A selection of recent publications from Ludwig Oxford researchers.
Epigenetic DNA modifications are essential for normal cell function in vertebrates, but they can also be hotspots of mutagenesis. Methylcytosine in particular has long been known to be less stable than other nucleotides and spontaneously deaminates to thymine. Beyond this well-established phenomenon, however, the influence of epigenetic marks on mutagenesis has recently become an active field of investigation. In this review, we summarize current knowledge of the interactions between different DNA modifications and other mutagenic processes. External mutagens, such as UV light or smoking carcinogens, affect modified cytosines differently from unmodified ones, and modified cytosine can in some cases be protective rather than mutagenic. Notably, cell-intrinsic processes, such as DNA replication, also appear to influence the mutagenesis of modified cytosines. Altogether, evidence is accumulating to show that epigenetic changes have a profound influence on tissue-specific mutation accumulation.
RIPK2 mediates inflammatory signaling by the bacteria-sensing receptors NOD1 and NOD2. Kinase inhibitors targeting RIPK2 are a proposed strategy to ameliorate NOD-mediated pathologies. Here, we reveal that RIPK2 kinase activity is dispensable for NOD2 inflammatory signaling and show that RIPK2 inhibitors function instead by antagonizing XIAP-binding and XIAP-mediated ubiquitination of RIPK2. We map the XIAP binding site on RIPK2 to the loop between β2 and β3 of the N-lobe of the kinase, which is in close proximity to the ATP-binding pocket. Through characterization of a new series of ATP pocket-binding RIPK2 inhibitors, we identify the molecular features that determine their inhibition of both the RIPK2-XIAP interaction, and of cellular and in vivoNOD2 signaling. Our study exemplifies how targeting of the ATP-binding pocket in RIPK2 can be exploited to interfere with the RIPK2-XIAP interaction for modulation of NOD signaling.
How cells coordinate the response to fluctuating carbon and nitrogen availability required to maintain effective homeostasis is a key issue. Amino acid limitation that inactivates mTORC1 promotes de-phosphorylation and nuclear translocation of Transcription Factor EB (TFEB), a key transcriptional regulator of lysosome biogenesis and autophagy that is deregulated in cancer and neurodegeneration. Beyond its cytoplasmic sequestration, how TFEB phosphorylation regulates its nuclear-cytoplasmic shuttling, and whether TFEB can coordinate amino acid supply with glucose availability is poorly understood. Here we show that TFEB phosphorylation on S142 primes for GSK3β phosphorylation on S138, and that phosphorylation of both sites but not either alone activates a previously unrecognized nuclear export signal (NES). Importantly, GSK3β is inactivated by AKT in response to mTORC2 signaling triggered by glucose limitation. Remarkably therefore, the TFEB NES integrates carbon (glucose) and nitrogen (amino acid) availability by controlling TFEB flux through a nuclear import-export cycle.
Desmosome components are frequently mutated in cardiac and cutaneous disorders in animals and humans and enhanced inflammation is a common feature of these diseases. Previous studies showed that inhibitor of Apoptosis Stimulating p53 Protein (iASPP) regulates desmosome integrity at cell-cell junctions and transcription in the nucleus, and its deficiency causes cardiocutaneous disorder in mice, cattle, and humans. As iASPP is a ubiquitously expressed shuttling protein with multiple functions, a key question is whether the observed cardiocutaneous phenotypes are caused by loss of a cell autonomous role of iASPP in cardiomyocytes and keratinocytes specifically or by a loss of iASPP in other cell types such as immune cells. To address this, we developed cardiomyocyte-specific and keratinocyte-specific iASPP-deficient mouse models and show that the cell-type specific loss of iASPP in cardiomyocytes or keratinocytes is sufficient to induce cardiac or cutaneous disorders, respectively. Additionally, keratinocyte-specific iASPP-deficient mice have delayed eyelid development and wound healing. In keratinocytes, junctional iASPP is critical for stabilizing desmosomes and iASPP deficiency results in increased and disorganized cell migration, as well as impaired cell adhesion, consistent with delayed wound healing. The identification of a cell autonomous role of iASPP deficiency in causing cardiocutaneous syndrome, impaired eyelid development and wound healing suggests that variants in the iASPP gene also may contribute to polygenic heart and skin diseases.
Indoleamine 2,3 dioxygenase-1 (IDO1) is a powerful immunoregulatory enzyme that is deficient in patients with type 1 diabetes (T1D). In this study, we present the first systematic evaluation of IDO1 expression and localization in human pancreatic tissue. Although IDO1 was constitutively expressed in β-cells from donors without diabetes, less IDO1 was expressed in insulin-containing islets from double autoantibody-positive donors and patients with recent-onset T1D, although it was virtually absent in insulin-deficient islets from donors with T1D. Scatter plot analysis suggested that IDO1 decay occurred in individuals with multiple autoantibodies, prior to β-cell demise. IDO1 impairment might therefore contribute to β-cell demise and could potentially emerge as a promising therapeutic target.
The carotid body is a peripheral chemoreceptor that plays a central role in mammalian oxygen homeostasis. In response to sustained hypoxia, it manifests rapid cellular proliferation and an associated increase in responsiveness to hypoxia. Understanding the cellular and molecular mechanisms underlying these processes is of interest both to specialised chemoreceptive functions of that organ and potentially to the general physiology and pathophysiology of cellular hypoxia. We have combined cell lineage tracing technology and conditionally inactivated alleles in recombinant mice to examine the role of components of the HIF hydroxylase pathway in specific cell types within the carotid body. We show that exposure to sustained hypoxia (10 % oxygen) drives rapid expansion of the Type I, tyrosine hydroxylase expressing cell lineage, with little transdifferentiation to or from that lineage. Inactivation of a specific HIF isoform, HIF-2α, in the Type I cells was associated with greatly reduced proliferation of Type I cells and hypoxic ventilatory responses, with ultrastructural evidence of an abnormality in the action of hypoxia on dense core secretory vesicles. We also show that inactivation of the principal HIF prolyl hydroxylase PHD2 within the Type I cell lineage is sufficient to cause multi-lineage expansion of the carotid body, with characteristics resembling paragangliomas. These morphological changes were dependent on the integrity of HIF-2α. These findings implicate specific components of the HIF hydroxylase pathway (PHD2 and HIF-2α) within Type I cells of the carotid body in the oxygen sensing and adaptive functions of that organ. This article is protected by copyright. All rights reserved.
Since the publication of this paper, the authors noticed that James E. East was assigned to the incorrect affiliation. The affiliation information is provided correctly, above.
Programmed cell death ligand 1 (PD-L1) interacts with programmed cell death protein-1 (PD-1) as an immune checkpoint. Reactivating the immune response by inhibiting PD-L1 using therapeutic antibodies provides substantial clinical benefits in many, though not all, melanoma patients. However, transcriptional suppression of PD-L1 expression as an alternative therapeutic anti-melanoma strategy has not been exploited. Here we provide biochemical evidence demonstrating that ultraviolet radiation (UVR) induction of PD-L1 in skin is directly controlled by nuclear factor E2-related transcription factor 2 (NRF2). Depletion of NRF2 significantly induces tumor infiltration by both CD8+ and CD4+ T cells to suppress melanoma progression, and combining NRF2 inhibition with anti-PD-1 treatment enhanced its anti-tumor function. Our studies identify a critical and targetable PD-L1 upstream regulator and provide an alternative strategy to inhibit the PD-1/PD-L1 signaling in melanoma treatment.
Preclinical work has long focused on male animals, though biological sex clearly influences risk for certain diseases, including many psychiatric disorders. Such disorders are often treated by drugs targeting the CNS norepinephrine system. Despite roles for noradrenergic neurons in behavior and neuropsychiatric disease models, their molecular characterization has lagged. We profiled mouse noradrenergic neurons in vivo, defining over 3,000 high-confidence transcripts expressed therein, including druggable receptors. We uncovered remarkable sex differences in gene expression, including elevated expression of the EP3 receptor in females-which we leverage to illustrate the behavioral and pharmacologic relevance of these findings-and of Slc6a15 and Lin28b, both major depressive disorder (MDD)-associated genes. Broadly, we present a means of transcriptionally profiling locus coeruleus under baseline and experimental conditions. Our findings underscore the need for preclinical work to include both sexes and suggest that sex differences in noradrenergic neurons may underlie behavioral differences relevant to disease.
Epidemiological evidence has long associated environmental mutagens with increased cancer risk. However, links between specific mutation-causing processes and the acquisition of individual driver mutations have remained obscure. Here we have used public cancer sequencing data from 11,336 cancers of various types to infer the independent effects of mutation and selection on the set of driver mutations in a cancer type. First, we detect associations between a range of mutational processes, including those linked to smoking, ageing, APOBEC and DNA mismatch repair (MMR) and the presence of key driver mutations across cancer types. Second, we quantify differential selection between well-known alternative driver mutations, including differences in selection between distinct mutant residues in the same gene. These results show that while mutational processes have a large role in determining which driver mutations are present in a cancer, the role of selection frequently dominates.
Adamantinomatous craniopharyngiomas (ACPs) are clinically challenging tumours, the majority of which have activating mutations in CTNNB1. They are histologically complex, showing cystic and solid components, the latter comprised of different morphological cell types (e.g. β-catenin-accumulating cluster cells and palisading epithelium), surrounded by a florid glial reaction with immune cells. Here, we have carried out RNA sequencing on 18 ACP samples and integrated these data with an existing ACP transcriptomic dataset. No studies so far have examined the patterns of gene expression within the different cellular compartments of the tumour. To achieve this goal, we have combined laser capture microdissection with computational analyses to reveal groups of genes that are associated with either epithelial tumour cells (clusters and palisading epithelium), glial tissue or immune infiltrate. We use these human ACP molecular signatures and RNA-Seq data from two ACP mouse models to reveal that cell clusters are molecularly analogous to the enamel knot, a critical signalling centre controlling normal tooth morphogenesis. Supporting this finding, we show that human cluster cells express high levels of several members of the FGF, TGFB and BMP families of secreted factors, which signal to neighbouring cells as evidenced by immunostaining against the phosphorylated proteins pERK1/2, pSMAD3 and pSMAD1/5/9 in both human and mouse ACP. We reveal that inhibiting the MAPK/ERK pathway with trametinib, a clinically approved MEK inhibitor, results in reduced proliferation and increased apoptosis in explant cultures of human and mouse ACP. Finally, we analyse a prominent molecular signature in the glial reactive tissue to characterise the inflammatory microenvironment and uncover the activation of inflammasomes in human ACP. We validate these results by immunostaining against immune cell markers, cytokine ELISA and proteome analysis in both solid tumour and cystic fluid from ACP patients. Our data support a new molecular paradigm for understanding ACP tumorigenesis as an aberrant mimic of natural tooth development and opens new therapeutic opportunities by revealing the activation of the MAPK/ERK and inflammasome pathways in human ACP.
© 2018 Elsevier B.V. The publisher regrets that some symbols were incorrectly typeset. All occurrences of “≫ >” have been corrected to “≫”. The publisher would like to apologise for any inconvenience caused.
Negative regulation of immune pathways is essential to achieve resolution of immune responses and to avoid excess inflammation. DNA stimulates type I IFN expression through the DNA sensor cGAS, the second messenger cGAMP, and the adaptor molecule STING Here, we report that STING degradation following activation of the pathway occurs through autophagy and is mediated by p62/SQSTM1, which is phosphorylated by TBK1 to direct ubiquitinated STING to autophagosomes. Degradation of STING was impaired in p62-deficient cells, which responded with elevated IFN production to foreign DNA and DNA pathogens. In the absence of p62, STING failed to traffic to autophagy-associated vesicles. Thus, DNA sensing induces the cGAS-STING pathway to activate TBK1, which phosphorylates IRF3 to induce IFN expression, but also phosphorylates p62 to stimulate STING degradation and attenuation of the response.
