Effects of in vitro and in vivo treatment of monoterpene perillyl alcohol on proliferation and gene expression control of high grade gliomas

AbstractStreptococcus pneumoniae can cause disease in various human tissues and organs, including the ear, the brain, the blood and the lung, and thus in highly diverse and dynamic environments. It is challenging to study how pneumococci control virulence factor expression, because cues of natural environments and the presence of an immune system are difficult to simulate in vitro. Here, we apply synthetic biology methods to reverse-engineer gene expression control in S. pneumoniae. A selection platform is described that allows for straightforward identification of transcriptional regulatory elements out of combinatorial libraries. We present TetR- and LacI-regulated promoters that show expression ranges of four orders of magnitude. Based on these promoters, regulatory networks of higher complexity are assembled, such as logic AND and IMPLY gates. Finally, we demonstrate single-copy genome-integrated toggle switches that give rise to bimodal population distributions. The tools described here can be used to mimic complex expression patterns, such as the ones found for pneumococcal virulence factors, paving the way for in vivo investigations of the importance of gene expression control on the pathogenicity of S. pneumoniae.

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Hedgehog inhibition impaired platinum response in high-grade serous ovarian cancer harboring high hedgehog ligand expression and mTOR pathway activation.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.15_suppl.5583 ◽

2017 ◽

Vol 35 (15_suppl) ◽

pp. 5583-5583

Author(s):

Gwo Yaw Ho ◽

Elizabeth Lieschke ◽

Elizabeth Kyran ◽

Kristy Shield-Artin ◽

Olga Kondrashova ◽

...

Keyword(s):

Cell Line ◽

Mtor Pathway ◽

High Grade ◽

Serous Ovarian Cancer ◽

Qrt Pcr ◽

Gli1 Expression ◽

Pathway Activation ◽

In Vivo Treatment

5583 Background: Elevated Glioma-associated Oncogene Homolog-1 (Gli1) protein expression is associated with Hedgehog (Hh) pathway activation in high-grade serous ovarian cancer (HGSOC). Inhibition of Hh signaling in Gli1-overexpressing HGSOC patient-derived xenograft (PDX) inhibited tumour growth, particularly in combination with chemotherapy. Early phase HGSOC clinical trials of vismodegib, a potent Hh inhibitor (SMO inhibitor), were disappointing. We identified a HGSOC PDX harboring both Indian Hh ligand-overexpression and bi-allelic deletion of TSC1, which latter event is reported to derepress the mTOR pathway, driving non-cannonical Gli1 expression. We explored the effect of vismodegib in combination with cisplatin or the mTOR inhibitor, everolimus, in this model. Methods: A cell-line was generated from the well-characterised PDX (identity confirmed by PDX-specific p53 mutation). In vitro response to vismodegib was assessed. qRT-PCR was performed to establish Hh-ligand and Gli1 expression with/without SMO inhibition. A PDX was generated from this cell-line and randomized to in vivo treatment with cisplatin, vismodegib, everolimus or vehicle alone, or vismodegib in combination with cisplatin or everolimus. Results: The HGSOC cell-line was sensitive to vismodegib in vitro (EC50 of 3.5µM) and qRT-PCR analysis revealed down-regulation of Hh-ligand and Gli1 expression following in vitro SMO inhibition, confirming on-target vismodegib activity. In vivo treatment with vismodegib or everolimus alone did not result in reproducible in vivo efficacy. The combination of vismodegib + everolimus caused short-lived responses in 3 of 6 mice. Strikingly, in vivo treatment with vismodegib in combination with cisplatin impaired median survival (19 days) when compared with cisplatin treatment alone (43 days; p = 0.039) due to rapid tumour progression. Conclusions: Combining chemotherapy with Hh inhibition in Hh ligand-overexpressing HGSOC PDX with mTOR pathway activation may be detrimental. These findings highlight the importance of an in-depth understanding of tumour biology in order to effectively combine therapeutic approaches.

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Study of the biodistribution of fluorescein in glioma-infiltrated mouse brain and histopathological correlation of intraoperative findings in high-grade gliomas resected under fluorescein fluorescence guidance

Journal of Neurosurgery ◽

10.3171/2015.2.jns132507 ◽

2015 ◽

Vol 122 (6) ◽

pp. 1360-1369 ◽

Cited By ~ 77

Author(s):

Roberto Jose Diaz ◽

Roberto Rey Dios ◽

Eyas M. Hattab ◽

Kelly Burrell ◽

Patricia Rakopoulos ◽

...

