Non-metabolic functions of phosphofructokinase-1 orchestrate tumor cellular invasion and genome maintenance under bevacizumab therapy.

BACKGROUND: Glioblastoma (GBM) is a highly lethal malignancy for which neoangiogenesis serves as a defining hallmark. The anti-VEGF antibody, bevacizumab, has been approved for the treatment of recurrent GBM, but resistance is universal. METHODS: We analyzed expression data of GBM patients treated with bevacizumab to discover potential resistance mechanisms. Patient-derived xenografts (PDXs) and cultures were interrogated for effects of phosphofructokinase-1, muscle isoform (PFKM) loss on tumor cell motility, migration, and invasion through genetic and pharmacologic targeting. RESULTS: We identified PFKM as a driver of bevacizumab resistance. PFKM functions dichotomize based on subcellular location: cytosolic PFKM interacted with KIF11, a tubular motor protein, to promote tumor invasion, whereas nuclear PFKM safeguarded genomic stability of tumor cells through interaction with NBS1. Leveraging differential transcriptional profiling, bupivacaine phenocopied genetic targeting of PFKM, and enhanced efficacy of bevacizumab in preclinical GBM models in vivo. CONCLUSION: PFKM drives novel molecular pathways in GBM, offering a translational path to a novel therapeutic paradigm.

Perspective: targeting VEGF-A and YKL-40 in glioblastoma - matter matters.

Glioblastomas (GBM) are heterogeneous highly vascular brain tumors exploiting the unique microenvironment in the brain to resist treatment and anti-tumor responses. Anti-angiogenic agents, immunotherapy, and targeted therapy have been studied extensively in GBM patients over a number of decades with minimal success. Despite maximal efforts, prognosis remains dismal with an overall survival of approximately 15 months.Bevacizumab, a humanized anti-vascular endothelial growth factor (VEGF) antibody, underwent accelerated approval by the U.S. Food and Drug Administration in 2009 for the treatment of recurrent GBM based on promising preclinical and early clinical studies. Unfortunately, subsequent clinical trials did not find overall survival benefit. Pursuing pleiotropic targets and leaning toward multitarget strategies may be a key to more effective therapeutic intervention in GBM, but preclinical evaluation requires careful consideration of model choices. In this study, we discuss bevacizumab resistance, dual targeting of pro-angiogenic modulators VEGF and YKL-40 in the context of brain tumor microenvironment, and how model choice impacts study conclusions and its translational significance.

A High-Affinity Peptide Ligand Targeting Syntenin Inhibits Glioblastoma.

Despite the recent advances in cancer therapeutics, highly aggressive cancer forms, such as glioblastoma (GBM), still have very low survival rates. The intracellular scaffold protein syntenin, comprising two postsynaptic density protein-95/discs-large/zona occludens-1 (PDZ) domains, has emerged as a novel therapeutic target in highly malignant phenotypes including GBM. Here, we report the development of a novel, highly potent, and metabolically stable peptide inhibitor of syntenin, KSL-128114, which binds the PDZ1 domain of syntenin with nanomolar affinity. KSL-128114 is resistant toward degradation in human plasma and mouse hepatic microsomes and displays a global PDZ domain selectivity for syntenin. An X-ray crystal structure reveals that KSL-128114 interacts with syntenin PDZ1 in an extended noncanonical binding mode. Treatment with KSL-128114 shows an inhibitory effect on primary GBM cell viability and significantly extends survival time in a patient-derived xenograft mouse model. Thus, KSL-128114 is a novel promising candidate with therapeutic potential for highly aggressive tumors, such as GBM.

Development of a highly sensitive chemiluminescence immunoassay for quantification of aggrecanase-generated ARGS aggrecan fragments in serum.