CD8+ cytolytic T lymphocytes are essential players of anti-tumor immune responses. On tumors, they recognize peptides of about 8-to-10 amino acids that generally result from the degradation of cellular proteins by the proteasome. Until a decade ago, these peptides were thought to solely correspond to linear fragments of proteins that were liberated after the hydrolysis of the peptide bonds located at their extremities. However, several examples of peptides containing two fragments originally distant in the protein sequence challenged this concept and demonstrated that proteasome could also splice peptides together by creating a new peptide bond between two distant fragments. Unexpectedly, peptide splicing emerges as an essential way to increase the peptide repertoire diversity as these spliced peptides were shown to represent up to 25% of the peptides presented on a cell by MHC class I. Here, we review the different steps that led to the discovery of peptide splicing by the proteasome as well as the lightening offered by the recent progresses of mass spectrometry and bioinformatics in the analysis of the spliced peptide repertoire.
Single-cell messenger RNA sequencing (scRNA-seq) has emerged as a powerful tool to study cellular heterogeneity within complex tissues. Subpopulations of cells with common gene expression profiles can be identified by applying unsupervised clustering algorithms. However, technical variance is a major confounding factor in scRNA-seq, not least because it is not possible to replicate measurements on the same cell. Here, we present BEARscc, a tool that uses RNA spike-in controls to simulate experiment-specific technical replicates. BEARscc works with a wide range of existing clustering algorithms to assess the robustness of clusters to technical variation. We demonstrate that the tool improves the unsupervised classification of cells and facilitates the biological interpretation of single-cell RNA-seq experiments.
A defect in indoleamine 2,3-dioxygenase 1 (IDO1), which is responsible for immunoregulatory tryptophan catabolism, impairs development of immune tolerance to autoantigens in NOD mice, a model for human autoimmune type 1 diabetes (T1D). Whether IDO1 function is also defective in T1D is still unknown. We investigated IDO1 function in sera and peripheral blood mononuclear cells (PBMCs) from children with T1D and matched controls. These children were further included in a discovery study to identify SNPs in IDO1 that might modify the risk of T1D. T1D in children was characterized by a remarkable defect in IDO1 function. A common haplotype, associated with dysfunctional IDO1, increased the risk of developing T1D in the discovery and also confirmation studies. In T1D patients sharing such a common IDO1 haplotype, incubation of PBMCs in vitro with tocilizumab (TCZ) - an IL-6 receptor blocker - would, however, rescue IDO1 activity. In an experimental setting with diabetic NOD mice, TCZ was found to restore normoglycemia via IDO1-dependent mechanisms. Thus, functional SNPs of IDO1 are associated with defective tryptophan catabolism in human T1D, and maneuvers aimed at restoring IDO1 function would be therapeutically effective in at least a subgroup of T1D pediatric patients.
BACKGROUND: Colorectal cancer (CRC) screening might be improved by using a measure of prior risk to modulate screening intensity or the faecal immunochemical test threshold. Intermediate molecular biomarkers could aid risk prediction by capturing both known and unknown risk factors. METHODS: We sampled normal bowel mucosa from the proximal colon, distal colon and rectum of 317 individuals undergoing colonoscopy. We defined cases as having a personal history of colorectal polyp(s)/cancer, and controls as having no history of colorectal neoplasia. Molecular analyses were performed for: telomere length (TL); global methylation; and the expression of genes in molecular pathways associated with colorectal tumourigenesis. We also calculated a polygenic risk score (PRS) based on CRC susceptibility polymorphisms. RESULTS: Bowel TL was significantly longer in cases than controls, but was not associated with blood TL. PRS was significantly and independently higher in cases. Hypermethylation showed a suggestive association with case:control status. No gene or pathway was differentially expressed between cases and controls. Gene expression often varied considerably between bowel locations. CONCLUSIONS: PRS and bowel TL (but not blood TL) may be clinically-useful predictors of CRC risk. Sample collection to assess these biomarkers is feasible in clinical practice, especially where population screening uses flexible sigmoidoscopy or colonoscopy.
CCN2 is a critical matricellular protein that is expressed in several cells with major implications in physiology and different pathologies. However, the transcriptional regulation of this gene remains obscure. We used the Encyclopaedia of DNA Elements browser (ENCODE) to visualise the region spanning from 300 kb upstream to the CCN2 start site in silico in order to identify enhancer regions that regulate transcription of this gene. Selection was based on three criteria associated with enhancer regions: 1) H3K4me1 and H3K27ac histone modifications, 2) DNase I hypersensitivity of chromatin and 3) inter-species conservation. Reporter constructs were created with sequences spanning each of the regions of interest placed upstream of an Hsp68 silent proximal promoter sequence in order to drive the expression of β-galactosidase transgene. Each of these constructs was subsequently used to create transgenic mice in which reporter gene production was assessed at the E15.5 developmental stage. Four functional enhancers were identified, with each driving distinct, tissue-specific patterns of transgene expression. An enhancer located -100 kb from the CCN2 transcription start site facilitated expression within vascular tissue. An enhancer -135 kb upstream of CCN2 drove expression within the articular chondrocytes of synovial joints. The other two enhancers, located at -198 kb and -229 kb, mediated transgene expression within dermal fibroblasts, however the most prevalent activity was found within hypertrophic chondrocytes and periosteal tissue, respectively. These findings suggest that the global expression of CCN2 during development results from the activity of several tissue-specific enhancer regions in addition to proximal regulatory elements that have previously been demonstrated to drive transcription of the gene during development.
Active regulation of protein abundance is an essential strategy to modulate cellular signaling pathways. Within the Wnt signaling cascade, regulated degradation of β-catenin by the ubiquitin-proteasome system (UPS) affects the outcome of canonical Wnt signaling. Here, we found that abundance of the Wnt cargo receptor Evi (Wls/GPR177), which is required for Wnt protein secretion, is also regulated by the UPS through endoplasmic reticulum (ER)-associated degradation (ERAD). In the absence of Wnt ligands, Evi is ubiquitinated and targeted for ERAD in a VCP-dependent manner. Ubiquitination of Evi involves the E2-conjugating enzyme UBE2J2 and the E3-ligase CGRRF1. Furthermore, we show that a triaging complex of Porcn and VCP determines whether Evi enters the secretory or the ERAD pathway. In this way, ERAD-dependent control of Evi availability impacts the scale of Wnt protein secretion by adjusting the amount of Evi to meet the requirement of Wnt protein export. As Wnt and Evi protein levels are often dysregulated in cancer, targeting regulatory ERAD components might be a useful approach for therapeutic interventions.
Transitions of cytosine to thymine in CpG dinucleotides are the most frequent type of mutations observed in cancer. This increased mutability is commonly explained by the presence of 5-methylcytosine (5mC) and its spontaneous hydrolytic deamination into thymine. Here, we describe observations that question whether spontaneous deamination alone causes the elevated mutagenicity of 5mC. Tumours with somatic mutations in DNA mismatch-repair genes or in the proofreading domain of DNA polymerase ε (Pol ε) exhibit more 5mC to T transitions than would be expected, given the kinetics of hydrolytic deamination. This enrichment is asymmetrical around replication origins with a preference for the leading strand template, in particular in methylated cytosines flanked by guanines (GCG). Notably, GCG to GTG mutations also exhibit strand asymmetry in mismatch-repair and Pol ε wild-type tumours. Together, these findings suggest that mis-incorporation of A opposite 5mC during replication of the leading strand might be a contributing factor in the mutagenesis of methylated cytosine.
Cancer development involves the stepwise accumulation of genetic lesions that overcome the normal regulatory pathways that prevent unconstrained cell division and tissue growth. Identification of the genetic changes that cause cancer has long been the subject of intensive study, leading to the identification of several RNA-binding proteins (RBPs) linked to cancer. Cross-reference of the complement of RBPs recently identified by RNA interactome capture with cancer-associated genes and biological processes led to the identification of a set of 411 proteins with potential implications in cancer biology. These involve a broad spectrum of cellular processes including response to stress, metabolism and cell adhesion. Future studies should aim to understand these proteins and their connection to cancer from an RNA-centred perspective, holding the promise of new mechanistic understanding of cancer formation and novel approaches to diagnosis and treatment.
Transition from pluripotency to differentiation is a pivotal yet poorly understood developmental step. Here, we show that the tumour suppressor RASSF1A is a key player driving the early specification of cell fate. RASSF1A acts as a natural barrier to stem cell self-renewal and iPS cell generation, by switching YAP from an integral component in the β-catenin-TCF pluripotency network to a key factor that promotes differentiation. We demonstrate that epigenetic regulation of the Rassf1A promoter maintains stemness by allowing a quaternary association of YAP-TEAD and β-catenin-TCF3 complexes on the Oct4 distal enhancer. However, during differentiation, promoter demethylation allows GATA1-mediated RASSF1A expression which prevents YAP from contributing to the TEAD/β-catenin-TCF3 complex. Simultaneously, we find that RASSF1A promotes a YAP-p73 transcriptional programme that enables differentiation. Together, our findings demonstrate that RASSF1A mediates transcription factor selection of YAP in stem cells, thereby acting as a functional "switch" between pluripotency and initiation of differentiation.
Insertion of proteins into membranes is an essential cellular process. The extensive biophysical and topological diversity of membrane proteins necessitates multiple insertion pathways that remain incompletely defined. Here we found that known membrane insertion pathways fail to effectively engage tail-anchored membrane proteins with moderately hydrophobic transmembrane domains. These proteins are instead shielded in the cytosol by calmodulin. Dynamic release from calmodulin allowed sampling of the endoplasmic reticulum (ER), where the conserved ER membrane protein complex (EMC) was shown to be essential for efficient insertion in vitro and in cells. Purified EMC in synthetic liposomes catalyzed the insertion of its substrates in a reconstituted system. Thus, EMC is a transmembrane domain insertase, a function that may explain its widely pleiotropic membrane-associated phenotypes across organisms.
The DOT1L histone H3 lysine 79 (H3K79) methyltransferase plays an oncogenic role in MLL-rearranged leukemogenesis. Here, we demonstrate that, in contrast to MLL-rearranged leukemia, DOT1L plays a protective role in ultraviolet radiation (UVR)-induced melanoma development. Specifically, the DOT1L gene is located in a frequently deleted region and undergoes somatic mutation in human melanoma. Specific mutations functionally compromise DOT1L methyltransferase enzyme activity leading to reduced H3K79 methylation. Importantly, in the absence of DOT1L, UVR-induced DNA damage is inefficiently repaired, so that DOT1L loss promotes melanoma development in mice after exposure to UVR. Mechanistically, DOT1L facilitates DNA damage repair, with DOT1L-methylated H3K79 involvement in binding and recruiting XPC to the DNA damage site for nucleotide excision repair (NER). This study indicates that DOT1L plays a protective role in UVR-induced melanomagenesis.
The transcriptional mechanism through which chondrocytes control the spatial and temporal composition of the cartilage tissue has remained largely elusive. The central aim of this study was to identify whether transcriptional enhancers played a role in the organisation of the chondrocytes in cartilaginous tissue. We focused on the Aggrecan gene (Acan) as it is essential for the normal structure and function of cartilage and it is expressed developmentally in different stages of chondrocyte maturation. Using transgenic reporter studies in mice we identified four elements, two of which showed individual chondrocyte developmental stage specificity. In particular, one enhancer (-80) distinguishes itself from the others by being predominantly active in adult cartilage. Furthermore, the -62 element uniquely drove reporter activity in early chondrocytes. The remaining chondrocyte specific enhancers, +28 and -30, showed no preference to chondrocyte type. The transcription factor SOX9 interacted with all the enhancers in vitro and mutation of SOX9 binding sites in one of the enhancers (-30) resulted in a loss of its chondrocyte specificity and ectopic enhancer reporter activity. Thus, the Acan enhancers orchestrate the precise spatiotemporal expression of this gene in cartilage types at different stages of development and adulthood.
Br J Cancer, 118 (1), pp. 1-2. | Read more2018. Cell and molecular biology: a new section joins the fight against cancer.
The proteasome is the major protease responsible for the production of antigenic peptides recognized by CD8+ cytolytic T cells (CTL). These peptides, generally 8-10 amino acids long, are presented at the cell surface by major histocompatibility complex (MHC) class I molecules. Originally, these peptides were believed to be solely derived from linear fragments of proteins, but this concept was challenged several years ago by the isolation of anti-tumor CTL that recognized spliced peptides, i.e. peptides composed of fragments distant in the parental protein. The splicing process was shown to occur in the proteasome through a transpeptidation reaction involving an acyl-enzyme intermediate. Here, we review the steps that led to the discovery of spliced peptides as well as the recent advances that uncover the unexpected importance of spliced peptides in the composition of the MHC class I repertoire.
