Targeting deoxycytidine kinase improves symptoms in mouse models of multiple sclerosis.

Multiple sclerosis (MS) is an autoimmune disease driven by lymphocyte activation against myelin autoantigens in the central nervous system leading to demyelination and neurodegeneration. The deoxyribonucleoside salvage pathway with the rate-limiting enzyme deoxycytidine kinase (dCK) captures extracellular deoxyribonucleosides for use in intracellular deoxyribonucleotide metabolism. Previous studies have shown that deoxyribonucleoside salvage activity is enriched in lymphocytes and required for early lymphocyte development. However, specific roles for the deoxyribonucleoside salvage pathway and dCK in autoimmune diseases such as MS are unknown. Here we demonstrate that dCK activity is necessary for the development of clinical symptoms in the MOG35-55 and MOG1-125 experimental autoimmune encephalomyelitis (EAE) mouse models of MS. During EAE disease, deoxyribonucleoside salvage activity is elevated in the spleen and lymph nodes. Targeting dCK with the small molecule dCK inhibitor TRE-515 limits disease severity when treatments are started at disease induction or when symptoms first appear. EAE mice treated with TRE-515 have significantly fewer infiltrating leukocytes in the spinal cord, and TRE-515 blocks activation-induced B and T cell proliferation and MOG35-55 -specific T cell expansion without affecting innate immune cells or naïve T and B cell populations. Our results demonstrate that targeting dCK limits symptoms in EAE mice and suggest that dCK activity is required for MOG35-55 -specific lymphocyte activation-induced proliferation.

DMRcaller: a versatile R/Bioconductor package for detection and visualization of differentially methylated regions in CpG and non-CpG contexts.

DNA methylation has been associated with transcriptional repression and detection of differential methylation is important in understanding the underlying causes of differential gene expression. Bisulfite-converted genomic DNA sequencing is the current gold standard in the field for building genome-wide maps at a base pair resolution of DNA methylation. Here we systematically investigate the underlying features of detecting differential DNA methylation in CpG and non-CpG contexts, considering both the case of mammalian systems and plants. In particular, we introduce DMRcaller, a highly efficient R/Bioconductor package, which implements several methods to detect differentially methylated regions (DMRs) between two samples. Most importantly, we show that different algorithms are required to compute DMRs and the most appropriate algorithm in each case depends on the sequence context and levels of methylation. Furthermore, we show that DMRcaller outperforms other available packages and we propose a new method to select the parameters for this tool and for other available tools. DMRcaller is a comprehensive tool for differential methylation analysis which displays high sensitivity and specificity for the detection of DMRs and performs entire genome wide analysis within a few hours.

Surface Immobilization of Redox-Labile Fluorescent Probes: Enabling Single-Cell Co-Profiling of Aerobic Glycolysis and Oncogenic Protein Signaling Activities.

An analytical method is described for profiling lactate production in single cells via the use of coupled enzyme reactions on surface-grafted resazurin molecules. The immobilization of the redox-labile probes was achieved through chemical modifications on resazurin, followed by bio-orthogonal click reactions. The lactate detection was demonstrated to be sensitive and specific. The method was incorporated into a single-cell barcode chip for simultaneous quantification of aerobic glycolysis activities and oncogenic signaling phosphoproteins in cancer. The interplay between glycolysis and oncogenic signaling activities was interrogated on a glioblastoma cell line. Results revealed a drug-induced oncogenic signaling reliance accompanying shifted metabolic paradigms. A drug combination that exploits this induced reliance exhibited synergistic effects in growth inhibition.

Picosecond-resolved fluorescence studies of substrate and cofactor-binding domain mutants in a thermophilic alcohol dehydrogenase uncover an extended network of communication.

