Local delivery of hrBMP4 as an anticancer therapy in patients with recurrent glioblastoma: a first-in-human phase 1 dose escalation trial.

BACKGROUND: This Phase 1 study evaluates the intra- and peritumoral administration by convection enhanced delivery (CED) of human recombinant Bone Morphogenetic Protein 4 (hrBMP4) - an inhibitory regulator of cancer stem cells (CSCs) - in recurrent glioblastoma. METHODS: In a 3 + 3 dose escalation design, over four to six days, fifteen recurrent glioblastoma patients received, by CED, one of five doses of hrBMP4 ranging from 0·5 to 18 mg. Patients were followed by periodic physical, neurological, blood testing, magnetic resonance imaging (MRI) and quality of life evaluations. The primary objective of this first-in-human study was to determine the safety, dose-limiting toxicities (DLT) and maximum tolerated dose (MTD) of hrBMP4. Secondary objectives were to assess potential efficacy and systemic exposure to hrBMP4 upon intracerebral infusion. RESULTS: Intra- and peritumoral infusion of hrBMP4 was safe and well-tolerated. We observed no serious adverse events related to this drug. Neither MTD nor DLT were reached. Three patients had increased hrBMP4 serum levels at the end of infusion, which normalized within 4 weeks, without sign of toxicity. One patient showed partial response and two patients a complete (local) tumor response, which was maintained until the most recent follow-up, 57 and 30 months post-hrBMP4. Tumor growth was inhibited in areas permeated by hrBMP4. CONCLUSION: Local delivery of hrBMP4 in and around recurring glioblastoma is safe and well-tolerated. Three patients responded to the treatment. A complete response and long-term survival occurred in two of them. This warrants further clinical studies on this novel treatment targeting glioblastoma CSCs. TRIAL REGISTRATION: ClinicaTrials.gov identifier: NCT02869243.

A comparative benchmark of classic DNA motif discovery tools on synthetic data.

Hundreds of human proteins were found to establish transient interactions with rather degenerated consensus DNA sequences or motifs. Identifying these motifs and the genomic sites where interactions occur represent one of the most challenging research goals in modern molecular biology and bioinformatics. The last twenty years witnessed an explosion of computational tools designed to perform this task, whose performance has been last compared fifteen years ago. Here, we survey sixteen of them, benchmark their ability to identify known motifs nested in twenty-nine simulated sequence datasets, and finally report their strengths, weaknesses, and complementarity.

MitImpact 3: modeling the residue interaction network of the Respiratory Chain subunits.

Numerous lines of evidence have shown that the interaction between the nuclear and mitochondrial genomes ensures the efficient functioning of the OXPHOS complexes, with substantial implications in bioenergetics, adaptation, and disease. Their interaction is a fascinating and complex trait of the eukaryotic cell that MitImpact explores with its third major release. MitImpact expands its collection of genomic, clinical, and functional annotations of all non-synonymous substitutions of the human mitochondrial genome with new information on putative Compensated Pathogenic Deviations and co-varying amino acid sites of the Respiratory Chain subunits. It further provides evidence of energetic and structural residue compensation by techniques of molecular dynamics simulation. MitImpact is freely accessible at http://mitimpact.css-mendel.it.

Multifunctionalized hydrogels foster hNSC maturation in 3D cultures and neural regeneration in spinal cord injuries.

Three-dimensional cell cultures are leading the way to the fabrication of tissue-like constructs useful to developmental biology and pharmaceutical screenings. However, their reproducibility and translational potential have been limited by biomaterial and culture media compositions, as well as cellular sources. We developed a construct comprising synthetic multifunctionalized hydrogels, serum-free media, and densely seeded good manufacturing practice protocol-grade human neural stem cells (hNSC). We tracked hNSC proliferation, differentiation, and maturation into GABAergic, glutamatergic, and cholinergic neurons, showing entangled electrically active neural networks. The neuroregenerative potential of the "engineered tissue" was assessed in spinal cord injuries, where hNSC-derived progenitors and predifferentiated hNSC progeny, embedded in multifunctionalized hydrogels, were implanted. All implants decreased astrogliosis and lowered the immune response, but scaffolds with predifferentiated hNSCs showed higher percentages of neuronal markers, better hNSC engraftment, and improved behavioral recovery. Our hNSC-construct enables the formation of 3D functional neuronal networks in vitro, allowing novel strategies for hNSC therapies in vivo.

