weeks. We hypothesized that dose-intensification of ONC201 may impact anti-tumor efficacy. We discovered that ONC201 exerts dose- and schedule-dependent effects on tumor progression and cell-death signaling in vivo. With dose intensification, we note a potent anti-metastasis effect and inhibition of cancer cell migration and invasion. Our preclinical results prompted a change in ONC201 dosing in all open clinical trials. We observe accumulation of activated NK+ and CD3+ cells within ONC201-treated tumors, and NK-cell depletion inhibits ONC201 efficacy in vivo, including against TRAIL/ONC201-resistant Bax–/– tumors. Immunocompetent NCR1-GFP mice with GFP-expressing NK-cells demonstrate GFP(+)-NK cell infiltration of syngeneic MC38 colorectal tumors. Activation of primary human NK cells and increased de-granulation occur in response to ONC201. Co-culture experiments identified a role for TRAIL in human NK-mediated anti-tumor cytotoxicity. Preclinical results indicate potential utility for ONC201 plus anti-PD-1 therapy. We observed an increase in activated TRAIL-secreting NK cells in the peripheral blood of patients after receiving ONC201 treatment. The results offer a unique pathway of immune stimulation for cancer therapy.
Jessica Wagner, C. Leah Kline, Lanlan Zhou, Kerry S. Campbell, Alexander W. MacFarlane, Anthony J. Olszanski, Kathy Q. Cai, Harvey H. Hensley, Eric A. Ross, Marie D. Ralff, Andrew Zloza, Charles B. Chesson, Jenna H. Newman, Howard Kaufman, Joseph R. Bertino, Mark N. Stein, Wafik El-Deiry
The remarkable regeneration capability of skeletal muscle depends on coordinated proliferation and differentiation of satellite cells. The self-renewal of satellite cells is critical for long-term maintenance of muscle regeneration potential. Hypoxia profoundly affects the proliferation, differentiation, and self-renewal of cultured myoblasts. However, the physiological relevance of hypoxia and hypoxia signaling in satellite cells in vivo remains largely unknown. Here, we report that satellite cells are in an intrinsic hypoxic state in vivo and express hypoxia-inducible factor 2A (HIF2A). HIF2A promotes the stemness and long-term homeostatic maintenance of satellite cells by maintaining the quiescence, increasing the self-renewal and blocking the myogenic differentiation of satellite cells. HIF2A stabilization in satellite cells cultured under normoxia augmented their engraftment potential in regenerative muscle. Reversely, HIF2A ablation led to the depletion of satellite cells and the consequent regenerative failure in the long-term. In contrast, transient pharmacological inhibition of HIF2A accelerated muscle regeneration by increasing satellite cell proliferation and differentiation. Mechanistically, HIF2A induces the quiescence/self-renewal of satellite cells by binding the promoter of Spry1 gene and activating Spry1 expression. These findings suggest that HIF2A is a pivotal mediator of hypoxia signaling in satellite cells and may be therapeutically targeted to improve muscle regeneration.
Liwei Xie, Amelia Yin, Anna S. Nichenko, Aaron M. Beedle, Jarrod A. Call, Hang Yin
Painful diabetic neuropathy (PDN) is an intractable complication of diabetes that affects 25% of patients. PDN is characterized by neuropathic pain and small-fiber degeneration, accompanied by dorsal root ganglion (DRG) nociceptor hyperexcitability and loss of their axons within the skin. The molecular mechanisms underlying DRG nociceptor hyperexcitability and small-fiber degeneration in PDN are unknown. We hypothesize that chemokine CXCL12/CXCR4 signaling is central to this mechanism, as we have shown that CXCL12/CXCR4 signaling is necessary for the development of mechanical allodynia, a pain hypersensitivity behavior common in PDN. Focusing on DRG neurons expressing the sodium channel Nav1.8, we applied transgenic, electrophysiological, imaging, and chemogenetic techniques to test this hypothesis. In the high-fat diet mouse model of PDN, we were able to prevent and reverse mechanical allodynia and small-fiber degeneration by limiting CXCR4 signaling or neuronal excitability. This study reveals that excitatory CXCR4/CXCL12 signaling in Nav1.8-positive DRG neurons plays a critical role in the pathogenesis of mechanical allodynia and small-fiber degeneration in a mouse model of PDN. Hence, we propose that targeting CXCR4-mediated DRG nociceptor hyperexcitability is a promising therapeutic approach for disease-modifying treatments for this currently intractable and widespread affliction.
Nirupa D. Jayaraj, Bula J. Bhattacharyya, Abdelhak A. Belmadani, Dongjun Ren, Craig A. Rathwell, Sandra Hackelberg, Brittany E. Hopkins, Herschel R. Gupta, Richard J. Miller, Daniela M. Menichella
Increasing evidence suggests a role for excessive intake of fructose in the Western diet as a contributor to the current epidemics of metabolic syndrome and obesity. Hereditary fructose intolerance (HFI) is a difficult and potentially lethal orphan disease associated with impaired fructose metabolism. In HFI, the deficiency of a particular aldolase, aldolase B, results in the accumulation of intracellular phosphorylated fructose thus leading to phosphate sequestration and depletion, increased ATP turnover and a plethora of conditions leading to clinical manifestations including fatty liver, hyperuricemia, Fanconi syndrome and severe hypoglycemia. Unfortunately, to date, there is no treatment for HFI and avoiding sugar and fructose in our society has become quite challenging. In this report, through use of genetically modified mice and pharmacological inhibitors, we demonstrate that the absence or inhibition of ketohexokinase (Khk), an enzyme upstream of aldolase B, is sufficient to prevent hypoglycemia and liver and intestinal injury associated with HFI using aldolase B knockout mice. We thus provide evidence for the first time of a potential therapeutic approach for this condition. Mechanistically, our studies suggest that it is the inhibition of the Khk C isoform, not the A isoform, that protects animals from HFI.