Objective: Cartilage degradation is a hallmark of osteoarthritis (OA). Aggrecan, a major proteoglycan of articular cartilage extracellular matrix (ECM), is degraded by ADAMTS-5 resulting in the release of ARGS-G2 fragments to synovial fluid and circulation. The aim was to quantify ARGS-G2 in the serum of OA patients using the huARGS immunoassay. Methods: The immunoassay was produced under GMP conditions and the technical performance was assessed. The biological relevance of the immunoassay was assessed in the conditioned media from a bovine full-depth cartilage explant (BEX) model. The diurnal and inter-day variations of ARGS levels were evaluated in OA patients' serum. Post-hoc analysis of huARGS was conducted in a sub-cohort of a phase III OA trial testing the safety and efficacy of oral salmon calcitonin. Results: Technical performance: huARGS demonstrated good technical performance. Biological relevance: ARGS release was induced by inflamatory facotrs stimulation compared to the vehicle group, reaching a peak at day 3 and gradually decreasing to base level at day 12. The ARGS release was suppressed by the addition of the ADAMTS-4/-5 activation inhibitor. Biological variation: No significant diurnal or inter-day effect was found. Phase III clinical trial: The participants in the lowest group (Q1) of baseline huARGS levels were more likely to progress radiographically than the highest group (Q4): OR 3.38[0.81-14.02]. Conclusions: The huARGS shows good technical performance and low biological variation. It has the potential to aid drug development in various stages, both as a PD biomarker and identifying progressors who might be likely to respond to an OA drug.

SPT6-driven error-free DNA repair safeguards genomic stability of glioblastoma cancer stem-like cells.

Glioblastoma cancer-stem like cells (GSCs) display marked resistance to ionizing radiation (IR), a standard of care for glioblastoma patients. Mechanisms underpinning radio-resistance of GSCs remain largely unknown. Chromatin state and the accessibility of DNA lesions to DNA repair machineries are crucial for the maintenance of genomic stability. Understanding the functional impact of chromatin remodeling on DNA repair in GSCs may lay the foundation for advancing the efficacy of radio-sensitizing therapies. Here, we present the results of a high-content siRNA microscopy screen, revealing the transcriptional elongation factor SPT6 to be critical for the genomic stability and self-renewal of GSCs. Mechanistically, SPT6 transcriptionally up-regulates BRCA1 and thereby drives an error-free DNA repair in GSCs. SPT6 loss impairs the self-renewal, genomic stability and tumor initiating capacity of GSCs. Collectively, our results provide mechanistic insights into how SPT6 regulates DNA repair and identify SPT6 as a putative therapeutic target in glioblastoma.

Children of Patients Undergoing Psychiatric Treatment: An Investigation of Statutory Support Services After Referrals to Child Protection Services.

AIMS: Preventive interventions for children of parents with mental illness are widely recommended. Mental health services entrust concern for patients' children by referrals to child protection services. We investigated service coverage for children following referrals. METHODS: Data from referrals regarding 376 children of adult psychiatry patients over 2008-2012 was linked to information from municipal records and Danish national registers. We conducted Cox regression and used Kaplan-Meier curves to show time to intervention and cumulative incidence of any child and family support services with one-year follow-up from referral date. RESULTS: At follow-up, 32% of children were provided with a child and family support service on average 73.4 days after referral. The most common services were family treatment (18%) and family counseling (11%). A statutory child assessment was conducted for 21% of children. Contents of the referrals suggested that 60% of children experienced adverse home environments and/or acute situations due to parents' psychiatric illness. Predictors of initiation of support services included a child living alone with the patient, hazard ratio 2.09 (1.41-3.08), the patient being the mother, hazard ratio 1.72 (1.11-2.65), and an adverse home environment presenting an acute situation specified in referral, hazard ratio 1.89 (1.01-3.51). CONCLUSION: Our finding that only one third of children receive support after referrals from psychiatry within an average of three months suggests an underserved population of at-risk children. These findings warrant reconsideration of resource allocation and creation of more efficient intervention strategies to protect at-risk children and prevent development of mental illness and adversity.

Benchmark datasets of immune receptor-epitope structural complexes.

