Promising results of a clinical feasibility study: CIRBP as a potential biomarker in pediatric cardiac surgery.

Objective: Cold-inducible RNA binding Protein (CIRBP) has been shown to be a potent inflammatory mediator and could serve as a novel biomarker for inflammation. Systemic inflammatory response syndrome (SIRS) and capillary leak syndrome (CLS) are frequent complications after pediatric cardiac surgery increasing morbidity, therefore early diagnosis and therapy is crucial. As CIRBP serum levels have not been analyzed in a pediatric population, we conducted a clinical feasibility establishing a customized magnetic bead panel analyzing CIRBP in pediatric patients undergoing cardiac surgery. Methods: A prospective hypothesis generating observational clinical study was conducted at the German Heart Center Berlin during a period of 9 months starting in May 2020 (DRKS00020885, https://drks.de/search/de/trial/DRKS00020885). Serum samples were obtained before the cardiac operation, upon arrival at the pediatric intensive care unit, 6 and 24 h after the operation in patients up to 18 years of age with congenital heart disease (CHD). Customized multiplex magnetic bead-based immunoassay panels were developed to analyze CIRBP, Interleukin-1ß (IL-1ß), Interleukin-6 (IL-6), Interleukin-8 (IL-8), Interleukin-10 (IL-10), Monocyte chemotactic protein 1 (MCP-1), Syndecan-1 (SDC-1), Thrombomodulin (TM), Vascular endothelial growth factor (VEGF-A), Angiopoietin-2 (Ang-2), and Fibroblast growth factor 23 (FGF-23) in 25 µl serum using the Luminex MagPix® system. Results: 19 patients representing a broad range of CHD (10 male patients, median age 2 years, 9 female patients, median age 3 years) were included in the feasibility study. CIRBP was detectable in the whole patient cohort. Relative to individual baseline values, CIRBP concentrations increased 6 h after operation and returned to baseline levels over time. IL-6, IL-8, IL-10, and MCP-1 concentrations were significantly increased after operation and except for MCP-1 concentrations stayed upregulated over time. SDC-1, TM, Ang-2, as well as FGF-23 concentrations were also significantly increased, whereas VEGF-A concentration was significantly decreased after surgery. Discussion: Using customized magnetic bead panels, we were able to detect CIRBP in a minimal serum volume (25 µl) in all enrolled patients. To our knowledge this is the first clinical study to assess CIRBP serum concentrations in a pediatric population.

Passenger Gene Coamplifications Create Collateral Therapeutic Vulnerabilities in Cancer.

DNA amplifications in cancer do not only harbor oncogenes. We sought to determine whether passenger coamplifications could create collateral therapeutic vulnerabilities. Through an analysis of >3,000 cancer genomes followed by the interrogation of CRISPR-Cas9 loss-of-function screens across >700 cancer cell lines, we determined that passenger coamplifications are accompanied by distinct dependency profiles. In a proof-of-principle study, we demonstrate that the coamplification of the bona fide passenger gene DEAD-Box Helicase 1 (DDX1) creates an increased dependency on the mTOR pathway. Interaction proteomics identified tricarboxylic acid (TCA) cycle components as previously unrecognized DDX1 interaction partners. Live-cell metabolomics highlighted that this interaction could impair TCA activity, which in turn resulted in enhanced mTORC1 activity. Consequently, genetic and pharmacologic disruption of mTORC1 resulted in pronounced cell death in vitro and in vivo. Thus, structurally linked coamplification of a passenger gene and an oncogene can result in collateral vulnerabilities. SIGNIFICANCE: We demonstrate that coamplification of passenger genes, which were largely neglected in cancer biology in the past, can create distinct cancer dependencies. Because passenger coamplifications are frequent in cancer, this principle has the potential to expand target discovery in oncology. This article is featured in Selected Articles from This Issue, p. 384.

Prdm16 mutation determines sex-specific cardiac metabolism and identifies two novel cardiac metabolic regulators.