Time-resolved fluorescence dynamics are investigated in two mutants of a thermophilic alcohol dehydrogenase (ht-ADH): Y25A (at the dimer interface) and V260A (at the cofactor-binding domain). These residues, ca. 32 Å apart, are shown to exhibit opposing low-temperature effects on the hydride tunneling step. Using single-tryptophan constructs at the active site (Trp87) and a remote, surface-exposed site (Trp167), time-dependent Stokes shifts and collisional quenching data allow an analysis of intra-protein dynamical communication. A double mutant, Y25A:V260A, was also inserted into each single-Trp construct and analyzed accordingly. None of the mutations affect fluorescence lifetimes, Stokes shift relaxation rates, and quenching data for the surface-exposed Trp167 to an appreciable extent. By contrast, fluorescent probes of the active-site tryptophan 87 reveal distinctive forms of dynamical communication. Stokes shifts show that the distal Y25A increases active-site flexibility, V260A introduces a temperature-dependent equilibration process not previously reported by such measurements, and the double mutant (Y25A:V260A) eliminates the temperature-dependent transition sensed by the active-site tryptophan in the presence of V260A. Collisional quenching data at Trp87 further show a structural change in the active-site environment/solvation for V260A. In the aggregate, the temperature dependencies of the fluorescence data are distinct from the breaks in behavior previously reported for catalysis and hydrogen/deuterium exchange, attributed to time scales for the interconversion of protein conformational substates that are slower and more global than the local motions monitored within. An extended network of dynamical communication between the protein dimer surface and substrate- and cofactor-binding domains emerges from the flourescent data.

18F-Labeled brain-penetrant EGFR tyrosine kinase inhibitors for PET imaging of glioblastoma.

Significant evidence suggests that the failure of clinically tested epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (e.g. erlotinib, lapatinib, gefitinib) in glioblastoma (GBM) patients is primarily attributed to insufficient brain penetration, resulting in inadequate exposure to the targeted cells. Molecular imaging tools can facilitate GBM drug development by visualizing drug biodistribution and confirming target expression and localization. To assess brain exposure via PET molecular imaging, we synthesized fluorine-18 isotopologues of two brain-penetrant EGFR tyrosine kinase inhibitors developed specifically for GBM. Adapting our recently reported radiofluorination of N-arylsydnones, we constructed an ortho-disubstituted [18F]fluoroarene as the key intermediate. The radiotracers were produced on an automated synthesis module in 7-8% activity yield with high molar activity. In vivo PET imaging revealed rapid brain uptake in rodents and tumor accumulation in an EGFR-driven orthotopic GBM xenograft model.

Glioblastoma Mesenchymal Transition and Invasion are Dependent on a NF-κB/BRD2 Chromatin Complex.

Glioblastoma (GBM) represents the most aggressive subtype of glioma, noted for its profound invasiveness and molecular heterogeneity. The mesenchymal (MES) transcriptomic subtype is frequently associated with therapy resistance, rapid recurrence, and increased tumor-associated macrophages. Notably, activation of the NF-κB pathway and alterations in the PTEN gene are both associated with this malignant transition. Although PTEN aberrations have been shown to be associated with enhanced NF-κB signaling, the relationships between PTEN, NF-κB and MES transition are poorly understood in GBM. Here, we show that PTEN regulates the chromatin binding of bromodomain and extraterminal (BET) family proteins, BRD2 and BRD4, mediated by p65/RelA localization to the chromatin. By utilizing patient-derived glioblastoma stem cells and CRISPR gene editing of the RELA gene, we demonstrate a crucial role for RelA lysine 310 acetylation in recruiting BET proteins to chromatin for MES gene expression and GBM cell invasion upon PTEN loss. Remarkably, we found that BRD2 is dependent on chromatin associated acetylated RelA for its recruitment to MES gene promoters and their expression. Furthermore, loss of BRD2 results in the loss of MES signature, accompanied by an enrichment of proneural signature and enhanced therapy responsiveness. Finally, we demonstrate that disrupting the NFκB/BRD2 interaction with a brain penetrant BET-BD2 inhibitor reduces mesenchymal gene expression, GBM invasion, and therapy resistance in GBM models. This study uncovers the role of hitherto unexplored PTEN-NF-κB-BRD2 pathway in promoting MES transition and suggests inhibiting this complex with BET-BD2 specific inhibitors as a therapeutic approach to target the MES phenotype in GBM.

M2 isoform of pyruvate kinase rewires glucose metabolism during radiation therapy to promote an antioxidant response and glioblastoma radioresistance.