Tumors use tryptophan-catabolizing enzymes such as indoleamine 2,3-dioxygenase (IDO-1) to induce an immunosuppressive environment. IDO-1 is induced in response to inflammatory stimuli and promotes immune tolerance through effector T-cell anergy and enhanced Treg function. As such, IDO-1 is a nexus for the induction of a key immunosuppressive mechanism and represents an important immunotherapeutic target in oncology. Starting from HTS hit 5, IDO-1 inhibitor 6 (EOS200271/PF-06840003) has been developed. The structure-activity relationship around 6 is described and rationalized using the X-ray crystal structure of 6 bound to human IDO-1, which shows that 6, differently from most of the IDO-1 inhibitors described so far, does not bind to the heme iron atom and has a novel binding mode. Clinical candidate 6 shows good potency in an IDO-1 human whole blood assay and also shows a very favorable ADME profile leading to favorable predicted human pharmacokinetic properties, including a predicted half-life of 16-19 h.
Histone lysine demethylases (KDMs) are of critical importance in the epigenetic regulation of gene expression, yet there are few selective, cell-permeable inhibitors or suitable tool compounds for these enzymes. We describe the discovery of a new class of inhibitor that is highly potent towards the histone lysine demethylases KDM2A/7A. A modular synthetic approach was used to explore the chemical space and accelerate the investigation of key structure-activity relationships, leading to the development of a small molecule with around 75-fold selectivity towards KDM2A/7A versus other KDMs, as well as cellular activity at low micromolar concentrations.
Within tumors, macrophage infiltration can promote cancer cell invasiveness and, consequently, metastatic dissemination. In this issue of Developmental Cell, Roh-Johnson et al. (2017) reveal that cytoplasmic transfer from macrophages to melanoma cells correlates with melanoma invasion and arises as a result of intimate cell-cell contact.
Inhibition of NK and effector T-cell functions and activation of regulatory cell populations are the main immunosuppressive effects of indoleamine-2,3-dioxygenase1 (IDO1). By converting tryptophan (Trp) into kynurenine (Kyn), IDO1 is involved in the immune response homeostasis, and its dysregulated expression is described in immune-related pathologies, as tumors that hijack it to evade immune destruction. Thereby, IDO1 inhibitors are being developed to stimulate antitumor immune responses. Existing and standard quantitation methods of IDO1 substrate and metabolite(s) are based on the total level of Trp and its metabolites determined by liquid chromatography tandem mass spectrometry analysis in human plasma, cerebrospinal fluid, and brain. Here, we describe the detection, localization, and absolute quantitation of Trp and Kyn by quantitative mass spectrometry imaging (qMSI) in transfected murine tumor models expressing various levels of IDO1. Myeloid, glycolysis metabolic signatures, and correlation between IDO1 expression and Trp to Kyn conversion are also shown. High-definition IDO1 and GCN2 immunostainings overlaid with Kyn molecular images underline the tumor metabolism and heterogeneity. The development of immunotherapies such as IDO1 inhibitors requires a deep understanding of the immune system, the interplay of cancer cells, and biomarker characterization. Our data underline that qMSI allows the study of the spatial distribution and quantitation of endogenous immune metabolites for biology and pharmacology studies.
Senescent cells may promote tumour progression through the activation of a senescence-associated secretory phenotype (SASP), whether these cells are capable of initiating tumourigenesis in vivo is not known. Expression of oncogenic β-catenin in Sox2+ young adult pituitary stem cells leads to formation of clusters of stem cells and induction of tumours resembling human adamantinomatous craniopharyngioma (ACP), derived from Sox2- cells in a paracrine manner. Here, we uncover the mechanisms underlying this paracrine tumourigenesis. We show that expression of oncogenic β-catenin in Hesx1+ embryonic precursors also results in stem cell clusters and paracrine tumours. We reveal that human and mouse clusters are analogous and share a common signature of senescence and SASP. Finally, we show that mice with reduced senescence and SASP responses exhibit decreased tumour-inducing potential. Together, we provide evidence that senescence and a stem cell-associated SASP drive cell transformation and tumour initiation in vivo in an age-dependent fashion.
Despite impressive clinical success, cancer immunotherapy based on immune checkpoint blockade remains ineffective in many patients due to tumoral resistance. Here we use the autochthonous TiRP melanoma model, which recapitulates the tumoral resistance signature observed in human melanomas. TiRP tumors resist immunotherapy based on checkpoint blockade, cancer vaccines or adoptive T-cell therapy. TiRP tumors recruit and activate tumor-specific CD8+ T cells, but these cells then undergo apoptosis. This does not occur with isogenic transplanted tumors, which are rejected after adoptive T-cell therapy. Apoptosis of tumor-infiltrating lymphocytes can be prevented by interrupting the Fas/Fas-ligand axis, and is triggered by polymorphonuclear-myeloid-derived suppressor cells, which express high levels of Fas-ligand and are enriched in TiRP tumors. Blocking Fas-ligand increases the anti-tumor efficacy of adoptive T-cell therapy in TiRP tumors, and increases the efficacy of checkpoint blockade in transplanted tumors. Therefore, tumor-infiltrating lymphocytes apoptosis is a relevant mechanism of immunotherapy resistance, which could be blocked by interfering with the Fas/Fas-ligand pathway.
BACKGROUND: Plasma cell-free RNA (cfRNA) encompasses a broad spectrum of RNA species that can be derived from both human cells and microbes. Because cfRNA is fragmented and of low concentration, it has been challenging to profile its transcriptome using standard RNA-seq methods. METHODS: We assessed several recently developed RNA-seq methods on cfRNA samples. We then analyzed the dynamic changes of both the human transcriptome and the microbiome of plasma during pregnancy from 60 women. RESULTS: cfRNA reflects a well-orchestrated immune modulation during pregnancy: an up-regulation of antiinflammatory genes and an increased abundance of antimicrobial genes. We observed that the plasma microbiome remained relatively stable during pregnancy. The bacteria Ureaplasma shows an increased prevalence and increased abundance at postpartum, which is likely to be associated with postpartum infection. We demonstrated that cfRNA-seq can be used to monitor viral infections. We detected a number of human pathogens in our patients, including an undiagnosed patient with a high load of human parvovirus B19 virus (B19V), which is known to be a potential cause of complications in pregnancy. CONCLUSIONS: Plasma cfRNA-seq demonstrates the potential to simultaneously monitor immune response and microbial infections during pregnancy.
Inhibition of the human 2-oxoglutarate (2OG) dependent hypoxia inducible factor (HIF) prolyl hydroxylases (human PHD1-3) causes upregulation of HIF, thus promoting erythropoiesis and is therefore of therapeutic interest. We describe cellular, biophysical, and biochemical studies comparing four PHD inhibitors currently in clinical trials for anaemia treatment, that describe their mechanisms of action, potency against isolated enzymes and in cells, and selectivities versus representatives of other human 2OG oxygenase subfamilies. The 'clinical' PHD inhibitors are potent inhibitors of PHD catalyzed hydroxylation of the HIF-α oxygen dependent degradation domains (ODDs), and selective against most, but not all, representatives of other human 2OG dependent dioxygenase subfamilies. Crystallographic and NMR studies provide insights into the different active site binding modes of the inhibitors. Cell-based results reveal the inhibitors have similar effects on the upregulation of HIF target genes, but differ in the kinetics of their effects and in extent of inhibition of hydroxylation of the N- and C-terminal ODDs; the latter differences correlate with the biophysical observations.
The linear ubiquitin chain assembly complex, LUBAC, is the only known mammalian ubiquitin ligase that makes methionine 1 (Met1)-linked polyubiquitin (also referred to as linear ubiquitin). A decade after LUBAC was discovered as a cellular activity of unknown function, there are now many lines of evidence connecting Met1-linked polyubiquitin to NF-κB signaling, cell death, inflammation, immunity, and cancer. We now know that Met1-linked polyubiquitin has potent signaling functions and that its deregulation is connected to disease. Indeed, mutations and deficiencies in several factors involved in conjugation and deconjugation of Met1-linked polyubiquitin have been implicated in immune-related disorders. Here, we discuss current knowledge and recent insights into the role and regulation of Met1-linked polyubiquitin, with an emphasis on the mechanisms controlling the function of LUBAC.
Development, 144 (20), pp. 3847-3848. | Read more2017. Correction: SoxF factors induce Notch1 expression via direct transcriptional regulation during early arterial development. Development doi: 10.1242/dev.146241.
5-Hydroxymethylcytosine (5hmC) is an important mammalian DNA epigenetic modification that has been linked to gene regulation and cancer pathogenesis. Here we explored the diagnostic potential of 5hmC in circulating cell-free DNA (cfDNA) using a sensitive chemical labeling-based low-input shotgun sequencing approach. We sequenced cell-free 5hmC from 49 patients of seven different cancer types and found distinct features that could be used to predict cancer types and stages with high accuracy. Specifically, we discovered that lung cancer leads to a progressive global loss of 5hmC in cfDNA, whereas hepatocellular carcinoma and pancreatic cancer lead to disease-specific changes in the cell-free hydroxymethylome. Our proof-of-principle results suggest that cell-free 5hmC signatures may potentially be used not only to identify cancer types but also to track tumor stage in some cancers.
The mammalian ubiquitin ligase Hrd1 is the central component of a complex facilitating degradation of misfolded proteins during the ubiquitin-proteasome-dependent process of ER-associated degradation (ERAD). Hrd1 associates with cofactors to execute ERAD, but their roles and how they assemble with Hrd1 are not well understood. Here, we identify crucial cofactor interaction domains within Hrd1 and report a previously unrecognised evolutionarily conserved segment within the intrinsically disordered cytoplasmic domain of Hrd1 (termed the HAF-H domain), which engages complementary segments in the cofactors FAM8A1 and Herp (also known as HERPUD1). This domain is required by Hrd1 to interact with both FAM8A1 and Herp, as well as to assemble higher-order Hrd1 complexes. FAM8A1 enhances binding of Herp to Hrd1, an interaction that is required for ERAD. Our findings support a model of Hrd1 complex formation, where the Hrd1 cytoplasmic domain and FAM8A1 have a central role in the assembly and activity of this ERAD machinery.
Cancer immunotherapy has recently emerged as a forefront strategy to fight cancer. Key players in antitumor responses are CD8+ cytolytic T lymphocytes (CTLs) that can detect tumor cells that carry antigens, in other words, small peptides bound to surface major histocompatibility complex (MHC) class I molecules. The success and safety of cancer immunotherapy strategies depends on the nature of the antigens recognized by the targeted T cells, their strict tumor specificity, and whether tumors and antigen-presenting cells can efficiently process the peptide. We review here the nature of the tumor antigens and their potential for the development of immunotherapeutic strategies. We also discuss the importance of proteasome in the production of these peptides in the context of immunotherapy and therapeutic cancer vaccines.
The melanocortin-1 receptor (MC1R), a G-protein-coupled receptor, has a crucial role in human and mouse pigmentation. Activation of MC1R in melanocytes by α-melanocyte-stimulating hormone (α-MSH) stimulates cAMP signalling and melanin production and enhances DNA repair after ultraviolet irradiation. Individuals carrying MC1R variants, especially those associated with red hair colour, fair skin and poor tanning ability (denoted as RHC variants), are associated with higher risk of melanoma. However, how MC1R activity is modulated by ultraviolet irradiation, why individuals with red hair are more prone to developing melanoma, and whether the activity of RHC variants might be restored for therapeutic benefit are unknown. Here we demonstrate a potential MC1R-targeted intervention strategy in mice to rescue loss-of-function MC1R in MC1R RHC variants for therapeutic benefit by activating MC1R protein palmitoylation. MC1R palmitoylation, primarily mediated by the protein-acyl transferase ZDHHC13, is essential for activating MC1R signalling, which triggers increased pigmentation, ultraviolet-B-induced G1-like cell cycle arrest and control of senescence and melanomagenesis in vitro and in vivo. Using C57BL/6J-Mc1re/eJ mice, in which endogenous MC1R is prematurely terminated, expressing Mc1r RHC variants, we show that pharmacological activation of palmitoylation rescues the defects of Mc1r RHC variants and prevents melanomagenesis. The results highlight a central role for MC1R palmitoylation in pigmentation and protection against melanoma.