Human Neural Stem Cell-Based Drug Product: Clinical and Nonclinical Characterization.

Translation of cell therapies into clinical practice requires the adoption of robust production protocols in order to optimize and standardize the manufacture and cryopreservation of cells, in compliance with good manufacturing practice regulations. Between 2012 and 2020, we conducted two phase I clinical trials (EudraCT 2009-014484-39, EudraCT 2015-004855-37) on amyotrophic lateral sclerosis secondary progressive multiple sclerosis patients, respectively, treating them with human neural stem cells. Our production process of a hNSC-based medicinal product is the first to use brain tissue samples extracted from fetuses that died in spontaneous abortion or miscarriage. It consists of selection, isolation and expansion of hNSCs and ends with the final pharmaceutical formulation tailored to a specific patient, in compliance with the approved clinical protocol. The cells used in these clinical trials were analyzed in order to confirm their microbiological safety; each batch was also tested to assess identity, potency and safety through morphological and functional assays. Preclinical, clinical and in vitro nonclinical data have proved that our cells are safe and stable, and that the production process can provide a high level of reproducibility of the cultures. Here, we describe the quality control strategy for the characterization of the hNSCs used in the above-mentioned clinical trials.

Known Drugs Identified by Structure-Based Virtual Screening Are Able to Bind Sigma-1 Receptor and Increase Growth of Huntington Disease Patient-Derived Cells.

Huntington disease (HD) is a devastating and presently untreatable neurodegenerative disease characterized by progressively disabling motor and mental manifestations. The sigma-1 receptor (σ1R) is a protein expressed in the central nervous system, whose 3D structure has been recently determined by X-ray crystallography and whose agonists have been shown to have neuroprotective activity in neurodegenerative diseases. To identify therapeutic agents against HD, we have implemented a drug repositioning strategy consisting of: (i) Prediction of the ability of the FDA-approved drugs publicly available through the ZINC database to interact with σ1R by virtual screening, followed by computational docking and visual examination of the 20 highest scoring drugs; and (ii) Assessment of the ability of the six drugs selected by computational analyses to directly bind purified σ1R in vitro by Surface Plasmon Resonance and improve the growth of fibroblasts obtained from HD patients, which is significantly impaired with respect to control cells. All six of the selected drugs proved able to directly bind purified σ1R in vitro and improve the growth of HD cells from both or one HD patient. These results support the validity of the drug repositioning procedure implemented herein for the identification of new therapeutic tools against HD.

Molecular dynamics recipes for genome research.

Molecular dynamics (MD) simulation allows one to predict the time evolution of a system of interacting particles. It is widely used in physics, chemistry and biology to address specific questions about the structural properties and dynamical mechanisms of model systems. MD earned a great success in genome research, as it proved to be beneficial in sorting pathogenic from neutral genomic mutations. Considering their computational requirements, simulations are commonly performed on HPC computing devices, which are generally expensive and hard to administer. However, variables like the software tool used for modeling and simulation or the size of the molecule under investigation might make one hardware type or configuration more advantageous than another or even make the commodity hardware definitely suitable for MD studies. This work aims to shed lights on this aspect.

A Link between Genetic Disorders and Cellular Impairment, Using Human Induced Pluripotent Stem Cells to Reveal the Functional Consequences of Copy Number Variations in the Central Nervous System-A Close Look at Chromosome 15.

