FGFR1 is a potential therapeutic target in neuroblastoma.

BACKGROUND: FGFR1 regulates cell-cell adhesion and extracellular matrix architecture and acts as oncogene in several cancers. Potential cancer driver mutations of FGFR1 occur in neuroblastoma (NB), a neural crest-derived pediatric tumor arising in sympathetic nervous system, but so far they have not been studied experimentally. We investigated the driver-oncogene role of FGFR1 and the implication of N546K mutation in therapy-resistance in NB cells. METHODS: Public datasets were used to predict the correlation of FGFR1 expression with NB clinical outcomes. Whole genome sequencing data of 19 paired diagnostic and relapse NB samples were used to find somatic mutations. In NB cell lines, silencing by short hairpin RNA and transient overexpression of FGFR1 were performed to evaluate the effect of the identified mutation by cell growth, invasion and cologenicity assays. HEK293, SHSY5Y and SKNBE2 were selected to investigate subcellular wild-type and mutated protein localization. FGFR1 inhibitor (AZD4547), alone or in combination with PI3K inhibitor (GDC0941), was used to rescue malignant phenotypes induced by overexpression of FGFR1 wild-type and mutated protein. RESULTS: High FGFR1 expression correlated with low relapse-free survival in two independent NB gene expression datasets. In addition, we found the somatic mutation N546K, the most recurrent point mutation of FGFR1 in all cancers and already reported in NB, in one out of 19 matched primary and recurrent tumors. Loss of FGFR1 function attenuated invasion and cologenicity in NB cells, whereas FGFR1 overexpression enhanced oncogenicity. The overexpression of FGFR1N546K protein showed a higher nuclear localization compared to wild-type protein and increased cellular invasion and cologenicity. Moreover, N546K mutation caused the failure in response to treatment with FGFR1 inhibitor by activation of ERK, STAT3 and AKT pathways. The combination of FGFR1 and PI3K pathway inhibitors was effective in reducing the invasive and colonigenic ability of cells overexpressing FGFR1 mutated protein. CONCLUSIONS: FGFR1 is an actionable driver oncogene in NB and a promising therapy may consist in targeting FGFR1 mutations in patients with therapy-resistant NB.

ZSCAN4+ mouse embryonic stem cells have an oxidative and flexible metabolic profile.

Cultured mouse embryonic stem cells are a heterogeneous population with diverse differentiation potential. In particular, the subpopulation marked by Zscan4 expression has high stem cell potency and shares with 2 cell stage preimplantation embryos both genetic and epigenetic mechanisms that orchestrate zygotic genome activation. Although embryonic de novo genome activation is known to rely on metabolites, a more extensive metabolic characterization is missing. Here we analyze the Zscan4+ mouse stem cell metabolic phenotype associated with pluripotency maintenance and cell reprogramming. We show that Zscan4+ cells have an oxidative and adaptable metabolism, which, on one hand, fuels a high bioenergetic demand and, on the other hand, provides intermediate metabolites for epigenetic reprogramming. Our findings enhance our understanding of the metastable Zscan4+ stem cell state with potential applications in regenerative medicine.

HIF-1 transcription activity: HIF1A driven response in normoxia and in hypoxia.

BACKGROUND: HIF1A (Hypoxia-Inducible-Factor 1A) expression in solid tumors is relevant to establish resistance to therapeutic approaches. The use of compounds direct against hypoxia signaling and HIF1A does not show clinical efficiency because of changeable oxygen concentrations in solid tumor areas. The identification of HIF1A targets expressed in both normoxia and hypoxia and of HIF1A/hypoxia signatures might meliorate the prognostic stratification and therapeutic successes in patients with high-risk solid tumors. METHODS: In this study, we conducted a combined analysis of RNA expression and DNA methylation of neuroblastoma cells silenced or unsilenced for HIF1A expression, grown in normoxia and hypoxia conditions. RESULTS: The analysis of pathways highlights HIF-1 (heterodimeric transcription factor 1) activity in normoxia in metabolic process and HIF-1 activity in hypoxia in neuronal differentiation process. HIF1A driven transcriptional response in hypoxia depends on epigenetic control at DNA methylation status of gene regulatory regions. Furthermore, low oxygen levels generate HIF1A-dependent or HIF1A-independent signatures, able to stratify patients according to risk categories. CONCLUSIONS: These findings may help to understand the molecular mechanisms by which low oxygen levels reshape gene signatures and provide new direction for hypoxia targeting in solid tumor.