Keyword(s):

Tumor Cell ◽

Low Dose ◽

Glioma Cells ◽

High Grade ◽

Fluorescein Sodium ◽

Tumor Margins ◽

Histopathological Correlation ◽

High Grade Gliomas

OBJECT Intravenous fluorescein sodium has been used during resection of high-grade gliomas to help the surgeon visualize tumor margins. Several studies have reported improved rates of gross-total resection (GTR) using high doses of fluorescein sodium under white light. The recent introduction of a fluorescein-specific camera that allows for high-quality intraoperative imaging and use of very low dose fluorescein has drawn new attention to this fluorophore. However, the ability of fluorescein to specifically stain glioma cells is not yet well understood. METHODS The authors designed an in vitro model to assess fluorescein uptake in normal human astrocytes and U251 malignant glioma cells. An in vivo experiment was also subsequently designed to study fluorescein uptake by intracranial U87 malignant glioma xenografts in male nonobese diabetic/severe combined immunodeficient mice. A genetically induced mouse glioma model was used to adjust for the possible confounding effect of an inflammatory response in the xenograft model. To assess the intraoperative application of this technology, the authors prospectively enrolled 12 patients who underwent fluorescein-guided resection of their high-grade gliomas using low-dose intravenous fluorescein and a microscope-integrated fluorescence module. Intraoperative fluorescent and nonfluorescent specimens at the tumor margins were randomly analyzed for histopathological correlation. RESULTS The in vitro and in vivo models suggest that fluorescein demarcation of glioma-invaded brain is the result of distribution of fluorescein into the extracellular space, most likely as a result of an abnormal blood-brain barrier. Glioblastoma tumor cell–specific uptake of fluorescein was not observed, and tumor cells appeared to mostly exclude fluorescein. For the 12 patients who underwent resection of their high-grade gliomas, the histopathological analysis of the resected specimens at the tumor margin confirmed the intraoperative fluorescent findings. Fluorescein fluorescence was highly specific (up to 90.9%) while its sensitivity was 82.2%. False negatives occurred due to lack of fluorescence in areas of diffuse, low-density cellular infiltration. Margins of contrast enhancement based on intraoperative MRI–guided StealthStation neuronavigation correlated well with fluorescent tumor margins. GTR of the contrast-enhancing area as guided by the fluorescent signal was achieved in 100% of cases based on postoperative MRI. CONCLUSIONS Fluorescein sodium does not appear to selectively accumulate in astrocytoma cells but in extracellular tumor cell-rich locations, suggesting that fluorescein is a marker for areas of compromised blood-brain barrier within high-grade astrocytoma. Fluorescein fluorescence appears to correlate intraoperatively with the areas of MR enhancement, thus representing a practical tool to help the surgeon achieve GTR of the enhancing tumor regions.

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In vivo involvement of mutated initiation factor IF1 in gene expression control at the translational level

FEBS Letters ◽

10.1016/j.febslet.2004.12.072 ◽

2005 ◽

Vol 579 (5) ◽

pp. 995-1000 ◽

Cited By ~ 10

Author(s):

Victor V. Croitoru ◽

Margarete Bucheli-Witschel ◽

Leif A. Isaksson

Keyword(s):

Gene Expression ◽

Initiation Factor ◽

Expression Control ◽

Gene Expression Control ◽

Translational Level

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Modifying the Epigenome as a Therapeutic Strategy in Myelodysplasia

Hematology ◽

10.1182/asheducation-2007.1.405 ◽

2007 ◽

Vol 2007 (1) ◽

pp. 405-411 ◽

Cited By ~ 16

Author(s):

Guillermo Garcia-Manero

Keyword(s):