Chromatin modifications and the promoter-associated epigenome are important for the regulation of gene expression. However, the mechanisms by which chromatin-modifying complexes are targeted to the appropriate gene promoters in vertebrates and how they influence gene expression have remained poorly defined. Here, using a combination of live-cell imaging and functional genomics, we discover that the vertebrate SET1 complex is targeted to actively transcribed gene promoters through CFP1, which engages in a form of multivalent chromatin reading that involves recognition of non-methylated DNA and histone H3 lysine 4 trimethylation (H3K4me3). CFP1 defines SET1 complex occupancy on chromatin, and its multivalent interactions are required for the SET1 complex to place H3K4me3. In the absence of CFP1, gene expression is perturbed, suggesting that normal targeting and function of the SET1 complex are central to creating an appropriately functioning vertebrate promoter-associated epigenome.
The oncogenic D816V mutation of the KIT receptor is well characterized in systemic mastocytosis and acute myeloid leukemia. Although KITD816V has been found in melanoma, its function and involvement in this malignancy is not understood. Here we show that KITD816V induces tyrosine phosphorylation of MITF through a triple protein complex formation between KIT, MITF, and SRC family kinases. In turn, phosphorylated MITF activates target genes that are involved in melanoma proliferation, cell-cycle progression, suppression of senescence, survival, and invasion. By blocking the triple protein complex formation, thus preventing MITF phosphorylation, the cells became hypersensitive to SRC inhibitors. We have therefore delineated a mechanism behind the oncogenic effects of KITD816V in melanoma and provided a rationale for the heightened SRC inhibitor sensitivity in KITD816V transformed cells.Implications: This study demonstrates that an oncogenic tyrosine kinase mutant, KITD816V, can alter the transcriptional program of the transcription factor MITF in melanoma Mol Cancer Res; 15(9); 1265-74. ©2017 AACR.
SCIENTIST, 31 (9), pp. 48-53.2017. DNA Extras
Cancer immunotherapy is experiencing a renaissance spearheaded by immune checkpoint inhibitors (ICIs). This has spurred interest in 'upgrading' existing immunotherapies that previously experienced only sporadic success, such as dendritic cells (DCs) vaccines. In this review, we discuss the major molecular, immunological, and clinical determinants of existing first- and second-generation DC vaccines. We also outline the future trends for next-generation DC vaccines and describe their major hallmarks and prerequisites necessary for high anticancer efficacy. In addition, using existing data we compare DC vaccines with ICIs targeting CTLA4, PD1, and PD-L1, and argue that in various contexts next-generation DC vaccines are ready to meet some challenges currently confronting ICIs, thereby raising the need to integrate DC vaccines in future combinatorial immunotherapy regimens.
Background: Never-smokers and never-drinkers patients (NSND) suffering from oral squamous cell carcinoma (OSCC) are epidemiologically different from smokers drinkers (SD). We therefore hypothesized that they harbored distinct targetable molecular alterations. Patients and methods: Data from The Cancer Genome Atlas (TCGA) (discovery set), Gene Expression Omnibus and Centre Léon Bérard (CLB) (three validation sets) with available gene expression profiles of HPV-negative OSCC from NSND and SD were mined. Protein expression profiles and genomic alterations were also analyzed from TCGA, and a functional pathway enrichment analysis was carried out. Formalin-fixed paraffin-embedded samples from 44 OSCC including 20 NSND and 24 SD treated at CLB were retrospectively collected to perform targeted-sequencing of 2559 transcripts (HTG EdgeSeq system), and CD3, CD4, CD8, IDO1, and PD-L1 expression analyses by immunohistochemistry (IHC). Enrichment of a six-gene interferon-γ signature of clinical response to pembrozulimab (PD-1 inhibitor) was evaluated in each sample from all cohorts, using the single sample gene set enrichment analysis method. Results: A total of 854 genes and 29 proteins were found to be differentially expressed between NSND and SD in TCGA. Functional pathway analysis highlighted an overall enrichment for immune-related pathways in OSCC from NSND, especially involving T-cell activation. Interferon-γ response and PD1 signaling were strongly enriched in NSND. IDO1 and PD-L1 were overexpressed and the score of response to pembrolizumab was higher in NSND than in SD, although the mutational load was lower in NSND. IHC analyses in the CLB cohort evidenced IDO1 and PD-L1 overexpression in tumor cells that was associated with a higher rate of tumor-infiltrating T-cells in NSND compared with SD. Conclusion: The main biological and actionable difference between OSCC from NSND and SD lies in the immune microenvironment, suggesting a higher clinical benefit of PD-L1 and IDO1 inhibition in OSCC from NSND.
Tumors use various mechanisms to avoid immune destruction. Cyclooxygenase-2 (COX-2) expression may be a driver of immune suppression in melanoma, but the mechanisms involved remain elusive. Here, we show that COX-2 expression drives constitutive expression of indoleamine 2,3-dioxygenase 1 (IDO1) in human tumor cells. IDO1 is an immunosuppressive enzyme that degrades tryptophan. In a series of seven human tumor lines, constitutive IDO1 expression depends on COX-2 and prostaglandin E2 (PGE2), which, upon autocrine signaling through the EP receptor, activates IDO1 via the PKC and PI3K pathways. COX-2 expression itself depends on the MAPK pathway, which therefore indirectly controls IDO1 expression. Most of these tumors carry PI3K or MAPK oncogenic mutations, which may favor constitutive IDO1 expression. Celecoxib treatment promoted immune rejection of IDO1-expressing human tumor xenografts in immunodeficient mice reconstituted with human allogeneic lymphocytes. This effect was associated with a reduced expression of IDO1 in those ovarian SKOV3 tumors and an increased infiltration of CD3+ and CD8+ cells. Our results highlight the role of COX-2 in constitutive IDO1 expression by human tumors and substantiate the use of COX-2 inhibitors to improve the efficacy of cancer immunotherapy, by reducing constitutive IDO1 expression, which contributes to the lack of T-cell infiltration in "cold" tumors, which fail to respond to immunotherapy. Cancer Immunol Res; 5(8); 695-709. ©2017 AACR.
Tumors use various mechanisms to avoid immune destruction. Cyclooxygenase-2 (COX-2) expression may be a driver of immune suppression in melanoma, but the mechanisms involved remain elusive. Here, we show that COX-2 expression drives constitutive expression of indoleamine 2,3-dioxygenase 1 (IDO1) in human tumor cells. IDO1 is an immunosuppressive enzyme that degrades tryptophan. In a series of seven human tumor lines, constitutive IDO1 expression depends on COX-2 and prostaglandin E2 (PGE2), which, upon autocrine signaling through the EP receptor, activates IDO1 via the PKC and PI3K pathways. COX-2 expression itself depends on the MAPK pathway, which therefore indirectly controls IDO1 expression. Most of these tumors carry PI3K or MAPK oncogenic mutations, which may favor constitutive IDO1 expression. Celecoxib treatment promoted immune rejection of IDO1-expressing human tumor xenografts in immunodeficient mice reconstituted with human allogeneic lymphocytes. This effect was associated with a reduced expression of IDO1 in those ovarian SKOV3 tumors and an increased infiltration of CD3+ and CD8+ cells. Our results highlight the role of COX-2 in constitutive IDO1 expression by human tumors and substantiate the use of COX-2 inhibitors to improve the efficacy of cancer immunotherapy, by reducing constitutive IDO1 expression, which contributes to the lack of T-cell infiltration in "cold" tumors, which fail to respond to immunotherapy. Cancer Immunol Res; 5(8); 1-15. ©2017 AACR.
Arterial specification and differentiation are influenced by a number of regulatory pathways. While it is known that the Vegfa-Notch cascade plays a central role, the transcriptional hierarchy controlling arterial specification has not been fully delineated. To elucidate the direct transcriptional regulators of Notch receptor expression in arterial endothelial cells, we used histone signatures, DNaseI hypersensitivity and ChIP-seq data to identify enhancers for the human NOTCH1 and zebrafish notch1b genes. These enhancers were able to direct arterial endothelial cell-restricted expression in transgenic models. Genetic disruption of SoxF binding sites established a clear requirement for members of this group of transcription factors (SOX7, SOX17 and SOX18) to drive the activity of these enhancers in vivo Endogenous deletion of the notch1b enhancer led to a significant loss of arterial connections to the dorsal aorta in Notch pathway-deficient zebrafish. Loss of SoxF function revealed that these factors are necessary for NOTCH1 and notch1b enhancer activity and for correct endogenous transcription of these genes. These findings position SoxF transcription factors directly upstream of Notch receptor expression during the acquisition of arterial identity in vertebrates.
Osteogenic-angiogenic coupling is promoted by the hypoxia-inducible factor 1-alpha (HIF-1α) transcription factor, provoking interest in HIF activation as a therapeutic strategy to improve osteoblast mineralization and treat pathological osteolysis. However, HIF also enhances the bone-resorbing activity of mature osteoclasts. It is therefore essential to determine the full effect(s) of HIF on both the formation and the bone-resorbing function of osteoclasts in order to understand how they might respond to such a strategy. Expression of HIF-1α mRNA and protein increased during osteoclast differentiation from CD14+ monocytic precursors, additionally inducing expression of the HIF-regulated glycolytic enzymes. However, HIF-1α siRNA only moderately affected osteoclast differentiation, accelerating fusion of precursor cells. HIF induction by inhibition of the regulatory prolyl-4-hydroxylase (PHD) enzymes reduced osteoclastogenesis, but was confirmed to enhance bone resorption by mature osteoclasts. Phd2+/- murine osteoclasts also exhibited enhanced bone resorption, associated with increased expression of resorption-associated Acp5, in comparison with wild-type cells from littermate controls. Phd3-/- bone marrow precursors displayed accelerated early fusion, mirroring results with HIF-1α siRNA. In vivo, Phd2+/- and Phd3-/- mice exhibited reduced trabecular bone mass, associated with reduced mineralization by Phd2+/- osteoblasts. These data indicate that HIF predominantly functions as a regulator of osteoclast-mediated bone resorption, with little effect on osteoclast differentiation. Inhibition of HIF might therefore represent an alternative strategy to treat diseases characterized by pathological levels of osteolysis. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
Cell Death Differ, 24 (7), pp. 1135-1136. | Citations: 1 (Web of Science Lite) | Read more2017. All roads lead to ubiquitin.
Mads Gyrd-Hansen studied biochemistry at the University of Copenhagen and received his PhD in 2005 under the supervision of Marja Jäättelä at the Danish Cancer Society Research Centre. He then joined the laboratory of Pascal Meier at the Institute of Cancer Research in London to work on the inhibitor of apoptosis (IAP) proteins. Mads returned to Copenhagen in 2008 to the Biotech Research and Innovation Centre (BRIC) in a senior postdoctoral position with Morten Frödin, and subsequently started his own research group with a career-development fellowship from the Danish Research Councils as part of the laboratory of Niels Mailand at the Novo Nordisk Foundation Centre for Protein Research. In 2013, he joined the Ludwig Institute for Cancer Research at the University of Oxford, where he is now an associate professor and holder of a Wellcome Trust Senior Research Fellowship and a Sapere Aude starting grant from the Danish Research Councils. Mads is interested in the non-degradative functions and regulation of ubiquitylation in pro-inflammatory signalling during innate immune responses.This article is part of a Minifocus on Ubiquitin Regulation and Function. For further reading, please see related articles: 'Mechanisms of regulation and diversification of deubiquitylating enzyme function' by Pawel Leznicki and Yogesh Kulathu (J. Cell Sci.130, 1997-2006). 'Exploitation of the host cell ubiquitin machinery by microbial effector' proteins by Yi-Han Lin and Matthias P. Machner (J. Cell Sci.130, 1985-1996).