Features, reason for testing, and changes with time of 583 paroxysmal nocturnal hemoglobinuria clones from 529 patients: a multicenter Italian study.

In this study, we aimed at disclosing the main features of paroxysmal nocturnal hemoglobinuria (PNH) clones, their association with presentation syndromes, and their changes during follow-up. A large-scale, cooperative collection (583 clones from 529 patients) of flow cytometric and clinical data was entered into a national repository. Reason for testing guidelines were provided to the 41 participating laboratories, which followed the 2010 technical recommendations for PNH testing by Borowitz. Subsequently, the 30 second-level laboratories adopted the 2012 guidelines for high-resolution PNH testing, both upon order by the local clinicians and as an independent laboratory initiative in selected cases. Type3 and Type2 PNH clones (total and partial absence of glycosyl-phosphatidyl-inositol-anchor, respectively) were simultaneously present in 54 patients. In these patients, Type3 component was sevenfold larger than Type2 (p < 0.001). Frequency distribution analysis of solitary Type3 clone size (N = 442) evidenced two discrete patterns: small (20% of peripheral neutrophils) and large (> 70%) clones. The first pattern was significantly associated with bone marrow failure and myelodysplastic syndromes, the second one with hemolysis, hemoglobinuria, and thrombosis. Pediatric patients (N = 34) showed significant preponderance of small clones and bone marrow failure. The majority of PNH clones involved neutrophils, monocytes, and erythrocytes. Nevertheless, we found clones made exclusively by white cells (N = 13) or erythrocytes (N = 3). Rare cases showed clonal white cells restricted only to monocytes (6 cases) or neutrophils (3 cases). Retesting over 1-year follow-up in 151 cases showed a marked clone size increase in 4 cases and a decrease in 13, demonstrating that early breaking-down of PNH clones is not a rare event (8.6% of cases). This collaborative nationwide study demonstrates a clear-cut difference in size between Type2 and Type3 clones, emphasizes the existence of just two classes of PNH presentations based on Type3 clone size, depicts an asymmetric cellular composition of PNH clones, and documents the possible occurrence of changes in clone size during the follow-up.

Lipopolysaccharide-Elicited TSLPR Expression Enriches a Functionally Discrete Subset of Human CD14+ CD1c+ Monocytes.

Thymic stromal lymphopoietin (TSLP) is a cytokine produced mainly by epithelial cells in response to inflammatory or microbial stimuli and binds to the TSLP receptor (TSLPR) complex, a heterodimer composed of TSLPR and IL-7 receptor α (CD127). TSLP activates multiple immune cell subsets expressing the TSLPR complex and plays a role in several models of disease. Although human monocytes express TSLPR and CD127 mRNAs in response to the TLR4 agonist LPS, their responsiveness to TSLP is poorly defined. We demonstrate that TSLP enhances human CD14+ monocyte CCL17 production in response to LPS and IL-4. Surprisingly, only a subset of CD14+ CD16- monocytes, TSLPR+ monocytes (TSLPR+ mono), expresses TSLPR complex upon LPS stimulation in an NF-κB- and p38-dependent manner. Phenotypic, functional, and transcriptomic analysis revealed specific features of TSLPR+ mono, including higher CCL17 and IL-10 production and increased expression of genes with important immune functions (i.e., GAS6, ALOX15B, FCGR2B, LAIR1). Strikingly, TSLPR+ mono express higher levels of the dendritic cell marker CD1c. This evidence led us to identify a subset of peripheral blood CD14+ CD1c+ cells that expresses the highest levels of TSLPR upon LPS stimulation. The translational relevance of these findings is highlighted by the higher expression of TSLPR and CD127 mRNAs in monocytes isolated from patients with Gram-negative sepsis compared with healthy control subjects. Our results emphasize a phenotypic and functional heterogeneity in an apparently homogeneous population of human CD14+ CD16- monocytes and prompt further ontogenetic and functional analysis of CD14+ CD1c+ and LPS-activated CD14+ CD1c+ TSLPR+ mono.