BACKGROUND: The development of accurate epitope prediction tools is important in facilitating disease diagnostics, treatment and vaccine development. The advent of new approaches making use of antibody and TCR sequence information to predict receptor-specific epitopes have the potential to transform the epitope prediction field. Development and validation of these new generation of epitope prediction methods would benefit from regularly updated high-quality receptor-antigen complex datasets. RESULTS: To address the need for high-quality datasets to benchmark performance of these new generation of receptor-specific epitope prediction tools, a webserver called SCEptRe (Structural Complexes of Epitope-Receptor) was created. SCEptRe extracts weekly updated 3D complexes of antibody-antigen, TCR-pMHC and MHC-ligand from the Immune Epitope Database and clusters them based on antigen, receptor and epitope features to generate benchmark datasets. SCEptRe also provides annotated information such as CDR sequences and VDJ genes on the receptors. Users can generate custom datasets based by selecting thresholds for structural quality and clustering parameters (e.g. resolution, R-free factor, antigen or epitope sequence identity) based on their need. CONCLUSIONS: SCEptRe provides weekly updated, user-customized comprehensive benchmark datasets of immune receptor-epitope structural complexes. These datasets can be used to develop and benchmark performance of receptor-specific epitope prediction tools in the future. SCEptRe is freely accessible at http://tools.iedb.org/sceptre .

TCRpMHCmodels: Structural modelling of TCR-pMHC class I complexes.

The interaction between the class I major histocompatibility complex (MHC), the peptide presented by the MHC and the T-cell receptor (TCR) is a key determinant of the cellular immune response. Here, we present TCRpMHCmodels, a method for accurate structural modelling of the TCR-peptide-MHC (TCR-pMHC) complex. This TCR-pMHC modelling pipeline takes as input the amino acid sequence and generates models of the TCR-pMHC complex, with a median Cα RMSD of 2.31 Å. TCRpMHCmodels significantly outperforms TCRFlexDock, a specialised method for docking pMHC and TCR structures. TCRpMHCmodels is simple to use and the modelling pipeline takes, on average, only two minutes. Thanks to its ease of use and high modelling accuracy, we expect TCRpMHCmodels to provide insights into the underlying mechanisms of TCR and pMHC interactions and aid in the development of advanced T-cell-based immunotherapies and rational design of vaccines. The TCRpMHCmodels tool is available at http://www.cbs.dtu.dk/services/TCRpMHCmodels/ .

NetSurfP-2.0: Improved prediction of protein structural features by integrated deep learning.

The ability to predict local structural features of a protein from the primary sequence is of paramount importance for unraveling its function in absence of experimental structural information. Two main factors affect the utility of potential prediction tools: their accuracy must enable extraction of reliable structural information on the proteins of interest, and their runtime must be low to keep pace with sequencing data being generated at a constantly increasing speed. Here, we present NetSurfP-2.0, a novel tool that can predict the most important local structural features with unprecedented accuracy and runtime. NetSurfP-2.0 is sequence-based and uses an architecture composed of convolutional and long short-term memory neural networks trained on solved protein structures. Using a single integrated model, NetSurfP-2.0 predicts solvent accessibility, secondary structure, structural disorder, and backbone dihedral angles for each residue of the input sequences. We assessed the accuracy of NetSurfP-2.0 on several independent test datasets and found it to consistently produce state-of-the-art predictions for each of its output features. We observe a correlation of 80% between predictions and experimental data for solvent accessibility, and a precision of 85% on secondary structure 3-class predictions. In addition to improved accuracy, the processing time has been optimized to allow predicting more than 1000 proteins in less than 2 hours, and complete proteomes in less than 1 day.

Author Correction: BRCA1-regulated RRM2 expression protects glioblastoma cells from endogenous replication stress and promotes tumorigenicity.

This Article contains an error in the spelling of the author Kjeld Møllgård, which is incorrectly given as Kjeld Møllgaard. The error has not been fixed in the original PDF and HTML versions of the Article.