Recent cutting-edge human genetics technology has allowed us to identify copy number variations (CNVs) and has provided new insights for understanding causative mechanisms of human diseases. A growing number of studies show that CNVs could be associated with physiological mechanisms linked to evolutionary trigger, as well as to the pathogenesis of various diseases, including cancer, autoimmune disease and mental disorders such as autism spectrum disorders, schizophrenia, intellectual disabilities or attention-deficit/hyperactivity disorder. Their incomplete penetrance and variable expressivity make diagnosis difficult and hinder comprehension of the mechanistic bases of these disorders. Additional elements such as co-presence of other CNVs, genomic background and environmental factors are involved in determining the final phenotype associated with a CNV. Genetically engineered animal models are helpful tools for understanding the behavioral consequences of CNVs. However, the genetic background and the biology of these animal model systems have sometimes led to confusing results. New cellular models obtained through somatic cellular reprogramming technology that produce induced pluripotent stem cells (iPSCs) from human subjects are being used to explore the mechanisms involved in the pathogenic consequences of CNVs. Considering the vast quantity of CNVs found in the human genome, we intend to focus on reviewing the current literature on the use of iPSCs carrying CNVs on chromosome 15, highlighting advantages and limits of this system with respect to mouse model systems.

Extracellular vesicles are independent metabolic units with asparaginase activity.

Extracellular vesicles (EVs) are membrane particles involved in the exchange of a broad range of bioactive molecules between cells and the microenvironment. Although it has been shown that cells can traffic metabolic enzymes via EVs, much remains to be elucidated with regard to their intrinsic metabolic activity. Accordingly, herein we assessed the ability of neural stem/progenitor cell (NSC)-derived EVs to consume and produce metabolites. Our metabolomics and functional analyses both revealed that EVs harbor L-asparaginase activity, catalyzed by the enzyme asparaginase-like protein 1 (Asrgl1). Critically, we show that Asrgl1 activity is selective for asparagine and is devoid of glutaminase activity. We found that mouse and human NSC EVs traffic Asrgl1. Our results demonstrate, for the first time, that NSC EVs function as independent metabolic units that are able to modify the concentrations of critical nutrients, with the potential to affect the physiology of their microenvironment.

Parkin Mutation Affects Clock Gene-Dependent Energy Metabolism.

Growing evidence highlights a tight connection between circadian rhythms, molecular clockworks, and mitochondrial function. In particular, mitochondrial quality control and bioenergetics have been proven to undergo circadian oscillations driven by core clock genes. Parkinson's disease (PD) is a chronic neurodegenerative disease characterized by a selective loss of dopaminergic neurons. Almost half of the autosomal recessive forms of juvenile parkinsonism have been associated with mutations in the PARK2 gene coding for parkin, shown to be involved in mitophagy-mediated mitochondrial quality control. The aim of this study was to investigate, in fibroblasts from genetic PD patients carrying parkin mutations, the interplay between mitochondrial bioenergetics and the cell autonomous circadian clock. Using two different in vitro synchronization protocols, we demonstrated that normal fibroblasts displayed rhythmic oscillations of both mitochondrial respiration and glycolytic activity. Conversely, in fibroblasts obtained from PD patients, a severe damping of the bioenergetic oscillatory patterns was observed. Analysis of the core clock genes showed deregulation of their expression patterns in PD fibroblasts, which was confirmed in induced pluripotent stem cells (iPSCs) and induced neural stem cells (iNSCs) derived thereof. The results from this study support a reciprocal interplay between the clockwork machinery and mitochondrial energy metabolism, point to a parkin-dependent mechanism of regulation, and unveil a hitherto unappreciated level of complexity in the pathophysiology of PD and eventually other neurodegenerative diseases.

EphB2 stem-related and EphA2 progression-related miRNA-based networks in progressive stages of CRC evolution: clinical significance and potential miRNA drivers.