Insight into Nephrocan Function in Mouse Endoderm Patterning.

Endoderm-derived organs as liver and pancreas are potential targets for regenerative therapies, and thus, there is great interest in understanding the pathways that regulate the induction and specification of this germ layer. Currently, the knowledge of molecular mechanisms that guide the in vivo endoderm specification is restricted by the lack of early endoderm specific markers. Nephrocan (Nepn) is a gene whose expression characterizes the early stages of murine endoderm specification (E7.5-11.5) and encodes a secreted N-glycosylated protein. In the present study, we report the identification of a new transcript variant that is generated through alternative splicing. The new variant was found to have differential and tissue specific expression in the adult mouse. In order to better understand Nepn role during endoderm specification, we generated Nepn knock-out (KO) mice. Nepn-/- mice were born at Mendelian ratios and displayed no evident phenotype compared to WT mice. In addition, we produced nullizygous mouse embryonic stem cell (mESC) line lacking Nepn by applying (CRISPR)/CRISPR-associated systems 9 (Cas9) and employed a differentiation protocol toward endoderm lineage. Our in vitro results revealed that Nepn loss affects the endoderm differentiation impairing the expression of posterior foregut-associated markers.

Stain-free identification of cell nuclei using tomographic phase microscopy in flow cytometry.

Quantitative Phase Imaging (QPI) has gained popularity in bioimaging because it can avoid the need for cell staining, which in some cases is difficult or impossible. However, as a result, QPI does not provide labelling of various specific intracellular structures. Here we show a novel computational segmentation method based on statistical inference that makes it possible for QPI techniques to identify the cell nucleus. We demonstrate the approach with refractive index tomograms of stain-free cells reconstructed through the tomographic phase microscopy in flow cytometry mode. In particular, by means of numerical simulations and two cancer cell lines, we demonstrate that the nucleus can be accurately distinguished within the stain-free tomograms. We show that our experimental results are consistent with confocal fluorescence microscopy (FM) data and microfluidic cytofluorimeter outputs. This is a significant step towards extracting specific three-dimensional intracellular structures directly from the phase-contrast data in a typical flow cytometry configuration.

Author Correction: Thyroid hormone induces progression and invasiveness of squamous cell carcinomas by promoting a ZEB-1/E-cadherin switch.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Rapid Affinity Maturation of Novel Anti-PD-L1 Antibodies by a Fast Drop of the Antigen Concentration and FACS Selection of Yeast Libraries.

The affinity engineering is a key step to increase the efficacy of therapeutic monoclonal antibodies and yeast surface display is the most widely used and powerful affinity maturation approach, achieving picomolar binding affinities. In this study, we provide an optimization of the yeast surface display methodology, applied to the generation of potentially therapeutic high affinity antibodies targeting the immune checkpoint PD-L1. In this approach, we coupled a 10-cycle error-prone mutagenesis of heavy chain complementarity determining region 3 of an anti-PD-L1 scFv, previously identified by phage display, with high-throughput sequencing, to generate scFv-yeast libraries with high mutant frequency and diversity. In addition, we set up a novel, faster and effective selection scheme by fluorescence-activated cell sorting, based on a fast drop of the antigen concentration between the first and the last selection cycles, unlike the gradual decrease typical of current selection protocols. In this way we isolated 6 enriched mutated scFv-yeast clones overall, showing an affinity improvement for soluble PD-L1 protein compared to the parental scFv. As a proof of the potency of the novel approach, we confirmed that the antibodies converted from all the mutated scFvs retained the affinity improvement. Remarkably, the best PD-L1 binder among them also bound with a higher affinity to PD-L1 expressed in its native conformation on human-activated lymphocytes, and it was able to stimulate lymphocyte proliferation in vitro more efficiently than its parental antibody. This optimized technology, besides the identification of a new potential checkpoint inhibitor, provides a tool for the quick isolation of high affinity binders.