Dna Methylation ◽

Clinical Information ◽

Histone Code ◽

Epigenetic Therapy ◽

Epigenetic Alterations ◽

Expression Control ◽

Gene Expression Control ◽

Clinical Interest

AbstractThe term epigenetics refers to a number of biochemical modifications of chromatin that, without altering the primary sequence of DNA, have a role in genomic regulation and in particular gene expression control. These modifications can occur at the DNA level (i.e., DNA methylation), and affect the chromatin protein scaffold (i.e., histone code modifications), among several others. The study of these modifications is a very active area of research both at the basic and clinical levels. Clinical interest in these epigenetic alterations stems mainly from two observations. First, detection of specific epigenetic alterations could be used to develop cancer biomarkers (e.g., for the early detection or prognostication of cancer). Second, most epigenetic alterations are reversible both in vitro and in vivo, leading the way to the development of new anticancer therapies. This review focuses on the current clinical information regarding different forms of epigenetic therapy in patients with myelodysplastic syndromes (MDS). Basic aspects of DNA methylation or histone code alterations are not covered in detail in this review.

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Noise-reducing optogenetic negative-feedback gene circuits in human cells

Nucleic Acids Research ◽

10.1093/nar/gkz556 ◽

2019 ◽

Vol 47 (14) ◽

pp. 7703-7714 ◽

Cited By ~ 11

Author(s):

Michael Tyler Guinn ◽

Gábor Balázsi

Keyword(s):

Gene Expression ◽

Noise Reduction ◽

Negative Feedback ◽

Mammalian Cells ◽

Cellular Proliferation ◽

Protein Domain ◽

Gene Circuits ◽

Expression Control ◽

Gene Expression Control

Abstract Gene autorepression is widely present in nature and is also employed in synthetic biology, partly to reduce gene expression noise in cells. Optogenetic systems have recently been developed for controlling gene expression levels in mammalian cells, but most have utilized activator-based proteins, neglecting negative feedback except for in silico control. Here, we engineer optogenetic gene circuits into mammalian cells to achieve noise-reduction for precise gene expression control by genetic, in vitro negative feedback. We build a toolset of these noise-reducing Light-Inducible Tuner (LITer) gene circuits using the TetR repressor fused with a Tet-inhibiting peptide (TIP) or a degradation tag through the light-sensitive LOV2 protein domain. These LITers provide a range of nearly 4-fold gene expression control and up to 5-fold noise reduction from existing optogenetic systems. Moreover, we use the LITer gene circuit architecture to control gene expression of the cancer oncogene KRAS(G12V) and study its downstream effects through phospho-ERK levels and cellular proliferation. Overall, these novel LITer optogenetic platforms should enable precise spatiotemporal perturbations for studying multicellular phenotypes in developmental biology, oncology and other biomedical fields of research.

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New Putative Long Non-Coding RNAs (lncRNA) Revealed by Pan-Transcriptome of the Emerging Human Pathogenic Fungus Talaromyces Marneffei

10.21203/rs.3.rs-1050608/v1 ◽

2021 ◽

Author(s):

David Aciole Barbosa ◽

Alexandre Santos Simeone ◽

Ana Carolina Humberto ◽

Yara Natercia Lima Faustino de Maria ◽

Regina Costa de Oliveira ◽

...

Keyword(s):

Gene Expression ◽

Transcriptome Assembly ◽

Pathogenic Fungus ◽

The Novel ◽

Talaromyces Marneffei ◽

Expression Control ◽

Gene Expression Control ◽

Human Pathogenic Fungus ◽

Non Coding Rnas

Abstract Previous genomic/transcriptomic analyses of Talaromyces marneffei (TM) unravelled relevant pathogenicity-related elements, as well as chromosomal regions potentially involved with the production of non-coding RNAs (ncRNAs), which have been parsimoniously reported in fungi. This manuscript describes a comprehensive pan-transcriptome assembly for TM that identifies a series of previously undetected genetic elements in this emerging pathogenic fungus. Our results confirm that ~58.28% of the 9,480 genes currently annotated in the TM genome are, in fact, transcribed in vivo and that ~23.6% of them may display alternative isomorphs. Moreover, we identified 585 transcripts that do not match any gene currently mapped in the genome, represented by 90 coding transcripts and 140 ncRNAs, including 48 long non-coding RNAs (lncRNAs). Overall, we expect that the novel elements described herein may contribute to improve the currently available Talaromyces databases and foster studies aiming at characterizing lncRNA-mediated gene expression control in fungi.