AIMS: Mutation of the PRDM16 gene causes human dilated and non-compaction cardiomyopathy. The PRDM16 protein is a transcriptional regulator that affects cardiac development via Tbx5 and Hand1, thus regulating myocardial structure. The biallelic inactivation of Prdm16 induces severe cardiac dysfunction with post-natal lethality and hypertrophy in mice. The early pathological events that occur upon Prdm16 inactivation have not been explored. METHODS AND RESULTS: This study performed in-depth pathophysiological and molecular analyses of male and female Prdm16csp1/wt mice that carry systemic, monoallelic Prdm16 gene inactivation. We systematically assessed early molecular changes through transcriptomics, proteomics, and metabolomics. Kinetic modelling of cardiac metabolism was performed in silico with CARDIOKIN. Prdm16csp1/wt mice are viable up to 8 months, develop hypoplastic hearts, and diminished systolic performance that is more pronounced in female mice. Prdm16csp1/wt cardiac tissue of both sexes showed reductions in metabolites associated with amino acid as well as glycerol metabolism, glycolysis, and the tricarboxylic acid cycle. Prdm16csp1/wt cardiac tissue revealed diminished glutathione (GSH) and increased inosine monophosphate (IMP) levels indicating oxidative stress and a dysregulated energetics, respectively. An accumulation of triacylglycerides exclusively in male Prdm16csp1/wt hearts suggests a sex-specific metabolic adaptation. Metabolic modelling using CARDIOKIN identified a reduction in fatty acid utilization in males as well as lower glucose utilization in female Prdm16csp1/wt cardiac tissue. On the level of transcripts and protein expression, Prdm16csp1/wt hearts demonstrate an up-regulation of pyridine nucleotide-disulphide oxidoreductase domain 2 (Pyroxd2) and the transcriptional regulator pre-B-cell leukaemia transcription factor interacting protein 1 (Pbxip1). The strongest concordant transcriptional up-regulation was detected for Prdm16 itself, probably through an autoregulatory mechanism. CONCLUSIONS: Monoallelic, global Prdm16 mutation diminishes cardiac performance in Prdm16csp1/wt mice. Metabolic alterations and transcriptional dysregulation in Prdm16csp1/wt affect cardiac tissue. Female Prdm16csp1/wt mice develop a more pronounced phenotype, indicating sexual dimorphism at this early pathological window. This study suggests that metabolic dysregulation is an early event in the PRDM16 associated cardiac pathology.

Neurofibromin 1 mutations impair the function of human induced pluripotent stem cell-derived microglia.

Neurofibromatosis type 1 (NF1) is an autosomal dominant condition caused by germline mutations in the neurofibromin 1 (NF1) gene. Children with NF1 are prone to the development of multiple nervous system abnormalities, including autism and brain tumors, which could reflect the effect of NF1 mutation on microglia function. Using heterozygous Nf1-mutant mice, we previously demonstrated that impaired purinergic signaling underlies deficits in microglia process extension and phagocytosis in situ. To determine whether these abnormalities are also observed in human microglia in the setting of NF1, we leveraged an engineered isogenic series of human induced pluripotent stem cells to generate human microglia-like (hiMGL) cells heterozygous for three different NF1 gene mutations found in patients with NF1. Whereas all NF1-mutant and isogenic control hiMGL cells expressed classical microglia markers and exhibited similar transcriptomes and cytokine/chemokine release profiles, only NF1-mutant hiMGL cells had defects in P2X receptor activation, phagocytosis and motility. Taken together, these findings indicate that heterozygous NF1 mutations impair a subset of the functional properties of human microglia, which could contribute to the neurological abnormalities seen in children with NF1.

Analysis of adherent cell culture lysates with low metabolite concentrations using the Biocrates AbsoluteIDQ p400 HR kit.