Miguel A. Lanaspa, Ana Andres-Hernando, David J. Orlicky, Christina Cicerchi, Cholsoon Jang, Nanxing Li, Tamara Milagres, Masanari Kuwabara, Michael F. Wempe, Joshua D. Rabinowitz, Richard J. Johnson, Dean R. Tolan
Obesity is a major risk factor for insulin resistance and type 2 diabetes. In adipose tissue, obesity-mediated insulin resistance correlates with the accumulation of proinflammatory macrophages and inflammation. However, the causal relationship of these events is unclear. Here, we report that obesity-induced insulin resistance in mice precedes macrophage accumulation and inflammation in adipose tissue. Using a mouse model that combines genetically induced, adipose-specific insulin resistance (mTORC2-knockout) and diet-induced obesity, we found that insulin resistance causes local accumulation of proinflammatory macrophages. Mechanistically, insulin resistance in adipocytes results in production of the chemokine monocyte chemoattractant protein 1 (MCP1), which recruits monocytes and activates proinflammatory macrophages. Finally, insulin resistance (high homeostatic model assessment of insulin resistance [HOMA-IR]) correlated with reduced insulin/mTORC2 signaling and elevated MCP1 production in visceral adipose tissue from obese human subjects. Our findings suggest that insulin resistance in adipose tissue leads to inflammation rather than vice versa.
Mitsugu Shimobayashi, Verena Albert, Bettina Woelnerhanssen, Irina C. Frei, Diana Weissenberger, Anne Christin Meyer-Gerspach, Nicolas Clement, Suzette Moes, Marco Colombi, Jerome A. Meier, Marta M. Swierczynska, Paul Jenö, Christoph Beglinger, Ralph Peterli, Michael N. Hall
Polypeptide vaccines effectively activate human T cells but suffer from poor biological stability, which confines both transport logistics and in vivo therapeutic activity. Synthetic biology has the potential to address these limitations through the generation of highly stable antigenic “mimics” using subunits that do not exist in the natural world. We developed a platform based on D–amino acid combinatorial chemistry and used this platform to reverse engineer a fully artificial CD8+ T cell agonist that mirrored the immunogenicity profile of a native epitope blueprint from influenza virus. This nonnatural peptide was highly stable in human serum and gastric acid, reflecting an intrinsic resistance to physical and enzymatic degradation. In vitro, the synthetic agonist stimulated and expanded an archetypal repertoire of polyfunctional human influenza virus–specific CD8+ T cells. In vivo, specific responses were elicited in naive humanized mice by subcutaneous vaccination, conferring protection from subsequent lethal influenza challenge. Moreover, the synthetic agonist was immunogenic after oral administration. This proof-of-concept study highlights the power of synthetic biology to expand the horizons of vaccine design and therapeutic delivery.
John J. Miles, Mai Ping Tan, Garry Dolton, Emily S.J. Edwards, Sarah A.E. Galloway, Bruno Laugel, Mathew Clement, Julia Makinde, Kristin Ladell, Katherine K. Matthews, Thomas S. Watkins, Katie Tungatt, Yide Wong, Han Siean Lee, Richard J. Clark, Johanne M. Pentier, Meriem Attaf, Anya Lissina, Ann Ager, Awen Gallimore, Pierre J. Rizkallah, Stephanie Gras, Jamie Rossjohn, Scott R. Burrows, David K. Cole, David A. Price, Andrew K. Sewell
UL18 is a human CMV (HCMV) MHC class I (MHCI) homolog that efficiently inhibits leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1)+ NK cells. We found an association of LILRB1 polymorphisms in the regulatory regions and ligand-binding domains with control of HCMV in transplant patients. Naturally occurring LILRB1 variants expressed in model NK cells showed functional differences with UL18 and classical MHCI, but not with HLA-G. The altered functional recognition was recapitulated in binding assays with the binding domains of LILRB1. Each of 4 nonsynonymous substitutions in the first 2 LILRB1 immunoglobulin domains contributed to binding with UL18, classical MHCI, and HLA-G. One of the polymorphisms controlled addition of an N-linked glycan, and that mutation of the glycosylation site altered binding to all ligands tested, including enhancing binding to UL18. Together, these findings indicate that specific LILRB1 alleles that allow for superior immune evasion by HCMV are restricted by mutations that limit LILRB1 expression selectively on NK cells. The polymorphisms also maintained an appropriate interaction with HLA-G, fitting with a principal role of LILRB1 in fetal tolerance.
Kang Yu, Chelsea L. Davidson, Agnieszka Wójtowicz, Luiz Lisboa, Ting Wang, Adriana M. Airo, Jean Villard, Jeremie Buratto, Tatyana Sandalova, Adnane Achour, Atul Humar, Katia Boggian, Alexia Cusini, Christian van Delden, Adrian Egli, Oriol Manuel, Nicolas Mueller, Pierre-Yves Bochud, Swiss Transplant Cohort Study, Deborah N. Burshtyn