OBJECTIVE: Patients with Niemann-Pick disease type C1 (NPC1), a lysosomal lipid storage disorder that causes neurodegeneration and liver damage, can present with IBD, but neither the significance nor the functional mechanism of this association is clear. We studied bacterial handling and antibacterial autophagy in patients with NPC1. DESIGN: We characterised intestinal inflammation in 14 patients with NPC1 who developed IBD. We investigated bacterial handling and cytokine production of NPC1 monocytes or macrophages in vitro and compared NPC1-associated functional defects to those caused by IBD-associated nucleotide-binding oligomerization domain-containing protein 2 (NOD2) variants or mutations in X-linked inhibitor of apoptosis (XIAP). RESULTS: Patients with the lysosomal lipid storage disorder NPC1 have increased susceptibility to early-onset fistulising colitis with granuloma formation, reminiscent of Crohn's disease (CD). Mutations in NPC1 cause impaired autophagy due to defective autophagosome function that abolishes NOD2-mediated bacterial handling in vitro similar to variants in NOD2 or XIAP deficiency. In contrast to genetic NOD2 and XIAP variants, NPC1 mutations do not impair NOD2-receptor-interacting kinase 2 (RIPK2)-XIAP-dependent cytokine production. Pharmacological activation of autophagy can rescue bacterial clearance in macrophages in vitro by increasing the autophagic flux and bypassing defects in NPC1. CONCLUSIONS: NPC1 confers increased risk of early-onset severe CD. Our data support the concept that genetic defects at different checkpoints of selective autophagy cause a shared outcome of CD-like immunopathology linking monogenic and polygenic forms of IBD. Muramyl dipeptide-driven cytokine responses and antibacterial autophagy induction are parallel and independent signalling cascades downstream of the NOD2-RIPK2-XIAP complex.
We here review primary methods used in quantifying and mapping 5-hydroxymethylcytosine (5hmC), including global quantification, restriction enzyme-based detection, and methods involving DNA-enrichment strategies and the genome-wide sequencing of 5hmC. As discovered in the mammalian genome in 2009, 5hmC, oxidized from 5-methylcytosine (5mC) by ten-eleven translocation (TET) dioxygenases, is increasingly being recognized as a biomarker in biological processes from development to pathogenesis, as its various detection methods have shown. We focus in particular on an ultrasensitive single-molecule imaging technique that can detect and quantify 5hmC from trace samples and thus offer information regarding the distance-based relationship between 5hmC and 5mC when used in combination with fluorescence resonance energy transfer.
BRAF inhibitor (BRAFi) therapy for melanoma patients harboring the V600E mutation is initially highly effective, but almost all patients relapse within a few months. Understanding the molecular mechanisms underpinning BRAFi-based therapy is therefore an important issue. Here we identified a previously unsuspected mechanism of BRAFi resistance driven by elevated Hedgehog (Hh) pathway activation that is observed in a cohort of melanoma patients after vemurafenib treatment. Specifically, we demonstrate that melanoma cell lines, with acquired in vitro-induced vemurafenib resistance, show increased levels of glioma-associated oncogene homolog 1 and 2 (GLI1/GLI2) compared with naïve cells. We also observed these findings in clinical melanoma specimens. Moreover, the increased expression of the transcription factors GLI1/GLI2 was independent of canonical Hh signaling and was instead correlated with the noncanonical Hh pathway, involving TGFβ/SMAD (transforming growth factor-β/Sma- and Mad-related family) signaling. Knockdown of GLI1 and GLI2 restored sensitivity to vemurafenib-resistant cells, an effect associated with both growth arrest and senescence. Treatment of vemurafenib-resistant cells with the GLI1/GLI2 inhibitor Gant61 led to decreased invasion of the melanoma cells in a three-dimensional skin reconstruct model and was associated with a decrease in metalloproteinase (MMP2/MMP9) expression and microphthalmia transcription factor upregulation. Gant61 monotherapy did not alter the drug sensitivity of naïve cells, but could reverse the resistance of melanoma cells chronically treated with vemurafenib. We further noted that alternating dosing schedules of Gant61 and vemurafenib prevented the onset of BRAFi resistance, suggesting that this could be a potential therapeutic strategy for the prevention of therapeutic escape. Our results suggest that targeting the Hh pathway in BRAFi-resistant melanoma may represent a viable therapeutic strategy to restore vemurafenib sensitivity, reducing or even inhibiting the acquired chemoresistance in melanoma patients.
Accurate assessment of TP53 gene status in sporadic tumors and in the germline of individuals at high risk of cancer due to Li-Fraumeni Syndrome (LFS) has important clinical implications for diagnosis, surveillance, and therapy. Genomic data from more than 20,000 cancer genomes provide a wealth of information on cancer gene alterations and have confirmed TP53 as the most commonly mutated gene in human cancer. Analysis of a database of 70,000 TP53 variants reveals that the two newly discovered exons of the gene, exons 9β and 9γ, generated by alternative splicing, are the targets of inactivating mutation events in breast, liver, and head and neck tumors. Furthermore, germline rearrange-ments in intron 1 of TP53 are associated with LFS and are frequently observed in sporadic osteosarcoma. In this context of constantly growing genomic data, we discuss how screening strategies must be improved when assessing TP53 status in clinical samples. Finally, we discuss how TP53 alterations should be described by using accurate nomenclature to avoid confusion in scientific and clinical reports. Cancer Res; 77(6); 1250-60. ©2017 AACR.
While invasion and metastasis of tumour cells are the principle factor responsible for cancer related deaths, the mechanisms governing the process remain poorly defined. Moreover, phenotypic divergence of sub-populations of tumour cells is known to underpin alternative behaviors linked to tumour progression such as proliferation, survival and invasion. In the context of melanoma, heterogeneity between two transcription factors, BRN2 and MITF, has been associated with phenotypic switching between predominantly invasive and proliferative behaviors respectively. Epigenetic changes, in response to external cues, have been proposed to underpin this process, however the mechanism by which the phenotypic switch occurs is unclear. Here we report the identification of the NFIB transcription factor as a novel downstream effector of BRN2 function in melanoma cells linked to the migratory and invasive characteristics of these cells. Furthermore, the function of NFIB appears to drive an invasive phenotype through an epigenetic mechanism achieved via the upregulation of the polycomb group protein EZH2. A notable target of NFIB mediated up-regulation of EZH2 is decreased MITF expression, which further promotes a less proliferative, more invasive phenotype. Together our data reveal that NFIB has the ability to promote dynamic changes in the chromatin state of melanoma cells to facilitate migration, invasion and metastasis.
Pharmacological targeting of transcription factors holds great promise for the development of new therapeutics, but strategies based on blockade of DNA binding, nuclear shuttling, or individual protein partner recruitment have yielded limited success to date. Transcription factors typically engage in complex interaction networks, likely masking the effects of specifically inhibiting single protein-protein interactions. Here, we used a combination of genomic, proteomic and biophysical methods to discover a suite of protein-protein interactions involving the SOX18 transcription factor, a known regulator of vascular development and disease. We describe a small-molecule that is able to disrupt a discrete subset of SOX18-dependent interactions. This compound selectively suppressed SOX18 transcriptional outputs in vitro and interfered with vascular development in zebrafish larvae. In a mouse pre-clinical model of breast cancer, treatment with this inhibitor significantly improved survival by reducing tumour vascular density and metastatic spread. Our studies validate an interactome-based molecular strategy to interfere with transcription factor activity, for the development of novel disease therapeutics.
In model organisms, classical genetic screening via random mutagenesis provides key insights into the molecular bases of genetic interactions, helping to define synthetic lethality, synthetic viability and drug-resistance mechanisms. The limited genetic tractability of diploid mammalian cells, however, precludes this approach. Here, we demonstrate the feasibility of classical genetic screening in mammalian systems by using haploid cells, chemical mutagenesis and next-generation sequencing, providing a new tool to explore mammalian genetic interactions.
The intratumor microenvironment generates phenotypically distinct but interconvertible malignant cell subpopulations that fuel metastatic spread and therapeutic resistance. Whether different microenvironmental cues impose invasive or therapy-resistant phenotypes via a common mechanism is unknown. In melanoma, low expression of the lineage survival oncogene microphthalmia-associated transcription factor (MITF) correlates with invasion, senescence, and drug resistance. However, how MITF is suppressed in vivo and how MITF-low cells in tumors escape senescence are poorly understood. Here we show that microenvironmental cues, including inflammation-mediated resistance to adoptive T-cell immunotherapy, transcriptionally repress MITF via ATF4 in response to inhibition of translation initiation factor eIF2B. ATF4, a key transcription mediator of the integrated stress response, also activates AXL and suppresses senescence to impose the MITF-low/AXL-high drug-resistant phenotype observed in human tumors. However, unexpectedly, without translation reprogramming an ATF4-high/MITF-low state is insufficient to drive invasion. Importantly, translation reprogramming dramatically enhances tumorigenesis and is linked to a previously unexplained gene expression program associated with anti-PD-1 immunotherapy resistance. Since we show that inhibition of eIF2B also drives neural crest migration and yeast invasiveness, our results suggest that translation reprogramming, an evolutionarily conserved starvation response, has been hijacked by microenvironmental stress signals in melanoma to drive phenotypic plasticity and invasion and determine therapeutic outcome.
An important question is how chemotherapy may (re-)activate tumor-specific immunity. In this study, we provide a phenotypic, functional and genomic analysis of tumor-specific CD8+ T cells in tumor (P815)-bearing mice, treated or not with cyclophosphamide. Our data show that chemotherapy favors the development of effector-type lymphocytes in tumor bed, characterized by higher KLRG-1 expression, lower PD-1 expression and increased cytotoxicity. This suggests re-engagement of T lymphocytes into the effector program. IFN-I appears involved in this remodeling. Our findings provide some insight into how cyclophosphamide regulates the amplitude and quality of tumor-specific immune responses.
PURPOSE: Although pancreatic ductal adenocarcinoma (PDAC) is an aggressive tumor, like other common cancers, it displays a wide range of biology. However, at present, there are no reliable tests to predict patients' cancer-specific outcomes and guide personalized treatment decisions. In this study, we aim to identify such biomarkers in resectable PDAC by studying SNPs in the CD44 gene, which drives the progression of pancreatic cancer. EXPERIMENTAL DESIGN: A total of 348 PDAC patients from three independent cohorts [Switzerland, Germany, The Cancer Genome Atlas (TCGA)] who underwent pancreatic resection are included in the study. Information on the haplotype structure of the CD44 gene is obtained using 1000 Genomes Project data, and the genotypes of the respective tagging SNPs are determined. Cox proportional hazards models are utilized to analyze the impact of SNP genotype on patients' survival. RESULTS: We identify an SNP in the CD44 gene (SNPrs187115) that independently associates with allelic differences in prognosis in all study cohorts. Specifically, in 121 Swiss patients, we observe an up to 2.38-fold (P = 0.020) difference in tumor-related death between the genotypes of SNPrs187115 We validate those results in both the German (HR = 2.32, P = 0.044, 101 patients) and the TCGA cohort (HR = 2.36, P = 0.044, 126 patients). CONCLUSIONS: CD44 SNPrs187115 can serve as a novel biomarker readily available at the time of PDAC diagnosis that identifies patients at risk for faster tumor progression and guide personalized treatment decisions. It has the potential to significantly expand the pool of patients that would benefit from tumor resection. Clin Cancer Res; 22(24); 6069-77. ©2016 AACR.
Elucidation of interactions involving DNA and histone post-translational-modifications (PTMs) is essential for providing insights into complex biological functions. Reader assemblies connected by flexible linkages facilitate avidity and increase affinity; however, little is known about the contribution to the recognition process of multiple PTMs because of rigidity in the absence of conformational flexibility. Here, we resolve the crystal structure of the triple reader module (PHD-BRD-PWWP) of ZMYND8, which forms a stable unit capable of simultaneously recognizing multiple histone PTMs while presenting a charged platform for association with DNA. Single domain disruptions destroy the functional network of interactions initiated by ZMYND8, impairing recruitment to sites of DNA damage. Our data establish a proof of principle that rigidity can be compensated by concomitant DNA and histone PTM interactions, maintaining multivalent engagement of transient chromatin states. Thus, our findings demonstrate an important role for rigid multivalent reader modules in nucleosome binding and chromatin function.