EphB2 and EphA2 control stemness and differentiation in the intestinal mucosa, but the way they cooperate with the complex mechanisms underlying tumor heterogeneity and how they affect the therapeutic outcome in colorectal cancer (CRC) patients, remain unclear. MicroRNA (miRNA) expression profiling along with pathway analysis provide comprehensive information on the dysregulation of multiple crucial pathways in CRC.Through a network-based approach founded on the characterization of progressive miRNAomes centered on EphA2/EphB2 signaling during tumor development in the AOM/DSS murine model, we found a miRNA-dependent orchestration of EphB2-specific stem-like properties in earlier phases of colorectal tumorigenesis and the EphA2-specific control of tumor progression in the latest CRC phases. Furthermore, two transcriptional signatures that are specifically dependent on the EphA2/EphB2 signaling pathways were identified, namely EphA2, miR-423-5p, CREB1, ADAMTS14, and EphB2, miR-31-5p, mir-31-3p, CRK, CXCL12, ARPC5, SRC.EphA2- and EphB2-related signatures were validated for their expression and clinical value in 1663 CRC patients. In multivariate analysis, both signatures were predictive of survival and tumor progression.The early dysregulation of miRs-31, as observed in the murine samples, was also confirmed on 49 human tissue samples including preneoplastic lesions and tumors. In light of these findings, miRs-31 emerged as novel potential drivers of CRC initiation.Our study evidenced a miRNA-dependent orchestration of EphB2 stem-related networks at the onset and EphA2-related cancer-progression networks in advanced stages of CRC evolution, suggesting new predictive biomarkers and potential therapeutic targets.

High-confidence assessment of functional impact of human mitochondrial non-synonymous genome variations by APOGEE.

24,189 are all the possible non-synonymous amino acid changes potentially affecting the human mitochondrial DNA. Only a tiny subset was functionally evaluated with certainty so far, while the pathogenicity of the vast majority was only assessed in-silico by software predictors. Since these tools proved to be rather incongruent, we have designed and implemented APOGEE, a machine-learning algorithm that outperforms all existing prediction methods in estimating the harmfulness of mitochondrial non-synonymous genome variations. We provide a detailed description of the underlying algorithm, of the selected and manually curated training and test sets of variants, as well as of its classification ability.

Phenotypic variation in Aicardi-Goutières syndrome explained by cell-specific IFN-stimulated gene response and cytokine release.

Aicardi-Goutières syndrome (AGS) is a monogenic inflammatory encephalopathy caused by mutations in TREX1, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, ADAR1, or MDA5. Mutations in those genes affect normal RNA/DNA intracellular metabolism and detection, triggering an autoimmune response with an increase in cerebral IFN-α production by astrocytes. Microangiopathy and vascular disease also contribute to the neuropathology in AGS. In this study, we report that AGS gene silencing of TREX1, SAMHD1, RNASEH2A, and ADAR1 by short hairpin RNAs in human neural stem cell-derived astrocytes, human primary astrocytes, and brain-derived endothelial cells leads to an antiviral status of these cells compared with nontarget short hairpin RNA-treated cells. We observed a distinct activation of the IFN-stimulated gene signature with a substantial increase in the release of proinflammatory cytokines (IL-6) and chemokines (CXCL10 and CCL5). A differential impact of AGS gene silencing was noted; silencing TREX1 gave rise to the most dramatic in both cell types. Our findings fit well with the observation that patients carrying mutations in TREX1 experience an earlier onset and fatal outcome. We provide in the present study, to our knowledge for the first time, insight into how astrocytic and endothelial activation of antiviral status may differentially lead to cerebral pathology, suggesting a rational link between proinflammatory mediators and disease severity in AGS.

Histone acetylation in astrocytes suppresses GFAP and stimulates a reorganization of the intermediate filament network.

Glial fibrillary acidic protein (GFAP) is the main intermediate filament in astrocytes and is regulated by epigenetic mechanisms during development. We demonstrate that histone acetylation also controls GFAP expression in mature astrocytes. Inhibition of histone deacetylases (HDACs) with trichostatin A or sodium butyrate reduced GFAP expression in primary human astrocytes and astrocytoma cells. Because splicing occurs co-transcriptionally, we investigated whether histone acetylation changes the ratio between the canonical isoform GFAPα and the alternative GFAPδ splice variant. We observed that decreased transcription of GFAP enhanced alternative isoform expression, as HDAC inhibition increased the GFAPδ∶GFAPα ratio. Expression of GFAPδ was dependent on the presence and binding of splicing factors of the SR protein family. Inhibition of HDAC activity also resulted in aggregation of the GFAP network, reminiscent of our previous findings of a GFAPδ-induced network collapse. Taken together, our data demonstrate that HDAC inhibition results in changes in transcription, splicing and organization of GFAP. These data imply that a tight regulation of histone acetylation in astrocytes is essential, because dysregulation of gene expression causes the aggregation of GFAP, a hallmark of human diseases like Alexander's disease.