BACKGROUND: Resistance to existing therapies is a significant challenge in improving outcomes for glioblastoma (GBM) patients. Metabolic plasticity has emerged as an important contributor to therapy resistance, including radiation therapy (RT). Here, we investigated how GBM cells reprogram their glucose metabolism in response to RT to promote radiation resistance. METHODS: Effects of radiation on glucose metabolism of human GBM specimens were examined in vitro and in vivo with the use of metabolic and enzymatic assays, targeted metabolomics, and FDG-PET. Radiosensitization potential of interfering with M2 isoform of pyruvate kinase (PKM2) activity was tested via gliomasphere formation assays and in vivo human GBM models. RESULTS: Here, we show that RT induces increased glucose utilization by GBM cells, and this is accompanied with translocation of GLUT3 transporters to the cell membrane. Irradiated GBM cells route glucose carbons through the pentose phosphate pathway (PPP) to harness the antioxidant power of the PPP and support survival after radiation. This response is regulated in part by the PKM2. Activators of PKM2 can antagonize the radiation-induced rewiring of glucose metabolism and radiosensitize GBM cells in vitro and in vivo. CONCLUSIONS: These findings open the possibility that interventions designed to target cancer-specific regulators of metabolic plasticity, such as PKM2, rather than specific metabolic pathways, have the potential to improve the radiotherapeutic outcomes in GBM patients.

Dopamine Receptor Antagonists, Radiation, and Cholesterol Biosynthesis in Mouse Models of Glioblastoma.

BACKGROUND: Glioblastoma is the deadliest brain tumor in adults, and the standard of care consists of surgery followed by radiation and treatment with temozolomide. Overall survival times for patients suffering from glioblastoma are unacceptably low indicating an unmet need for novel treatment options. METHODS: Using patient-derived HK-157, HK-308, HK-374, and HK-382 glioblastoma lines, the GL261 orthotopic mouse models of glioblastoma, and HK-374 patient-derived orthotopic xenografts, we tested the effect of radiation and the dopamine receptor antagonist quetiapine on glioblastoma self-renewal in vitro and survival in vivo. A possible resistance mechanism was investigated using RNA-sequencing. The blood-brain-barrier-penetrating statin atorvastatin was used to overcome this resistance mechanism. All statistical tests were 2-sided. RESULTS: Treatment of glioma cells with the dopamine receptor antagonist quetiapine reduced glioma cell self-renewal in vitro, and combined treatment of mice with quetiapine and radiation prolonged the survival of glioma-bearing mice. The combined treatment induced the expression of genes involved in cholesterol biosynthesis. This rendered GL261 and HK-374 orthotopic tumors vulnerable to simultaneous treatment with atorvastatin and further statistically significantly prolonged the survival of C57BL/6 (n = 10 to 16 mice per group; median survival not reached; log-rank test, P < .001) and NOD Scid gamma mice (n = 8 to 21 mice per group; hazard ratio = 3.96, 95% confidence interval = 0.29 to 12.40; log-rank test, P < .001), respectively. CONCLUSIONS: Our results indicate promising therapeutic efficacy with the triple combination of quetiapine, atorvastatin, and radiation treatment against glioblastoma without increasing the toxicity of radiation. With both drugs readily available for clinical use, our study could be rapidly translated into a clinical trial.

Development of a Potent Brain-Penetrant EGFR Tyrosine Kinase Inhibitor against Malignant Brain Tumors.

The epidermal growth factor receptor (EGFR) is genetically altered in nearly 60% of glioblastoma tumors; however, tyrosine kinase inhibitors (TKIs) against EGFR have failed to show efficacy for patients with these lethal brain tumors. This failure is attributed to the inability of clinically tested EGFR TKIs to cross the blood-brain barrier (BBB) and achieve adequate pharmacological levels to inhibit various oncogenic forms of EGFR that drive glioblastoma. Through SAR analysis, we developed compound 5 (JCN037) from an anilinoquinazoline scaffold by ring fusion of the 6,7-dialkoxy groups to reduce the number of rotatable bonds and polar surface area and by introduction of an ortho-fluorine and meta-bromine on the aniline ring for improved potency and BBB penetration. Relative to the conventional EGFR TKIs erlotinib and lapatinib, JCN037 displayed potent activity against EGFR amplified/mutant patient-derived cell cultures, significant BBB penetration (2:1 brain-to-plasma ratio), and superior efficacy in an EGFR-driven orthotopic glioblastoma xenograft model.