T cell receptor fingerprinting enables in-depth characterization of the interactions governing recognition of peptide-MHC complexes.

The promiscuous nature of T-cell receptors (TCRs) allows T cells to recognize a large variety of pathogens, but makes it challenging to understand and control T-cell recognition. Existing technologies provide limited information about the key requirements for T-cell recognition and the ability of TCRs to cross-recognize structurally related elements. Here we present a 'one-pot' strategy for determining the interactions that govern TCR recognition of peptide-major histocompatibility complex (pMHC). We measured the relative affinities of TCRs to libraries of barcoded peptide-MHC variants and applied this knowledge to understand the recognition motif, here termed the TCR fingerprint. The TCR fingerprints of 16 different TCRs were identified and used to predict and validate cross-recognized peptides from the human proteome. The identified fingerprints differed among TCRs recognizing the same epitope, demonstrating the value of this strategy for understanding T-cell interactions and assessing potential cross-recognition before selection of TCRs for clinical development.

Author Correction: Genome-wide association and HLA fine-mapping studies identify risk loci and genetic pathways underlying allergic rhinitis.

In the version of this article initially published, in Fig. 3, the y-axis numbering did not match the log scale indicated in the axis label. The error has been corrected in the HTML and PDF version of the article.

Genome-wide association and HLA fine-mapping studies identify risk loci and genetic pathways underlying allergic rhinitis.

Allergic rhinitis is the most common clinical presentation of allergy, affecting 400 million people worldwide, with increasing incidence in westernized countries1,2. To elucidate the genetic architecture and understand the underlying disease mechanisms, we carried out a meta-analysis of allergic rhinitis in 59,762 cases and 152,358 controls of European ancestry and identified a total of 41 risk loci for allergic rhinitis, including 20 loci not previously associated with allergic rhinitis, which were confirmed in a replication phase of 60,720 cases and 618,527 controls. Functional annotation implicated genes involved in various immune pathways, and fine mapping of the HLA region suggested amino acid variants important for antigen binding. We further performed genome-wide association study (GWAS) analyses of allergic sensitization against inhalant allergens and nonallergic rhinitis, which suggested shared genetic mechanisms across rhinitis-related traits. Future studies of the identified loci and genes might identify novel targets for treatment and prevention of allergic rhinitis.

VEGF-C sustains VEGFR2 activation under bevacizumab therapy and promotes glioblastoma maintenance.

Background: Glioblastoma ranks among the most lethal cancers, with current therapies offering only palliation. Paracrine vascular endothelial growth factor (VEGF) signaling has been targeted using anti-angiogenic agents, whereas autocrine VEGF/VEGF receptor 2 (VEGFR2) signaling is poorly understood. Bevacizumab resistance of VEGFR2-expressing glioblastoma cells prompted interrogation of autocrine VEGF-C/VEGFR2 signaling in glioblastoma. Methods: Autocrine VEGF-C/VEGFR2 signaling was functionally investigated using RNA interference and exogenous ligands in patient-derived xenograft lines and primary glioblastoma cell cultures in vitro and in vivo. VEGF-C expression and interaction with VEGFR2 in a matched pre- and post-bevacizumab treatment cohort were analyzed by immunohistochemistry and proximity ligation assay. Results: VEGF-C was expressed by patient-derived xenograft glioblastoma lines, primary cells, and matched surgical specimens before and after bevacizumab treatment. VEGF-C activated autocrine VEGFR2 signaling to promote cell survival, whereas targeting VEGF-C expression reprogrammed cellular transcription to attenuate survival and cell cycle progression. Supporting potential translational significance, targeting VEGF-C impaired tumor growth in vivo, with superiority to bevacizumab treatment. Conclusions: Our results demonstrate VEGF-C serves as both a paracrine and an autocrine pro-survival cytokine in glioblastoma, promoting tumor cell survival and tumorigenesis. VEGF-C permits sustained VEGFR2 activation and tumor growth, where its inhibition appears superior to bevacizumab therapy in improving tumor control.