Lung cancer is the leading cause of cancer deaths, and effective treatments are urgently needed. Loss-of-function mutations in the DNA damage response kinase ATM are common in lung adenocarcinoma but directly targeting these with drugs remains challenging. Here we report that ATM loss-of-function is synthetic lethal with drugs inhibiting the central growth factor kinases MEK1/2, including the FDA-approved drug trametinib. Lung cancer cells resistant to MEK inhibition become highly sensitive upon loss of ATM both in vitro and in vivo. Mechanistically, ATM mediates crosstalk between the prosurvival MEK/ERK and AKT/mTOR pathways. ATM loss also enhances the sensitivity of KRAS- or BRAF-mutant lung cancer cells to MEK inhibition. Thus, ATM mutational status in lung cancer is a mechanistic biomarker for MEK inhibitor response, which may improve patient stratification and extend the applicability of these drugs beyond RAS and BRAF mutant tumours.
The Jumonji-C (JmjC) subfamily of 2-oxoglutarate (2OG)-dependent oxygenases are of biomedical interest because of their roles in the regulation of gene expression and protein biosynthesis. Human JmjC 2OG oxygenases catalyze oxidative modifications to give either chemically stable alcohol products, or in the case of Nɛ-methyl lysine demethylation, relatively unstable hemiaminals that fragment to give formaldehyde and the demethylated product. Recent work has yielded conflicting reports as to whether some JmjC oxygenases catalyze N-methyl group demethylation or hydroxylation reactions. We review JmjC oxygenase-catalyzed reactions within the context of structural knowledge, highlighting key differences between hydroxylases and demethylases, which have the potential to inform on the possible type(s) of reactions catalyzed by partially characterized or un-characterized JmjC oxygenases in humans and other organisms.
Chromosomal abnormalities are implicated in a substantial number of human developmental syndromes, but for many such disorders little is known about the causative genes. The recently described 1q41q42 microdeletion syndrome is characterized by characteristic dysmorphic features, intellectual disability and brain morphological abnormalities, but the precise genetic basis for these abnormalities remains unknown. Here, our detailed analysis of the genetic abnormalities of 1q41q42 microdeletion cases identified TP53BP2, which encodes apoptosis-stimulating protein of p53 2 (ASPP2), as a candidate gene for brain abnormalities. Consistent with this, Trp53bp2-deficient mice show dilation of lateral ventricles resembling the phenotype of 1q41q42 microdeletion patients. Trp53bp2 deficiency causes 100% neonatal lethality in the C57BL/6 background associated with a high incidence of neural tube defects and a range of developmental abnormalities such as congenital heart defects, coloboma, microphthalmia, urogenital and craniofacial abnormalities. Interestingly, abnormalities show a high degree of overlap with 1q41q42 microdeletion-associated abnormalities. These findings identify TP53BP2 as a strong candidate causative gene for central nervous system (CNS) defects in 1q41q42 microdeletion syndrome, and open new avenues for investigation of the mechanisms underlying CNS abnormalities.
Nuclear 3'-end-polyadenylation is essential for the transport, stability and translation of virtually all eukaryotic mRNAs. Poly(A) tail extension can also occur in the cytoplasm, but the transcripts involved are incompletely understood, particularly in cancer. Here we identify a lineage-specific requirement of the cytoplasmic polyadenylation binding protein 4 (CPEB4) in malignant melanoma. CPEB4 is upregulated early in melanoma progression, as defined by computational and histological analyses. Melanoma cells are distinct from other tumour cell types in their dependency on CPEB4, not only to prevent mitotic aberrations, but to progress through G1/S cell cycle checkpoints. RNA immunoprecipitation, sequencing of bound transcripts and poly(A) length tests link the melanoma-specific functions of CPEB4 to signalling hubs specifically enriched in this disease. Essential in these CPEB4-controlled networks are the melanoma drivers MITF and RAB7A, a feature validated in clinical biopsies. These results provide new mechanistic links between cytoplasmic polyadenylation and lineage specification in melanoma.
© 2016 Elsevier Inc. (Cancer Cell 23, 618–633; May 13, 2013) In the original Figure 4G, two of the upper panels (His-iASPP(625-828)-FITC; the two rightmost panels) were inadvertently duplicated in the lower set of panels (His-ASPP2(905-1128)-FITC; third and fourth from left). This was a mistake made by the authors during the assembly of the figure. This error does not affect any of the findings reported in the paper. The corrected Figure 4 is presented below. The authors apologize for any confusion that this error may have caused. [figure presented]
Ubiquitin chains assembled via the N-terminal methionine (Met1 or linear ubiquitin), conjugated by the linear ubiquitin chain assembly complex (LUBAC), participate in NF-κΒ-dependent inflammatory signaling and immune responses. A recent report in Cell finds that OTULIN, a deubiquitinase that selectively cleaves Met1-linked ubiquitin chains, is essential for restraining inflammation in vivo.
Angiogenesis, the fundamental process by which new blood vessels form from existing ones, depends on precise spatial and temporal gene expression within specific compartments of the endothelium. However, the molecular links between proangiogenic signals and downstream gene expression remain unclear. During sprouting angiogenesis, the specification of endothelial cells into the tip cells that lead new blood vessel sprouts is coordinated by vascular endothelial growth factor A (VEGFA) and Delta-like ligand 4 (Dll4)/Notch signaling and requires high levels of Notch ligand DLL4. Here, we identify MEF2 transcription factors as crucial regulators of sprouting angiogenesis directly downstream from VEGFA. Through the characterization of a Dll4 enhancer directing expression to endothelial cells at the angiogenic front, we found that MEF2 factors directly transcriptionally activate the expression of Dll4 and many other key genes up-regulated during sprouting angiogenesis in both physiological and tumor vascularization. Unlike ETS-mediated regulation, MEF2-binding motifs are not ubiquitous to all endothelial gene enhancers and promoters but are instead overrepresented around genes associated with sprouting angiogenesis. MEF2 target gene activation is directly linked to VEGFA-induced release of repressive histone deacetylases and concurrent recruitment of the histone acetyltransferase EP300 to MEF2 target gene regulatory elements, thus establishing MEF2 factors as the transcriptional effectors of VEGFA signaling during angiogenesis.
Isocitrate dehydrogenase 1 mutations drive human gliomagenesis, probably through neomorphic enzyme activity that produces D-2-hydroxyglutarate. To model this disease, we conditionally expressed Idh1R132H in the subventricular zone (SVZ) of the adult mouse brain. The mice developed hydrocephalus and grossly dilated lateral ventricles, with accumulation of 2-hydroxyglutarate and reduced α-ketoglutarate. Stem and transit amplifying/progenitor cell populations were expanded, and proliferation increased. Cells expressing SVZ markers infiltrated surrounding brain regions. SVZ cells also gave rise to proliferative subventricular nodules. DNA methylation was globally increased, while hydroxymethylation was decreased. Mutant SVZ cells overexpressed Wnt, cell-cycle and stem cell genes, and shared an expression signature with human gliomas. Idh1R132H mutation in the major adult neurogenic stem cell niche causes a phenotype resembling gliomagenesis.
Hum Mutat, 37 (10), pp. 989. | Read more2016. Cancer Genetics May Aid Diagnostics of Developmental Disorders.
Forkhead box A1 (FOXA1) is an FKHD family protein that plays pioneering roles in lineage-specific enhancer activation and gene transcription. Through genome-wide location analyses, here we show that FOXA1 expression and occupancy are, in turn, required for the maintenance of these epigenetic signatures, namely DNA hypomethylation and histone 3 lysine 4 methylation. Mechanistically, this involves TET1, a 5-methylcytosine dioxygenase. We found that FOXA1 induces TET1 expression via direct binding to its cis-regulatory elements. Further, FOXA1 physically interacts with the TET1 protein through its CXXC domain. TET1 thus co-occupies FOXA1-dependent enhancers and mediates local DNA demethylation and concomitant histone 3 lysine 4 methylation, further potentiating FOXA1 recruitment. Consequently, FOXA1 binding events are markedly reduced following TET1 depletion. Together, our results suggest that FOXA1 is not only able to recognize but also remodel the epigenetic signatures at lineage-specific enhancers, which is mediated, at least in part, by a feed-forward regulatory loop between FOXA1 and TET1.
The linear ubiquitin chain assembly complex (LUBAC) regulates immune signaling, and its function is regulated by the deubiquitinases OTULIN and CYLD, which associate with the catalytic subunit HOIP. However, the mechanism through which CYLD interacts with HOIP is unclear. We here show that CYLD interacts with HOIP via spermatogenesis-associated protein 2 (SPATA2). SPATA2 interacts with CYLD through its non-canonical PUB domain, which binds the catalytic CYLD USP domain in a CYLD B-box-dependent manner. Significantly, SPATA2 binding activates CYLD-mediated hydrolysis of ubiquitin chains. SPATA2 also harbors a conserved PUB-interacting motif that selectively docks into the HOIP PUB domain. In cells, SPATA2 is recruited to the TNF receptor 1 signaling complex and is required for CYLD recruitment. Loss of SPATA2 increases ubiquitination of LUBAC substrates and results in enhanced NOD2 signaling. Our data reveal SPATA2 as a high-affinity binding partner of CYLD and HOIP, and a regulatory component of LUBAC-mediated NF-κB signaling.
Bidirectional interactions between astrocytes and neurons have physiological roles in the central nervous system and an altered state or dysfunction of such interactions may be associated with neurodegenerative diseases, such as Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS). Astrocytes exert structural, metabolic and functional effects on neurons, which can be either neurotoxic or neuroprotective. Their neurotoxic effect is mediated via the senescence-associated secretory phenotype (SASP) involving pro-inflammatory cytokines (e.g., IL-6), while their neuroprotective effect is attributed to neurotrophic growth factors (e.g., NGF). We here demonstrate that the p53 isoforms Δ133p53 and p53β are expressed in astrocytes and regulate their toxic and protective effects on neurons. Primary human astrocytes undergoing cellular senescence upon serial passaging in vitro showed diminished expression of Δ133p53 and increased p53β, which were attributed to the autophagic degradation and the SRSF3-mediated alternative RNA splicing, respectively. Early-passage astrocytes with Δ133p53 knockdown or p53β overexpression were induced to show SASP and to exert neurotoxicity in co-culture with neurons. Restored expression of Δ133p53 in near-senescent, otherwise neurotoxic astrocytes conferred them with neuroprotective activity through repression of SASP and induction of neurotrophic growth factors. Brain tissues from AD and ALS patients possessed increased numbers of senescent astrocytes and, like senescent astrocytes in vitro, showed decreased Δ133p53 and increased p53β expression, supporting that our in vitro findings recapitulate in vivo pathology of these neurodegenerative diseases. Our finding that Δ133p53 enhances the neuroprotective function of aged and senescent astrocytes suggests that the p53 isoforms and their regulatory mechanisms are potential targets for therapeutic intervention in neurodegenerative diseases.
Patterns of somatic mutations in cancer genes provide information about their functional role in tumourigenesis, and thus indicate their potential for therapeutic exploitation. Yet, the classical distinction between oncogene and tumour suppressor may not always apply. For instance, TP53 has been simultaneously associated with tumour suppressing and promoting activities. Here, we uncover a similar phenomenon for GATA3, a frequently mutated, yet poorly understood, breast cancer gene. We identify two functional classes of frameshift mutations that are associated with distinct expression profiles in tumours, differential disease-free patient survival and gain- and loss-of-function activities in a cell line model. Furthermore, we find an estrogen receptor-independent synthetic lethal interaction between a GATA3 frameshift mutant with an extended C-terminus and the histone methyltransferases G9A and GLP, indicating perturbed epigenetic regulation. Our findings reveal important insights into mutant GATA3 function and breast cancer, provide the first potential therapeutic strategy and suggest that dual tumour suppressive and oncogenic activities are more widespread than previously appreciated.
Many proteins originally identified as cytoplasmic - including many associated with the cytoskeleton or cell junctions - are increasingly being found in the nucleus, where they have specific functions. Here, we focus on proteins that translocate from the cytoplasm to the nucleus in response to external signals and regulate transcription without binding to DNA directly (for example, through interaction with transcription factors). We propose that proteins with such characteristics are classified as a distinct group of extracellular signalling effectors, and we suggest the term STRaND (shuttling transcriptional regulators and non-DNA binding) to refer to this group. Crucial roles of STRaNDs include linking cell morphology and adhesion with changes in transcriptional programmes in response to signals such as mechanical stresses.