Retinoic Acid Induces Embryonic Stem Cells (ESCs) Transition to 2 Cell-Like State Through a Coordinated Expression of Dux and Duxbl1.

Embryonic stem cells (ESCs) are derived from inner cell mass (ICM) of the blastocyst. In serum/LIF culture condition, they show variable expression of pluripotency genes that mark cell fluctuation between pluripotency and differentiation metastate. The ESCs subpopulation marked by zygotic genome activation gene (ZGA) signature, including Zscan4, retains a wider differentiation potency than epiblast-derived ESCs. We have recently shown that retinoic acid (RA) significantly enhances Zscan4 cell population. However, it remains unexplored how RA initiates the ESCs to 2-cell like reprogramming. Here we found that RA is decisive for ESCs to 2C-like cell transition, and reconstructed the gene network surrounding Zscan4. We revealed that RA regulates 2C-like population co-activating Dux and Duxbl1. We provided novel evidence that RA dependent ESCs to 2C-like cell transition is regulated by Dux, and antagonized by Duxbl1. Our suggested mechanism could shed light on the role of RA on ESC reprogramming.

Thyroid hormone induces progression and invasiveness of squamous cell carcinomas by promoting a ZEB-1/E-cadherin switch.

Epithelial tumor progression often involves epithelial-mesenchymal transition (EMT). We report that increased intracellular levels of thyroid hormone (TH) promote the EMT and malignant evolution of squamous cell carcinoma (SCC) cells. TH induces the EMT by transcriptionally up-regulating ZEB-1, mesenchymal genes and metalloproteases and suppresses E-cadherin expression. Accordingly, in human SCC, elevated D2 (the T3-producing enzyme) correlates with tumor grade and is associated with an increased risk of postsurgical relapse and shorter disease-free survival. These data provide the first in vivo demonstration that TH and its activating enzyme, D2, play an effective role not only in the EMT but also in the entire neoplastic cascade starting from tumor formation up to metastatic transformation, and supports the concept that TH is an EMT promoter. Our studies indicate that tumor progression relies on precise T3 availability, suggesting that pharmacological inactivation of D2 and TH signaling may suppress the metastatic proclivity of SCC.

High-Throughput Screening Identifies Kinase Inhibitors That Increase Dual Adeno-Associated Viral Vector Transduction In Vitro and in Mouse Retina.

Retinal gene therapy based on adeno-associated viral (AAV) vectors is safe and efficient in humans. The low intrinsic DNA transfer capacity of AAV has been expanded by dual vectors where a large expression cassette is split in two halves independently packaged in two AAV vectors. Dual AAV transduction efficiency, however, is greatly reduced compared to that obtained with a single vector. As AAV intracellular trafficking and processing are negatively affected by phosphorylation, this study set to identify kinase inhibitors that can increase dual AAV vector transduction. By high-throughput screening of a kinase inhibitors library, three compounds were identified that increase AAV transduction in vitro, one of which has a higher effect on dual than on single AAV vectors. Importantly, the transduction enhancement is exerted on various AAV serotypes and is not transgene dependent. As kinase inhibitors are promiscuous, siRNA-mediated silencing of targeted kinases was performed, and AURKA and B, PLK1, and PTK2 were among those involved in the increase of AAV transduction levels. The study shows that kinase inhibitor administration reduces AAV serotype 2 (AAV2) capsid phosphorylation and increases the activity of DNA-repair pathways involved in AAV DNA processing. Importantly, the kinase inhibitor PF-00562271 improves dual AAV8 transduction in photoreceptors following sub-retinal delivery in mice. The study identifies kinase inhibitors that increase dual and single AAV transduction by modulating AAV entry and post-entry steps.