Targeting glioma stem-like cell survival and chemoresistance through inhibition of lysine-specific histone demethylase KDM2B.

Glioblastoma (GBM) ranks among the most lethal cancers, with current therapies offering only palliation. Inter- and intrapatient heterogeneity is a hallmark of GBM, with epigenetically distinct cancer stem-like cells (CSCs) at the apex. Targeting GSCs remains a challenging task because of their unique biology, resemblance to normal neural stem/progenitor cells, and resistance to standard cytotoxic therapy. Here, we find that the chromatin regulator, JmjC domain histone H3K36me2/me1 demethylase KDM2B, is highly expressed in glioblastoma surgical specimens compared to normal brain. Targeting KDM2B function genetically or pharmacologically impaired the survival of patient-derived primary glioblastoma cells through the induction of DNA damage and apoptosis, sensitizing them to chemotherapy. KDM2B loss decreased the GSC pool, which was potentiated by coadministration of chemotherapy. Collectively, our results demonstrate KDM2B is crucial for glioblastoma maintenance, with inhibition causing loss of GSC survival, genomic stability, and chemoresistance.

Improved methods for predicting peptide binding affinity to MHC class II molecules.

Major histocompatibility complex class II (MHC-II) molecules are expressed on the surface of professional antigen-presenting cells where they display peptides to T helper cells, which orchestrate the onset and outcome of many host immune responses. Understanding which peptides will be presented by the MHC-II molecule is therefore important for understanding the activation of T helper cells and can be used to identify T-cell epitopes. We here present updated versions of two MHC-II-peptide binding affinity prediction methods, NetMHCII and NetMHCIIpan. These were constructed using an extended data set of quantitative MHC-peptide binding affinity data obtained from the Immune Epitope Database covering HLA-DR, HLA-DQ, HLA-DP and H-2 mouse molecules. We show that training with this extended data set improved the performance for peptide binding predictions for both methods. Both methods are publicly available at www.cbs.dtu.dk/services/NetMHCII-2.3 and www.cbs.dtu.dk/services/NetMHCIIpan-3.2.

Patients' experience of thirst while being conscious and mechanically ventilated in the intensive care unit.

BACKGROUND: Because of changes in sedation strategies, more patients in the intensive care unit (ICU) are conscious. Therefore, new and challenging tasks in nursing practice have emerged, which require a focus on the problems that patients experience. Thirst is one such major problem, arising because the mechanical ventilator prevents the patients from drinking when they have the urge to do so. To gain a deeper understanding of the patients' experiences and to contribute new knowledge in nursing care, this study focuses on the patients' experiences of thirst during mechanical ventilation (MV) while being conscious. AIMS: To explore patients' experience of thirst while being conscious and mechanically ventilated. DESIGN: This hermeneutic study used qualitative interviews of 12 patients. METHOD: Data were analyzed based on content analysis. Interviews were conducted between September and October 2014 in two large ICUs in Denmark. RESULT: Four themes relating to the patients' experiences of thirst during MV were identified: a paramount thirst, a different sense in the mouth, deprivation of the opportunity to quench thirst and difficulties associated with thirst. CONCLUSION: Patients associate feelings of desperation, anxiety and powerlessness with the experience of thirst. These feelings have a negative impact on their psychological well-being. A strategy in the ICU that includes no sedation for critically ill patients in need of MV introduces new demands on the nurses who must care for patients who are struggling with thirst. RELEVANCE TO CLINICAL PRACTICE: This study shows that despite several practical attempts to relieve thirst, it remains a paramount problem for the patients. ICU nurses need to increase their focus on issues of thirst and dry mouth, which are two closely related issues for the patients. Communication may be a way to involve the patients, recognize and draw attention to their problem.

The collagen receptor uPARAP/Endo180 as a novel target for antibody-drug conjugate mediated treatment of mesenchymal and leukemic cancers.