Intraspinal stem cell transplantation for amyotrophic lateral sclerosis: Ready for efficacy clinical trials?

Intraspinal stem cell (SC) transplantation represents a new therapeutic approach for amyotrophic lateral sclerosis (ALS) clinical trials. There are considerable difficulties in designing future efficacy trials, some related to the field of ALS and some that are specific to SCs or the mode of delivery. In October 2015, the most controversial points on SC transplantation were addressed during an international workshop intended to bring together international SC and ALS researchers in a public discussion on a topic for which expertise is limited. During the meeting, a discussion was started on the basic structure of the ideal clinical trial testing the efficacy and safety of SC transplantation. The current document includes a number of consensus points reflecting the design of phase II/III clinical trials.

Human neural stem cell transplantation in ALS: initial results from a phase I trial.

BACKGROUND: We report the initial results from a phase I clinical trial for ALS. We transplanted GMP-grade, fetal human neural stem cells from natural in utero death (hNSCs) into the anterior horns of the spinal cord to test for the safety of both cells and neurosurgical procedures in these patients. The trial was approved by the Istituto Superiore di Sanità and the competent Ethics Committees and was monitored by an external Safety Board. METHODS: Six non-ambulatory patients were treated. Three of them received 3 unilateral hNSCs microinjections into the lumbar cord tract, while the remaining ones received bilateral (n = 3 + 3) microinjections. None manifested severe adverse events related to the treatment, even though nearly 5 times more cells were injected in the patients receiving bilateral implants and a much milder immune-suppression regimen was used as compared to previous trials. RESULTS: No increase of disease progression due to the treatment was observed for up to18 months after surgery. Rather, two patients showed a transitory improvement of the subscore ambulation on the ALS-FRS-R scale (from 1 to 2). A third patient showed improvement of the MRC score for tibialis anterior, which persisted for as long as 7 months. The latter and two additional patients refused PEG and invasive ventilation and died 8 months after surgery due to the progression of respiratory failure. The autopsies confirmed that this was related to the evolution of the disease. CONCLUSIONS: We describe a safe cell therapy approach that will allow for the treatment of larger pools of patients for later-phase ALS clinical trials, while warranting good reproducibility. These can now be carried out under more standardized conditions, based on a more homogenous repertoire of clinical grade hNSCs. The use of brain tissue from natural miscarriages eliminates the ethical concerns that may arise from the use of fetal material. TRIAL REGISTRATION: EudraCT:2009-014484-39 .

Cool-1-mediated inhibition of c-Cbl modulates multiple critical properties of glioblastomas, including the ability to generate tumors in vivo.

We discovered that glioblastoma (GBM) cells use Cool-1/ß-pix to inhibit normal activation of the c-Cbl ubiquitin ligase via the redox/Fyn/c-Cbl pathway and that c-Cbl inhibition is critical for GBM cell function. Restoring normal c-Cbl activity by Cool-1 knockdown in vitro reduced GBM cell division, almost eliminated generation of adhesion-independent spheroids, reduced the representation of cells expressing antigens thought to identify tumor initiating cells (TICs), reduced levels of several proteins of critical importance in TIC function (such as Notch-1 and Sox2), and increased sensitivity to BCNU (carmustine) and temozolomide (TMZ). In vivo, Cool-1 knockdown greatly suppressed the ability of GBM cells to generate tumors, an outcome that was c-Cbl dependent. In contrast, Cool-1 knockdown did not reduce division or increase BCNU or TMZ sensitivity in primary glial progenitor cells and Cool-1/c-Cbl complexes were not found in normal brain tissue. Our studies provide the first evidence that Cool-1 may be critical in the biology of human tumors, that suppression of c-Cbl by Cool-1 may be critical for generation of at least a subset of GBMs and offer a novel target that appears to be selectively necessary for TIC function and modulates chemoresistance in GBM cells. Targeting such proteins that inhibit c-Cbl offers potentially attractive opportunities for therapeutic development.