Glioblastoma in adults: a Society for Neuro-Oncology (SNO) and European Society of Neuro-Oncology (EANO) consensus review on current management and future directions.

Glioblastomas are the most common form of malignant primary brain tumor and an important cause of morbidity and mortality. In recent years there have been important advances in understanding the molecular pathogenesis and biology of these tumors, but this has not translated into significantly improved outcomes for patients. In this consensus review from the Society for Neuro-Oncology (SNO) and the European Association of Neuro-Oncology (EANO), the current management of isocitrate dehydrogenase wildtype (IDHwt) glioblastomas will be discussed. In addition, novel therapies such as targeted molecular therapies, agents targeting DNA damage response and metabolism, immunotherapies, and viral therapies will be reviewed, as well as the current challenges and future directions for research.

Regulation of endothelial cell function by GRGDSP peptide grafted on interpenetrating polymers.

Vascular endothelium plays an important role in preventing thrombogenesis. Bioactive molecules such as fibronectin-derived peptide Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) can be used to modify the surface of cardiovascular implants such as vascular grafts to promote endothelialization. Here we conjugated GRGDSP peptide to the nonfouling surface of an interpenetrating polymer network (IPN), and investigated the effects of the immobilized GRGDSP molecules on EC functions under static and flow conditions at well-defined GRGDSP surface densities (approximately 0 to 3 pmol/cm2). EC adhesion and spreading increased with GRGDSP surface density, reached a plateau at 1.5 pmol/cm2, and increased further beyond 2.8 pmol/cm2. Cell adhesion and spreading on GRGDSP induced two waves of extracellular signal-regulated kinase (ERK) activation, and 0.2 pmol/cm2 density of GRGDSP was sufficient to activate ERK. EC proliferation rate was not sensitive to GRGDSP surface density, suggesting that cell spreading at low-density of GRGDSP is sufficient to maintain EC proliferation. EC migration on lower-density GRGDSP-IPN surfaces was faster under static condition. With the increase of GRGDSP density, the speed and persistence of EC migration dropped quickly (0.2-0.8 pmol/cm2) and reached a plateau, followed by a slower and gradual decrease (1.5-3.0 pmol/cm2). These data suggest that the changes of EC functions were more sensitive to the increase of GRGDSP density at lower range. Under flow condition with shear stress at 12 dyn/cm2, EC migration was inhibited on GRGDSP-IPN surfaces, which may be attributed to the assembly of large focal adhesions induced by shear stress, suggesting a catch-bond characteristic for RGD-integrin binding. This study provides a rational base for surface engineering of cardiovascular implants.

Cytoplasmic p53 couples oncogene-driven glucose metabolism to apoptosis and is a therapeutic target in glioblastoma.

Cross-talk among oncogenic signaling and metabolic pathways may create opportunities for new therapeutic strategies in cancer. Here we show that although acute inhibition of EGFR-driven glucose metabolism induces only minimal cell death, it lowers the apoptotic threshold in a subset of patient-derived glioblastoma (GBM) cells. Mechanistic studies revealed that after attenuated glucose consumption, Bcl-xL blocks cytoplasmic p53 from triggering intrinsic apoptosis. Consequently, targeting of EGFR-driven glucose metabolism in combination with pharmacological stabilization of p53 with the brain-penetrant small molecule idasanutlin resulted in synthetic lethality in orthotopic glioblastoma xenograft models. Notably, neither the degree of EGFR-signaling inhibition nor genetic analysis of EGFR was sufficient to predict sensitivity to this therapeutic combination. However, detection of rapid inhibitory effects on [18F]fluorodeoxyglucose uptake, assessed through noninvasive positron emission tomography, was an effective predictive biomarker of response in vivo. Together, these studies identify a crucial link among oncogene signaling, glucose metabolism, and cytoplasmic p53, which may potentially be exploited for combination therapy in GBM and possibly other malignancies.