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Small Molecule-Inducible RNA-Targeting Systems for Temporal Control of RNA Regulation in Vivo

10.26434/chemrxiv.11673711.v1 ◽

2020 ◽

Cited By ~ 1

Author(s):

Simone Rauch ◽

Krysten A. Jones ◽

Bryan Dickinson

Keyword(s):

Gene Expression ◽

Small Molecule ◽

Rna Regulation ◽

Base Editing ◽

Expression Control ◽

Gene Expression Control ◽

Rna Targeting ◽

Dynamics And Control ◽

And Control

RNA regulation is critical for gene expression control, but tools to temporally manipulate RNA regulatory mechanisms are lacking. Here, we present small molecule-inducible RNA-targeting effectors based on our previously developed CRISPR/Cas-inspired RNA targeting system (CIRTS), which can trigger RNA editing, degradation, or translation on target transcripts. We go on to show the inducible CIRTS editor can be deployed for RNA base editing in vivo, providing a new set of tools to probe RNA regulatory dynamics and control gene expression.

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Oncolytic adenovirus and gene therapy with EphA2-BiTE for the treatment of pediatric high-grade gliomas

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-001930 ◽

2021 ◽

Vol 9 (5) ◽

pp. e001930

Author(s):

Claudia Manuela Arnone ◽

Vinicia Assunta Polito ◽

Angela Mastronuzzi ◽

Andrea Carai ◽

Francesca Camassei Diomedi ◽

...

Keyword(s):

Gene Therapy ◽

T Cells ◽

T Cell ◽

Tumor Cells ◽

Tumor Control ◽

Malignant Neoplasms ◽

High Grade ◽

High Grade Gliomas

BackgroundPediatric high-grade gliomas (pHGGs) are among the most common and incurable malignant neoplasms of childhood. Despite aggressive, multimodal treatment, the outcome of children with high-grade gliomas has not significantly improved over the past decades, prompting the development of innovative approaches.MethodsTo develop an effective treatment, we aimed at improving the suboptimal antitumor efficacy of oncolytic adenoviruses (OAs) by testing the combination with a gene-therapy approach using a bispecific T-cell engager (BiTE) directed towards the erythropoietin-producing human hepatocellular carcinoma A2 receptor (EphA2), conveyed by a replication-incompetent adenoviral vector (EphA2 adenovirus (EAd)). The combinatorial approach was tested in vitro, in vivo and thoroughly characterized at a molecular level.ResultsAfter confirming the relevance of EphA2 as target in pHGGs, documenting a significant correlation with worse clinical outcome of the patients, we showed that the proposed strategy provides significant EphA2-BiTE amplification and enhanced tumor cell apoptosis, on coculture with T cells. Moreover, T-cell activation through an agonistic anti-CD28 antibody further increased the activation/proliferation profiles and functional response against infected tumor cells, inducing eradication of highly resistant, primary pHGG cells. The gene-expression analysis of tumor cells and T cells, after coculture, revealed the importance of both EphA2-BiTE and costimulation in the proposed system. These in vitro observations translated into significant tumor control in vivo, in both subcutaneous and a more challenging orthotopic model.ConclusionsThe combination of OA and EphA2-BiTE gene therapy strongly enhances the antitumor activity of OA, inducing the eradication of highly resistant tumor cells, thus supporting the clinical translation of the approach.

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Small Molecule-Inducible RNA-Targeting Systems for Temporal Control of RNA Regulation in Vivo

10.26434/chemrxiv.11673711.v2 ◽

2020 ◽

Author(s):

Simone Rauch ◽

Krysten A. Jones ◽

Bryan Dickinson

Keyword(s):

Gene Expression ◽

Small Molecule ◽

Rna Regulation ◽

Base Editing ◽

Expression Control ◽

Gene Expression Control ◽

Rna Targeting ◽

Dynamics And Control ◽

And Control

RNA regulation is critical for gene expression control, but tools to temporally manipulate RNA regulatory mechanisms are lacking. Here, we present small molecule-inducible RNA-targeting effectors based on our previously developed CRISPR/Cas-inspired RNA targeting system (CIRTS), which can trigger RNA editing, degradation, or translation on target transcripts. We go on to show the inducible CIRTS editor can be deployed for RNA base editing in vivo, providing a new set of tools to probe RNA regulatory dynamics and control gene expression.

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