Reverse genetic screens have driven gene annotation and target discovery in model organisms. However, many disease-relevant genotypes and phenotypes cannot be studied in lower organisms. It is therefore essential to overcome technical hurdles associated with large-scale reverse genetics in human cells. Here, we establish a reverse genetic approach based on highly robust and sensitive multiplexed RNA sequencing of mutant human cells. We conduct 10 parallel screens using a collection of engineered haploid isogenic cell lines with knockouts covering tyrosine kinases and identify known and unexpected effects on signaling pathways. Our study provides proof of concept for a scalable approach to link genotype to phenotype in human cells, which has broad applications. In particular, it clears the way for systematic phenotyping of still poorly characterized human genes and for systematic study of uncharacterized genomic features associated with human disease.
Apolipoproteins L (ApoLs) are Bcl-2-like proteins expressed under inflammatory conditions in myeloid and endothelial cells. We found that Toll-like receptor (TLR) stimuli, particularly the viral mimetic polyinosinic:polycytidylic acid (poly(I:C)), specifically induce ApoLs7/11 subfamilies in murine CD8α(+) dendritic cells (DCs). This induction requires the TLR3/TRIF (where TRIF is TIR domain containing adapter-inducing interferon β) signaling pathway and is dependent on IFN-β in all ApoLs subfamilies except for ApoL7c. Poly(I:C) treatment of DCs is also associated with induction of both cell death and autophagy. ApoLs expression is related to promotion of DC death by poly(I:C), as ApoLs7/11 knockdown increases DC survival and ApoLs7 are associated with the anti-apoptotic protein Bcl-xL (where Bcl-xL is B-cell lymphoma extra large). Similarly, in human monocyte-derived DCs poly(I:C) induces both cell death and the expression of ApoLs, principally ApoL3. Finally, the BH3-like peptide of ApoLs appears to be involved in the DC death-promoting activity. We would like to propose that ApoLs are involved in cell death linked to activation of DCs by viral stimuli.
Skp1-Cul1-F-box protein (SCF) ubiquitin ligases direct cell survival decisions by controlling protein ubiquitylation and degradation. Sufu (Suppressor of fused) is a central regulator of Hh (Hedgehog) signaling and acts as a tumor suppressor by maintaining the Gli (Glioma-associated oncogene homolog) transcription factors inactive. Although Sufu has a pivotal role in Hh signaling, the players involved in controlling Sufu levels and their role in tumor growth are unknown. Here, we show that Fbxl17 (F-box and leucine-rich repeat protein 17) targets Sufu for proteolysis in the nucleus. The ubiquitylation of Sufu, mediated by Fbxl17, allows the release of Gli1 from Sufu for proper Hh signal transduction. Depletion of Fbxl17 leads to defective Hh signaling associated with an impaired cancer cell proliferation and medulloblastoma tumor growth. Furthermore, we identify a mutation in Sufu, occurring in medulloblastoma of patients with Gorlin syndrome, which increases Sufu turnover through Fbxl17-mediated polyubiquitylation and leads to a sustained Hh signaling activation. In summary, our findings reveal Fbxl17 as a novel regulator of Hh pathway and highlight the perturbation of the Fbxl17-Sufu axis in the pathogenesis of medulloblastoma.
Clearing misfolded proteins from the cytoplasm is essential to maintain cellular homeostasis. Now, a parallel clearance system is described that uses the deubiquitylase USP19 to enable secretion of misfolded cytoplasmic proteins when conventional proteasomal degradation is compromised. Misfolding-associated protein secretion (MAPS) has important implications for protein quality control and prion-like transmission.
OBJECTIVE: The vascular endothelial growth factor (VEGF) receptor Flk1 is essential for vascular development, but the signaling and transcriptional pathways by which its expression is regulated in endothelial cells remain unclear. Although previous studies have identified 2 Flk1 regulatory enhancers, these are dispensable for Flk1 expression, indicating that additional enhancers contribute to Flk1 regulation in endothelial cells. In the present study, we sought to identify Flk1 enhancers contributing to expression in endothelial cells. APPROACH AND RESULTS: A region of the 10th intron of the Flk1 gene (Flk1in10) was identified as a putative enhancer and tested in mouse and zebrafish transgenic models. This region robustly directed reporter gene expression in arterial endothelial cells. Using a combination of targeted mutagenesis of transcription factor-binding sites and gene silencing of transcription factors, we found that Gata and Ets factors are required for Flk1in10 enhancer activity in all endothelial cells. Furthermore, we showed that activity of the Flk1in10 enhancer is restricted to arteries through repression of gene expression in venous endothelial cells by the Notch pathway transcriptional regulator Rbpj. CONCLUSIONS: This study demonstrates a novel mechanism of arterial-venous identity acquisition, indicates a direct link between the Notch and VEGF signaling pathways, and illustrates how cis-regulatory diversity permits differential expression outcomes from a limited repertoire of transcriptional regulators.
INTRODUCTION: Indoleamine 2,3-dioxygenase (IDO) catalyzes the initial step in the catabolism of l-tryptophan along the kynurenine pathway and exerts immunosuppressive properties in inflammatory and tumor tissues by blocking locally T-lymphocyte proliferation. Recently, 1-(2-[(19)F]fluoroethyl)-dl-tryptophan (1-[(19)F]FE-dl-Trp) was reported as a good and specific substrate of this enzyme. Herein, the radiosynthesis of its radioactive isotopomer (1-[(18)F]FE-dl-Trp, dl-[(18)F]5) is presented along with in vitro enzymatic and cellular uptake studies. METHODS: The one-pot n.c.a. radiosynthesis of this novel potential PET imaging tracer, including HPLC purification and formulation, has been fully automated on a FASTlab™ synthesizer. Chiral separation of both isomers and their formulation were implemented on a second cassette. In vitro enzymatic and cellular uptake studies were then conducted with the d-, l- and dl-radiotracers. RESULTS: The radiolabeling of the tosylate precursor was performed in DMF (in 5min; RCY: 57% (d.c.), n=3). After hydrolysis, HPLC purification and formulation, dl-[(18)F]5 was obtained with a global radiochemical yield of 18±3% (not decay corrected, n=7, in 80min) and a specific activity of 600±180GBq/μmol (n=5). The subsequent separation of l- and d-enantiomers was performed by chiral HPLC and both were obtained after formulation with an RCY (d.c.) of 6.1% and 5.8%, respectively. In vitro enzymatic assays reveal that l-[(18)F]5 is a better substrate than d-[(18)F]5 for human IDO. In vitro cellular assays show an IDO-specific uptake of the racemate varying from 30% to 50% of that of l-[(18)F]5, and a negligible uptake of d-[(18)F]5. CONCLUSION: In vitro studies show that l-[(18)F]5 is a good and specific substrate of hIDO, while presenting a very low efflux. These results confirm that l-[(18)F]5 could be a very useful PET radiotracer for IDO expressing cells in cancer imaging.
CpG dinucleotides are the main mutational hot-spot in most cancers. The characteristic elevated C>T mutation rate in CpG sites has been related to 5-methylcytosine (5mC), an epigenetically modified base which resides in CpGs and plays a role in transcription silencing. In brain nearly a third of 5mCs have recently been found to exist in the form of 5-hydroxymethylcytosine (5hmC), yet the effect of 5hmC on mutational processes is still poorly understood. Here we show that 5hmC is associated with an up to 53% decrease in the frequency of C>T mutations in a CpG context compared to 5mC. Tissue specific 5hmC patterns in brain, kidney and blood correlate with lower regional CpG>T mutation frequency in cancers originating in the respective tissues. Together our data reveal global and opposing effects of the two most common cytosine modifications on the frequency of cancer causing somatic mutations in different cell types.
Numerous developmentally regulated genes in mouse embryonic stem cells (ESCs) are marked by both active (H3K4me3)- and polycomb group (PcG)-mediated repressive (H3K27me3) histone modifications. This bivalent state is thought to be important for transcriptional poising, but the mechanisms that regulate bivalent genes and the bivalent state remain incompletely understood. Examining the contribution of microRNAs (miRNAs) to the regulation of bivalent genes, we found that the miRNA biogenesis enzyme DICER was required for the binding of the PRC2 core components EZH2 and SUZ12, and for the presence of the PRC2-mediated histone modification H3K27me3 at many bivalent genes. Genes that lost bivalency were preferentially upregulated at the mRNA and protein levels. Finally, reconstituting Dicer-deficient ESCs with ESC miRNAs restored bivalent gene repression and PRC2 binding at formerly bivalent genes. Therefore, miRNAs regulate bivalent genes and the bivalent state itself.
In a recent study, Leucci et al. report a role for the long non-coding RNA SAMMSON in driving mitochondrial function in melanoma. Targeting SAMMSON, the gene of which is frequently co-amplified with MITF, highlights a new cell-type-specific therapeutic vulnerability in melanoma irrespective of BRAF, NRAS, or p53 status.
The NRF2/sMAF protein complex regulates the oxidative stress response by occupying cis-acting enhancers containing an antioxidant response element (ARE). Integrating genome-wide maps of NRF2/sMAF occupancy with disease-susceptibility loci, we discovered eight polymorphic AREs linked to 14 highly ranked disease-risk SNPs in individuals of European ancestry. Among these SNPs was rs242561, located within a regulatory region of the MAPT gene (encoding microtubule-associated protein Tau). It was consistently occupied by NRF2/sMAF in multiple experiments and its strong-binding allele associated with higher mRNA levels in cell lines and human brain tissue. Induction of MAPT transcription by NRF2 was confirmed using a human neuroblastoma cell line and a Nrf2-deficient mouse model. Most importantly, rs242561 displayed complete linkage disequilibrium with a highly protective allele identified in multiple GWASs of progressive supranuclear palsy, Parkinson's disease, and corticobasal degeneration. These observations suggest a potential role for NRF2/sMAF in tauopathies and a possible role for NRF2 pathway activators in disease prevention.
The modifications 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are the two major DNA epigenetic modifications in mammalian genomes and play crucial roles in development and pathogenesis. Little is known about the colocalization or potential correlation of these two modifications. Here we present an ultrasensitive single-molecule imaging technology capable of detecting and quantifying 5hmC and 5mC from trace amounts of DNA. We used this approach to perform single-molecule fluorescence resonance energy transfer (smFRET) experiments which measure the proximity between 5mC and 5hmC in the same DNA molecule. Our results reveal high levels of adjacent and opposing methylated and hydroxymethylated CpG sites (5hmC/5mCpGs) in mouse genomic DNA across multiple tissues. This identifies the previously undetectable and unappreciated 5hmC/5mCpGs as one of the major states for 5hmC in the mammalian genome and suggest that they could function in promoting gene expression.
Cancer vaccine development has been vigorously pursued for 40 years. Immunity to tumor antigens can be elicited by most vaccines tested, but their clinical efficacy remains modest. We argue that a concerted international effort is necessary to understand the human antitumor immune response and achieve clinically effective cancer vaccines.
Proteasome-catalyzed peptide splicing represents an additional catalytic activity of proteasomes contributing to the pool of MHC-class I-presented epitopes. We here biochemically and functionally characterized a new melanoma gp100 derived spliced epitope. We demonstrate that the gp100(mel)47-52/40-42 antigenic peptide is generated in vitro and in cellulo by a not yet described proteasomal condensation reaction. gp100(mel)47-52/40-42 generation is enhanced in the presence of the β5i/LMP7 proteasome-subunit and elicits a peptide-specific CD8(+) T cell response. Importantly, we demonstrate that different gp100(mel)-derived spliced epitopes are generated and presented to CD8(+) T cells with efficacies comparable to non-spliced canonical tumor epitopes and that gp100(mel)-derived spliced epitopes trigger activation of CD8(+) T cells found in peripheral blood of half of the melanoma patients tested. Our data suggest that both transpeptidation and condensation reactions contribute to the frequent generation of spliced epitopes also in vivo and that their immune relevance may be comparable to non-spliced epitopes.