The AbsoluteIDQ p400 HR kit is a commercial product for targeted metabolomics. While the kit has been validated for human plasma and serum, adherent cell lysates have not yet been evaluated. We have optimized the detection of polar and lipid metabolites in cell lysates using the kit to enable robust and repeatable analysis of the detected metabolites. Parameters optimized include total cell mass, loading volume and extraction solvent. We present a cell preparation and analytical method and report on the performance of the kit with regard to detectability of the targeted metabolites and their repeatability. The kit can be successfully used for a relative quantification analysis of cell lysates from adherent cells although validated only for human plasma and serum. Most metabolites are below the limit of the Biocrates' set quantification limits and we confirmed that this relative quantification can be used for further statistical analysis. Using this approach, up to 45% of the total metabolites in the kit can be detected with a reasonable analytical performance (lowest median RSD 9% and 13% for LC and FIA, respectively) dependent on the method used. We recommend using ethanol as the extraction solvent for cell lysates of osteosarcoma cell lines for the broadest metabolite coverage and 25 mg of cell mass with a loading volume of 20 µL per sample.

Optimized Workflow for On-Line Derivatization for Targeted Metabolomics Approach by Gas Chromatography-Mass Spectrometry.

Using manual derivatization in gas chromatography-mass spectrometry samples have varying equilibration times before analysis which increases technical variability and limits the number of potential samples analyzed. By contrast, automated derivatization methods can derivatize and inject each sample in an identical manner. We present a fully automated (on-line) derivatization method used for targeted analysis of different matrices. We describe method optimization and compare results from using off-line and on-line derivatization protocols, including the robustness and reproducibility of the methods. Our final parameters for the derivatization process were 20 µL of methoxyamine (MeOx) in pyridine for 60 min at 30 °C followed by 80 µL N-Methyl-N-trimethylsilyltrifluoracetamide (MSTFA) for 30 min at 30 °C combined with 4 h of equilibration time. The repeatability test in plasma and liver revealed a median relative standard deviation (RSD) of 16% and 10%, respectively. Serum samples showed a consistent intra-batch median RSD of 20% with an inter-batch variability of 27% across three batches. The direct comparison of on-line versus off-line demonstrated that on-line was fit for purpose and improves repeatability with a measured median RSD of 11% compared to 17% using the same method off-line. In summary, we recommend that optimized on-line methods may improve results for metabolomics and should be used where available.

Progression-Dependent Altered Metabolism in Osteosarcoma Resulting in Different Nutrient Source Dependencies.

Osteosarcoma (OS) is a primary malignant bone tumor and OS metastases are mostly found in the lung. The limited understanding of the biology of metastatic processes in OS limits the ability for effective treatment. Alterations to the metabolome and its transformation during metastasis aids the understanding of the mechanism and provides information on treatment and prognosis. The current study intended to identify metabolic alterations during OS progression by using a targeted gas chromatography mass spectrometry approach. Using a female OS cell line model, malignant and metastatic cells increased their energy metabolism compared to benign OS cells. The metastatic cell line showed a faster metabolic flux compared to the malignant cell line, leading to reduced metabolite pools. However, inhibiting both glycolysis and glutaminolysis resulted in a reduced proliferation. In contrast, malignant but non-metastatic OS cells showed a resistance to glycolytic inhibition but a strong dependency on glutamine as an energy source. Our in vivo metabolic approach hinted at a potential sex-dependent metabolic alteration in OS patients with lung metastases (LM), although this will require validation with larger sample sizes. In line with the in vitro results, we found that female LM patients showed a decreased central carbon metabolism compared to metastases from male patients.

Modified Protocol of Harvesting, Extraction, and Normalization Approaches for Gas Chromatography Mass Spectrometry-Based Metabolomics Analysis of Adherent Cells Grown Under High Fetal Calf Serum Conditions.