A key task in developing the field of personalized cancer therapy is the identification of novel molecular targets that enable treatment of cancers not susceptible to other means of specific therapy. The collagen receptor uPARAP/Endo180 is overexpressed by malignant cells in several non-epithelial cancers, notably including sarcomas, glioblastomas and subsets of acute myeloid leukemia. In contrast, in healthy adult individuals, expression is restricted to minor subsets of mesenchymal cells. Functionally, uPARAP/Endo180 is a rapidly recycling endocytic receptor that delivers its cargo directly into the endosomal-lysosomal system, thus opening a potential route of entry into receptor-positive cells. This combination of specific expression and endocytic function appears well suited for targeting of uPARAP/Endo180-positive cancers by antibody-drug conjugate (ADC) mediated drug delivery. Therefore, we utilized a specific monoclonal antibody against uPARAP/Endo180, raised through immunization of a uPARAP/Endo180 knock-out mouse, which reacts with both the human and the murine receptor, to construct a uPARAP-directed ADC. This antibody was coupled to the highly toxic dolastatin derivative, monomethyl auristatin E, via a cathepsin-labile valine-citrulline linker. With this ADC, we show strong and receptor-dependent cytotoxicity in vitro in uPARAP/Endo180-positive cancer cell lines of sarcoma, glioblastoma and leukemic origin. Furthermore, we demonstrate the potency of the ADC in vivo in a xenograft mouse model with human uPARAP/Endo180-positive leukemic cells, obtaining a complete cure of all tested mice following intravenous ADC treatment with no sign of adverse effects. Our study identifies uPARAP/Endo180 as a promising target for novel therapy against several highly malignant cancer types.

Attitudes towards people with depression and schizophrenia among social service workers in Denmark.

BACKGROUND: Mental health-related stigma is a major public health issue, and is an obstacle to the possibility for successful treatment, recovery, and reintegration. AIM: To examine attitudes towards mental illness among employees in the social services. METHODS: The study design was part of a large randomized trial, and data presented in this study are baseline data from this trial. Respondents completed a baseline questionnaire to assess the respondents' attitudes. RESULTS: A significant difference was found between employees' personal attitudes towards depression and schizophrenia. The same significant difference was found in the employees' perceived attitudes. Furthermore, a significant difference was found between the employees' personal and perceived attitudes. A significant difference was found between the respondents wish for social distance towards depression and schizophrenia in all cases, except regarding the willingness to provide a job at one's own workplace. CONCLUSION: Employees in the social services are comparable to the general public concerning attitudes towards mental illness. IMPLICATIONS: The results indicate that the employees in social services could have great use of gaining more knowledge about mental illness and ways in which to recognize a mental illness, in order to be able to offer the right kind of help and reduce the treatment gap concerning people suffering from mental illness.

BRCA1-regulated RRM2 expression protects glioblastoma cells from endogenous replication stress and promotes tumorigenicity.

Oncogene-evoked replication stress (RS) fuels genomic instability in diverse cancer types. Here we report that BRCA1, traditionally regarded a tumour suppressor, plays an unexpected tumour-promoting role in glioblastoma (GBM), safeguarding a protective response to supraphysiological RS levels. Higher BRCA1 positivity is associated with shorter survival of glioma patients and the abrogation of BRCA1 function in GBM enhances RS, DNA damage (DD) accumulation and impairs tumour growth. Mechanistically, we identify a novel role of BRCA1 as a transcriptional co-activator of RRM2 (catalytic subunit of ribonucleotide reductase), whereby BRCA1-mediated RRM2 expression protects GBM cells from endogenous RS, DD and apoptosis. Notably, we show that treatment with a RRM2 inhibitor triapine reproduces the BRCA1-depletion GBM-repressive phenotypes and sensitizes GBM cells to PARP inhibition. We propose that GBM cells are addicted to the RS-protective role of the BRCA1-RRM2 axis, targeting of which may represent a novel paradigm for therapeutic intervention in GBM.