Decades of research have shown that mutations in the p53 stress response pathway affect the incidence of diverse cancers more than mutations in other pathways. However, most evidence is limited to somatic mutations and rare inherited mutations. Using newly abundant genomic data, we demonstrate that commonly inherited genetic variants in the p53 pathway also affect the incidence of a broad range of cancers more than variants in other pathways. The cancer-associated single nucleotide polymorphisms (SNPs) of the p53 pathway have strikingly similar genetic characteristics to well-studied p53 pathway cancer-causing somatic mutations. Our results enable insights into p53-mediated tumour suppression in humans and into p53 pathway-based cancer surveillance and treatment strategies.
Innate immune signaling relies on the deposition of non-degradative polyubiquitin at receptor-signaling complexes, but how these ubiquitin modifications are regulated by deubiquitinases remains incompletely understood. Met1-linked ubiquitin (Met1-Ub) is assembled by the linear ubiquitin assembly complex (LUBAC), and this is counteracted by the Met1-Ub-specific deubiquitinase OTULIN, which binds to the catalytic LUBAC subunit HOIP. In this study, we report that HOIP also interacts with the deubiquitinase CYLD but that CYLD does not regulate ubiquitination of LUBAC components. Instead, CYLD limits extension of Lys63-Ub and Met1-Ub conjugated to RIPK2 to restrict signaling and cytokine production. Accordingly, Met1-Ub and Lys63-Ub were individually required for productive NOD2 signaling. Our study thus suggests that LUBAC, through its associated deubiquitinases, coordinates the deposition of not only Met1-Ub but also Lys63-Ub to ensure an appropriate response to innate immune receptor activation.
Expression of functionally important genes is often tightly regulated at both transcriptional and post-transcriptional levels. We reported previously that TET1, the founding member of the TET methylcytosine dioxygenase family, plays an essential oncogenic role in MLL-rearranged acute myeloid leukemia (AML), where it is overexpressed owing to MLL-fusion-mediated direct up-regulation at the transcriptional level. Here we show that the overexpression of TET1 in MLL-rearranged AML also relies on the down-regulation of miR-26a, which directly negatively regulates TET1 expression at the post-transcriptional level. Through inhibiting expression of TET1 and its downstream targets, forced expression of miR-26a significantly suppresses the growth/viability of human MLL-rearranged AML cells, and substantially inhibits MLL-fusion-mediated mouse hematopoietic cell transformation and leukemogenesis. Moreover, c-Myc, an oncogenic transcription factor up-regulated in MLL-rearranged AML, mediates the suppression of miR-26a expression at the transcriptional level. Collectively, our data reveal a previously unappreciated signaling pathway involving the MLL-fusion/MYC⊣miR-26a⊣TET1 signaling circuit, in which miR-26a functions as an essential tumor-suppressor mediator and its transcriptional repression is required for the overexpression and oncogenic function of TET1 in MLL-rearranged AML. Thus, restoration of miR-26a expression/function holds therapeutic potential to treat MLL-rearranged AML.
Ventilatory sensitivity to hypoxia increases in response to continued hypoxic exposure as part of acute acclimatisation. Although this process is incompletely understood, insights have been gained through studies of the hypoxia-inducible factor (HIF) hydroxylase system. Genetic studies implicate these pathways widely in the integrated physiology of hypoxia, through effects on developmental or adaptive processes. In keeping with this, mice that are heterozygous for the principal HIF prolyl hydroxylase, PHD2, show enhanced ventilatory sensitivity to hypoxia and carotid body hyperplasia. Here we have sought to understand this process better through comparative analysis of inducible and constitutive inactivation of PHD2 and its principal targets HIF-1α and HIF-2α. We demonstrate that general inducible inactivation of PHD2 in tamoxifen-treated Phd2(f/f);Rosa26(+/CreERT2) mice, like constitutive, heterozygous PHD2 deficiency, enhances hypoxic ventilatory responses (HVRs: 7.2 ± 0.6 vs. 4.4 ± 0.4 ml min(-1) g(-1) in controls, P < 0.01). The ventilatory phenotypes associated with both inducible and constitutive inactivation of PHD2 were strongly compensated for by concomitant inactivation of HIF-2α, but not HIF-1α. Furthermore, inducible inactivation of HIF-2α strikingly impaired ventilatory acclimatisation to chronic hypoxia (HVRs: 4.1 ± 0.5 vs. 8.6 ± 0.5 ml min(-1) g(-1) in controls, P < 0.0001), as well as carotid body cell proliferation (400 ± 81 vs. 2630 ± 390 bromodeoxyuridine-positive cells mm(-2) in controls, P < 0.0001). The findings demonstrate the importance of the PHD2/HIF-2α enzyme-substrate couple in modulating ventilatory sensitivity to hypoxia.
MOTIVATION: Calling changes in DNA, e.g. as a result of somatic events in cancer, requires analysis of multiple matched sequenced samples. Events in low-mappability regions of the human genome are difficult to encode in variant call files and have been under-reported as a result. However, they can be described accurately through thesaurus annotation-a technique that links multiple genomic loci together to explicate a single variant. RESULTS: We here describe software and benchmarks for using thesaurus annotation to detect point changes in DNA from matched samples. In benchmarks on matched normal/tumor samples we show that the technique can recover between five and ten percent more true events than conventional approaches, while strictly limiting false discovery and being fully consistent with popular variant analysis workflows. We also demonstrate the utility of the approach for analysis of de novo mutations in parents/child families. AVAILABILITY AND IMPLEMENTATION: Software performing thesaurus annotation is implemented in java; available in source code on github at GeneticThesaurus (https://github.com/tkonopka/GeneticThesaurus) and as an executable on sourceforge at geneticthesaurus (https://sourceforge.net/projects/geneticthesaurus). Mutation calling is implemented in an R package available on github at RGeneticThesaurus (https://github.com/tkonopka/RGeneticThesaurus). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online. CONTACT: email@example.com.
Human mouse double minute 2 (Mdm2) plays an essential role in the regulation of the tumor suppressor p53. The G/G variant of SNP309 was shown to increase Mdm2 mRNA/protein expression and to be associated with an increased risk and earlier onset of different cancers in Asian populations. However, the frequency and impact of these G/G variants have not been studied in Caucasian renal cell carcinoma (RCC) patients. Therefore, we analyzed an unselected German cohort of 197 consecutive RCC patients and detected the G/G variant in 18 (9.1%) patients, the G/T variant in 116 (58.9%) patients and the T/T variant in 63 (32.0%) patients. Studying the association between age at tumor onset and SNP309 genotypes, no correlation was detected in the entire RCC cohort or among the male RCC patients. However, the female G/G patients (median age 59.5 years) were diagnosed 13.5 years earlier than the T/T females (median age 73 years). When separating all females into two groups at their median age (68 years), 7 and 1 patients with the G/G variant and 9 and 13 patients with the T/T variant were noted in these age groups (P=0.024). To study the age dependency of tumor onset further, a second, age-selected cohort of 205 RCC patients was investigated, which comprised especially young and old patients. Interestingly, the G/G type occurred more often at lower tumor stages and tumor grades compared with higher stages (P=0.039 and 0.004, respectively). In females, the percentage of the G/G variant was only slightly higher in the younger age group, whereas in males, the percentage of the G/G variant was remarkably higher in the younger age group (19.4% vs 8.0%). In summary, female Caucasian RCC patients with the MDM2 SNP309 G/G genotype showed significantly earlier tumor onset than patients with the wild-type T/T genotype.
Cross-presentation enables dendritic cells to present on their MHC class I molecules antigenic peptides derived from exogenous material, through a mechanism that remains partly unclear. It is particularly efficient with long peptides, which are used in cancer vaccines. We studied the mechanism of long-peptide cross-presentation using human dendritic cells and specific CTL clones against melanoma Ags gp100 and Melan-A/MART1. We found that cross-presentation of those long peptides does not depend on the proteasome or the transporter associated with Ag processing, and therefore follows a vacuolar pathway. We also observed that it makes use of newly synthesized MHC class I molecules, through peptide exchange in vesicles distinct from the endoplasmic reticulum and classical secretory pathway, in an SEC22b- and CD74-independent manner. Our results indicate a nonclassical secretion pathway followed by nascent HLA-I molecules that are used for cross-presentation of those long melanoma peptides in the vacuolar pathway. Our results may have implications for the development of vaccines based on long peptides.
Epithelial to mesenchymal transition (EMT) of cancer cells involves loss of epithelial polarity and adhesiveness, and gain of invasive and migratory mesenchymal behaviours. EMT occurs in prostate cancer (PCa) but it is unknown whether this is in specific areas of primary tumours. We examined whether any of eleven EMT-related proteins have altered expression or subcellular localisation within the extraprostatic extension component of locally advanced PCa compared with other localisations, and whether similar changes may occur in in vitro organotypic PCa cell cultures and in vivo PCa models. Expression profiles of three proteins (E-cadherin, Snail, and α-smooth muscle actin) were significantly different in extraprostatic extension PCa compared with intra-prostatic tumour, and 18/27 cases had an expression change of at least one of these three proteins. Of the three significantly altered EMT proteins in pT3 samples, one showed similar significantly altered expression patterns in in vitro organotypic culture models, and two in in vivo Pten-/- model samples. These results suggest that changes in EMT protein expression can be observed in the extraprostatic extension component of locally invasive PCa. The biology of some of these changes in protein expression may be studied in certain in vitro and in vivo PCa models.
Pulmonary neuroepithelial bodies (NEBs), presumed polymodal airway sensors, consist of innervated clusters of amine (serotonin) and peptide-producing cells. While NEB responses to acute hypoxia are mediated by a membrane-bound O2 sensor complex, responses to sustained and/or chronic hypoxia involve a prolyl hydroxylase (PHD)-hypoxia-inducible factor-dependent mechanism. We have previously reported hyperplasia of NEBs in the lungs of Phd1-/- mice associated with enhanced serotonin secretion. Here we use a novel multilabel immunofluorescence method to assess NEB distribution, frequency, and size, together with the number and size of NEB cell nuclei, and to colocalize multiple cytoplasmic and nuclear epitopes in the lungs of Phd1-/-, Phd2+/-, and Phd3-/- mice and compare them with wild-type controls. To define the mechanisms of NEB cell hyperplasia, we used antibodies against Mash1 and Prox1 (neurogenic genes involved in NEB cell differentiation/maturation), hypoxia-inducible factor-1alpha, and the cell proliferation marker Ki67. Morphometric analysis of (% total lung area) immunostaining for synaptophysin (% synaptophysin), a cytoplasmic marker of NEB cells, was significantly increased in Phd1-/- and Phd3-/- mice compared to wild-type mice. In addition, NEB size and the number and size of NEB nuclei were also significantly increased, indicating that deficiency of Phds is associated with striking hyperplasia and hypertrophy of NEBs. In Phd2+/- mice, while mean % synaptophysin was comparable to wild-type controls, the NEB size was moderately increased, suggesting an effect even in heterozygotes. NEBs in all Phd-deficient mice showed increased expression of Mash1, Prox1, Ki67, and hypoxia-inducible factor-1alpha, in keeping with enhanced differentiation from precursor cells and a minor component of cell proliferation. Since the loss of PHD activity mimics chronic hypoxia, our data provide critical information on the potential role of PHDs in the pathobiology and mechanisms of NEB cell hyperplasia that is relevant to a number of pediatric lung disorders.
Genome engineering has been greatly enhanced by the availability of Cas9 endonuclease that can be targeted to almost any genomic locus using so called guide RNAs (gRNAs). However, the introduction of foreign DNA sequences to tag an endogenous gene is still cumbersome as it requires the synthesis or cloning of homology templates. Here we present a strategy that enables the tagging of endogenous loci using one generic donor plasmid. It contains the tag of interest flanked by two gRNA recognition sites that allow excision of the tag from the plasmid. Co-transfection of cells with Cas9, a gRNA specifying the genomic locus of interest, the donor plasmid and a cassette-specific gRNA triggers the insertion of the tag by a homology-independent mechanism. The strategy is efficient and delivers clones that display a predictable integration pattern. As showcases we generated NanoLuc luciferase- and TurboGFP-tagged reporter cell lines.
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