A gas chromatography mass spectrometry (GC-MS) metabolomics protocol was modified for quenching, harvesting, and extraction of metabolites from adherent cells grown under high (20%) fetal calf serum conditions. The reproducibility of using either 50% or 80% methanol for quenching of cells was compared for sample harvest. To investigate the efficiency and reproducibility of intracellular metabolite extraction, different volumes and ratios of chloroform were tested. Additionally, we compared the use of total protein amount versus cell mass as normalization parameters. We demonstrate that the method involving 50% methanol as quenching buffer followed by an extraction step using an equal ratio of methanol:chloroform:water (1:1:1, v/v/v) followed by the collection of 6 mL polar phase for GC-MS measurement was superior to the other methods tested. Especially for large sample sets, its comparative ease of measurement leads us to recommend normalization to protein amount for the investigation of intracellular metabolites of adherent human cells grown under high (or standard) fetal calf serum conditions. To avoid bias, care should be taken beforehand to ensure that the ratio of total protein to cell number are consistent among the groups tested. For this reason, it may not be suitable where culture conditions or cell types have very different protein outputs (e.g., hypoxia vs. normoxia). The full modified protocol is available in the Supplementary Materials.

Sex-specific metabolic and functional differences in human umbilical vein endothelial cells from twin pairs.

BACKGROUND AND AIMS: Gonadal hormones are mainly thought to account for sex and gender differences in the incidence, clinical manifestation and therapy of many cardiovascular diseases. However, intrinsic sex differences at the cellular level are mostly overlooked. Here, we assessed sex-specific metabolic and functional differences between male and female human umbilical vein endothelial cells (HUVECs). METHODS: Cellular metabolism was investigated by bioenergetic studies (Seahorse Analyser) and a metabolomic approach. Protein levels were determined by Western blots and proteome analysis. Vascular endothelial growth factor (VEGF)-stimulated cellular migration was assessed by gap closure. HUVECs from dizygotic twin pairs were used for most experiments. RESULTS: No sex differences were observed in untreated cells. However, sexual dimorphisms appeared after stressing the cells by serum starvation and treatment with VEGF. Under both conditions, female cells had higher intracellular ATP and metabolite levels. A significant decline in ATP levels was observed in male cells after serum starvation. After VEGF, the ratio of glycolysis/mitochondrial respiration was higher in female cells and migration was more pronounced. CONCLUSIONS: These results point to an increased stress tolerance of female cells. We therefore propose that female cells have an energetic advantage over male cells under conditions of diminished nutrient supply. A more favourable energy balance of female HUVECs after serum starvation and VEGF could potentially explain their stronger migratory capacity.

SorCS2 Controls Functional Expression of Amino Acid Transporter EAAT3 and Protects Neurons from Oxidative Stress and Epilepsy-Induced Pathology.

VPS10P domain receptors emerge as central regulators of intracellular protein sorting in neurons with relevance for various brain pathologies. Here, we identified a role for the family member SorCS2 in protection of neurons from oxidative stress and epilepsy-induced cell death. We show that SorCS2 acts as sorting receptor that sustains cell surface expression of the neuronal amino acid transporter EAAT3 to facilitate import of cysteine, required for synthesis of the reactive oxygen species scavenger glutathione. Lack of SorCS2 causes depletion of EAAT3 from the plasma membrane and impairs neuronal cysteine uptake. As a consequence, SorCS2-deficient mice exhibit oxidative brain damage that coincides with enhanced neuronal cell death and increased mortality during epilepsy. Our findings highlight a protective role for SorCS2 in neuronal stress response and provide a possible explanation for upregulation of this receptor seen in surviving neurons of the human epileptic brain.

Alterations of mTOR signaling impact metabolic stress resistance in colorectal carcinomas with BRAF and KRAS mutations.

Metabolic reprogramming is as a hallmark of cancer, and several studies have reported that BRAF and KRAS tumors may be accompanied by a deregulation of cellular metabolism. We investigated how BRAFV600E and KRASG12V affect cell metabolism, stress resistance and signaling in colorectal carcinoma cells driven by these mutations. KRASG12V expressing cells are characterized by the induction of glycolysis, accumulation of lactic acid and sensitivity to glycolytic inhibition. Notably mathematical modelling confirmed the critical role of MCT1 designating the survival of KRASG12V cells. Carcinoma cells harboring BRAFV600E remain resistant towards alterations of glucose supply or application of signaling or metabolic inhibitors. Altogether these data demonstrate that an oncogene-specific decoupling of mTOR from AMPK or AKT signaling accounts for alterations of resistance mechanisms and metabolic phenotypes. Indeed the inhibition of mTOR in BRAFV600E cells counteracts the metabolic predisposition and demonstrates mTOR as a potential target in BRAFV600E-driven colorectal carcinomas.