Risk Management in Biologics Technology Transfer.

Technology transfer of biological products is a complex process that is important for product commercialization. To achieve a successful technology transfer, the risks that arise from changes throughout the project must be managed. Iterative risk analysis and mitigation tools can be used to both evaluate and reduce risk. The technology transfer stage gate model is used as an example tool to help manage risks derived from both designed process change and unplanned changes that arise due to unforeseen circumstances. The strategy of risk assessment for a change can be tailored to the type of change. In addition, a cross-functional team and centralized documentation helps maximize risk management efficiency to achieve a successful technology transfer.

Overview of Best Practices for Biopharmaceutical Technology Transfers.

Technology transfer is a key foundational component in product commercialization. It is more than just the transfer of documents; it relates to all aspects of the transfer of knowledge and experience to the commercial manufacturing unit to ensure consistent, safe, and high-quality product. This is the first in a series of articles from the BioPhorum Operations Group (BPOG) member companies discussing best practices and benchmarking of biopharmaceutical technology transfer. In this article, we provide the common terminology developed by BPOG to accommodate both transferring and receiving organizations. We also review the key elements of a robust technology transfer business process, including critical milestones. Finally, we provide a brief overview of the articles in this series.

The relationship between dopamine receptor D1 and cognitive performance.

BACKGROUND: Cognitive impairment cuts across traditional diagnostic boundaries and is one of the most typical symptoms in various psychiatric and neurobiological disorders. AIMS: The objective of this study was to examine the genetic association between 94 candidate genes, including receptors and enzymes that participate in neurotransmission, with measures of cognition. METHODS: The Clinical Dementia Rating (CDR), a global measure of cognition, and genotypes derived from a custom array of 1,536 single-nucleotide polymorphisms (SNPs) in 94 genes were available for a large postmortem cohort of Caucasian cases with Alzheimer's disease (AD), schizophrenia and controls (n=727). A cohort of healthy young males (n=1,493) originating from the LOGOS project (Learning On Genetics Of Schizophrenia Spectrum) profiled across multiple cognitive domains was available for targeted SNP genotyping. Gene expression was quantified in the superior temporal gyrus of control samples (n=109). The regulatory effect on transcriptional activity was assessed using the luciferase reporter system. RESULTS: The rs5326-A allele at the promoter region of dopamine receptor D1 (DRD1) locus was associated with: (i) poorer cognition (higher CDR) in the postmortem cohort (P=9.325×10(-4)); (ii) worse cognitive performance relevant to strategic planning in the LOGOS cohort (P=0.008); (iii) lower DRD1 gene expression in the superior temporal gyrus of controls (P=0.038); and (iv) decreased transcriptional activity in human neuroblastoma (SH-SY5Y) cells (P=0.026). CONCLUSIONS: An interdisciplinary approach combining genetics with cognitive and molecular neuroscience provided a possible mechanistic link among DRD1 and alterations in cognitive performance.

Erratum: The relationship between dopamine receptor D1 and cognitive performance.

[This corrects the article DOI: 10.1038/npjschz.2014.2.][This corrects the article DOI: 10.1038/npjschz.2014.2.].

A role for noncoding variation in schizophrenia.

A large portion of common variant loci associated with genetic risk for schizophrenia reside within noncoding sequence of unknown function. Here, we demonstrate promoter and enhancer enrichment in schizophrenia variants associated with expression quantitative trait loci (eQTL). The enrichment is greater when functional annotations derived from the human brain are used relative to peripheral tissues. Regulatory trait concordance analysis ranked genes within schizophrenia genome-wide significant loci for a potential functional role, based on colocalization of a risk SNP, eQTL, and regulatory element sequence. We identified potential physical interactions of noncontiguous proximal and distal regulatory elements. This was verified in prefrontal cortex and -induced pluripotent stem cell-derived neurons for the L-type calcium channel (CACNA1C) risk locus. Our findings point to a functional link between schizophrenia-associated noncoding SNPs and 3D genome architecture associated with chromosomal loopings and transcriptional regulation in the brain.