Brain tumour stem cells.

The dogma that the genesis of new cells is a negligible event in the adult mammalian brain has long influenced our perception and understanding of the origin and development of CNS tumours. The discovery that new neurons and glia are produced throughout life from neural stem cells provides new possibilities for the candidate cells of origin of CNS neoplasias. The emerging hypothesis is that alterations in the cellular and genetic mechanisms that control adult neurogenesis might contribute to brain tumorigenesis, thereby allowing the identification of new therapeutic strategies.

The Big Bluff of Amyotrophic Lateral Sclerosis Diagnosis: The Role of Neurodegenerative Disease Mimics.

Neurodegenerative diseases include a significant number of pathologies affecting the nervous system. Generally, the primary cause of each disease is specific; however, recently, it was shown that they may be correlated at molecular level. This aspect, together with the exhibition of similar symptoms, renders the diagnosis of these disorders difficult. Amyotrophic lateral sclerosis is one of these pathologies. Herein, we report several cases of amyotrophic lateral sclerosis misdiagnosed as a consequence of features that are common to several neurodegenerative diseases, such as Parkinson's, Huntington's and Alzheimer's disease, spinal muscular atrophy, progressive bulbar palsy, spastic paraplegia and frontotemporal dementia, and mostly with the lysosomal storage disorder GM2 gangliosidosis. Overall reports highlight that the differential diagnosis for amyotrophic lateral sclerosis should include correlated mechanisms.

PDGF receptor alpha inhibition induces apoptosis in glioblastoma cancer stem cells refractory to anti-Notch and anti-EGFR treatment.

BACKGROUND: Cancer stem cells (CSC) represent a rare fraction of cancer cells characterized by resistance to chemotherapy and radiation, therefore nowadays there is great need to develop new targeted therapies for brain tumors and our study aim to target pivotal transmembrane receptors such as Notch, EGFR and PDGFR, which are already under investigation in clinical trials setting for the treatment of Glioblastoma Multiforme (GBM). METHODS: MTS assay was performed to evaluate cells response to pharmacological treatments. Quantitative RT-PCR and Western blots were performed to state the expression of Notch1, EGFR and PDGFRα/ß and the biological effects exerted by either single or combined targeted therapy in GBM CSC. GBM CSC invasive ability was tested in vitro in absence or presence of Notch and/or EGFR signaling inhibitors. RESULTS: In this study, we investigated gene expression and function of Notch1, EGFR and PDGFR to determine their role among GBM tumor core- (c-CSC) vs. peritumor tissue-derived cancer stem cells (p-CSC) of six cases of GBM. Notch inhibition significantly impaired cell growth of c-CSC compared to p-CSC pools, with no effects observed in cell cycle distribution, apoptosis and cell invasion assays. Instead, anti-EGFR therapy induced cell cycle arrest, sometimes associated with apoptosis and reduction of cell invasiveness in GBM CSC. In two cases, c-CSC pools were more sensitive to simultaneous anti-Notch and anti-EGFR treatment than either therapy alone compared to p-CSC, which were mostly resistant to treatment. We reported the overexpression of PDGFRα and its up-regulation following anti-EGFR therapy in GBM p-CSC compared to c-CSC. RNA interference of PDGFRα significantly reduced cell proliferation rate of p-CSC, while its pharmacological inhibition with Crenolanib impaired survival of both CSC pools, whose effects in combination with EGFR inhibition were maximized. CONCLUSIONS: We have used different drugs combination to identify the more effective therapeutic targets for GBM CSC, particularly against GBM peritumor tissue-derived CSC, which are mostly resistant to treatments. Overall, our results provide the rationale for simultaneous targeting of EGFR and PDGFR, which would be beneficial in the treatment of GBM.