An immediate-late gene expression module decodes ERK signal duration.

The RAF-MEK-ERK signalling pathway controls fundamental, often opposing cellular processes such as proliferation and apoptosis. Signal duration has been identified to play a decisive role in these cell fate decisions. However, it remains unclear how the different early and late responding gene expression modules can discriminate short and long signals. We obtained both protein phosphorylation and gene expression time course data from HEK293 cells carrying an inducible construct of the proto-oncogene RAF By mathematical modelling, we identified a new gene expression module of immediate-late genes (ILGs) distinct in gene expression dynamics and function. We find that mRNA longevity enables these ILGs to respond late and thus translate ERK signal duration into response amplitude. Despite their late response, their GC-rich promoter structure suggested and metabolic labelling with 4SU confirmed that transcription of ILGs is induced immediately. A comparative analysis shows that the principle of duration decoding is conserved in PC12 cells and MCF7 cells, two paradigm cell systems for ERK signal duration. Altogether, our findings suggest that ILGs function as a gene expression module to decode ERK signal duration.

Effects of RAF inhibitors on PI3K/AKT signalling depend on mutational status of the RAS/RAF signalling axis.

Targeted therapies within the RAS/RAF/MEK/ERK signalling axis become increasingly popular, yet cross-talk and feedbacks in the signalling network lead to unexpected effects. Here we look systematically into how inhibiting RAF and MEK with clinically relevant inhibitors result in changes in PI3K/AKT activation. We measure the signalling response using a bead-based ELISA, and use a panel of three cell lines, and isogenic cell lines that express mutant forms of the oncogenes KRAS and BRAF to interrogate the effects of the MEK and RAF inhibitors on signalling. We find that treatment with the RAF inhibitors have opposing effects on AKT phosphorylation depending on the mutational status of two important oncogenes, KRAS and BRAF. If these two genes are in wildtype configuration, RAF inhibitors reduce AKT phosphorylation. In contrast, if BRAF or KRAS are mutant, RAF inhibitors will leave AKT phosphorylation unaffected or lead to an increase of AKT phosphorylation. Down-regulation of phospho-AKT by RAF inhibitors also extends to downstream transcription factors, and correlates with apoptosis induction. Our results show that oncogenes rewire signalling such that targeted therapies can have opposing effects on parallel pathways, which depend on the mutational status of the cell.

Analysis of impedance-based cellular growth assays.

MOTIVATION: Impedance-based technologies are advancing methods for measuring proliferation of adherent cell cultures non-invasively and in real time. The analysis of the resulting data has so far been hampered by inappropriate computational methods and the lack of systematic data to evaluate the characteristics of the assay. RESULTS: We used a commercially available system for impedance-based growth measurement (xCELLigence) and compared the reported cell index with data from microscopy. We found that the measured signal correlates linearly with the cell number throughout the time of an experiment with sufficient accuracy in subconfluent cell cultures. The resulting growth curves for various colon cancer cells could be well described with the empirical Richards growth model, which allows for extracting quantitative parameters (such as characteristic cycle times). We found that frequently used readouts like the cell index at a specific time or the area under the growth curve cannot be used to faithfully characterize growth inhibition. We propose to calculate the average growth rate of selected time intervals to accurately estimate time-dependent IC50 values of drugs from growth curves. CONTACT: nils.bluethgen@charite.de SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Network quantification of EGFR signaling unveils potential for targeted combination therapy.

The epidermal growth factor receptor (EGFR) signaling network is activated in most solid tumors, and small-molecule drugs targeting this network are increasingly available. However, often only specific combinations of inhibitors are effective. Therefore, the prediction of potent combinatorial treatments is a major challenge in targeted cancer therapy. In this study, we demonstrate how a model-based evaluation of signaling data can assist in finding the most suitable treatment combination. We generated a perturbation data set by monitoring the response of RAS/PI3K signaling to combined stimulations and inhibitions in a panel of colorectal cancer cell lines, which we analyzed using mathematical models. We detected that a negative feedback involving EGFR mediates strong cross talk from ERK to AKT. Consequently, when inhibiting MAPK, AKT activity is increased in an EGFR-dependent manner. Using the model, we predict that in contrast to single inhibition, combined inactivation of MEK and EGFR could inactivate both endpoints of RAS, ERK and AKT. We further could demonstrate that this combination blocked cell growth in BRAF- as well as KRAS-mutated tumor cells, which we confirmed using a xenograft model.

Strong negative feedback from Erk to Raf confers robustness to MAPK signalling.

Protein levels within signal transduction pathways vary strongly from cell to cell. Here, we analysed how signalling pathways can still process information quantitatively despite strong heterogeneity in protein levels. We systematically perturbed the protein levels of Erk, the terminal kinase in the MAPK signalling pathway in a panel of human cell lines. We found that the steady-state phosphorylation of Erk is very robust against perturbations of Erk protein level. Although a multitude of mechanisms exist that may provide robustness against fluctuating protein levels, we found that one single feedback from Erk to Raf-1 accounts for the observed robustness. Surprisingly, robustness is provided through a fast post-translational mechanism although variation of Erk levels occurs on a timescale of days.

De novo expression of EphA2 in osteosarcoma modulates activation of the mitogenic signalling pathway.

AIMS: In osteosarcoma patients the development of metastases, often to the lungs, is the most frequent cause of death. The aim of this study was to elucidate the molecular mechanisms governing osteosarcoma development and dissemination and, thereby, to identify possible novel drug targets for improved treatment. METHODS AND RESULTS: Osteosarcoma samples were characterized using genome-wide microarrays: increased expression of the EphA2 receptor and its ligand EFNA1 was detected. In addition, increased expression of EFNB1, EFNB3 and EphA3 was suggested. Immunohistochemistry revealed an absence of EphA2 in normal bone, and de novo expression in osteosarcomas. EFNA1 was expressed in normal bone, but was significantly elevated in tumours. Further in vitro investigations on the functional role of EphA2 and EFNA1 showed that EFNA1 ligand binding induced increased tyrosine phosphorylation, receptor degradation and downstream mitogen-activated protein kinase (MAPK) activation. Interference with the MAPK pathway unravelled a potential autoregulatory loop governing mainly EFNA1 expression via the same pathway. CONCLUSION: Upregulation and de novo expression of ephrins in osteosarcomas are involved in oncogenic signalling and thus might stimulate osteosarcoma metastasis.

Therapeutic potential of CAMPATH-1H in skeletal tumours.

AIMS: CD 52 is a glycosylphosphatidylinositol (GPI)-anchored glycoprotein that is expressed abundantly on all lymphocytes, monocytes, macrophages, eosinophils and in the male genital tract. To date, the physiological role of CD52 on lymphocytes has not been elucidated. However, an antibody directed to CD52 called CAMPATH-1H has been shown to be capable of depleting lymphocytes. The aim of this study was to analyse tissue and cell lines of non-neoplastic bone, cartilage and skeletal tumours for CD52 expression. METHODS AND RESULTS: The expression of CD52 mRNA and protein both in vivo and in vitro was detected. Malignant tumours showed higher CD52 expression compared to benign tumours, suggesting a role in the development and progression of bone tumours. Interestingly, immunohistochemistry and flow cytometry revealed that CD52 was expressed not only on the surface of tumour cells, but also in the cytoplasm. The results obtained in osteosarcoma cells showed that CAMPATH-1H leads to a complement-independent reduction of viable cells. CONCLUSION: CD52 is expressed in a variety of bone tumours and the in vitro studies presented herein suggest that CAMPATH-1H treatment might have therapeutic potential for osteosarcoma patients.