Interaction of mammalian DNA repair mechanisms for the repair of O6-methylguanine damages

O6-Methylguanin (O6-MeG) ist ein DNA-Schaden, der durch endogene und exogene alkylierende Substanzen oder tumortherapeutisch eingesetzte Verbindungen hervorgerufen wird und sowohl mutagen als auch zytotoxisch wirken kann. Biochemische Analysen dieser Arbeitsgruppe ließen vermuten, dass es in Säugerzellen zusätzlich zu der direkten Demethylierung durch das spezifische Reparatur-Protein MGMT einen weiteren Mechanismus zur Entfernung von O6-MeG gibt, bei dem offenbar Komponenten aus bereits bekannten Reparaturwegen verwendet werden. Der neue Mechanismus entfernt durch duale Einschnitte das alkylierte Guanin und weist auf eine Beteiligung des Nukleotid Exzisions Reparatur (NER)-Systems hin. In dieser Dissertation wurde der neu beschriebene Reparatur-Mechanismus weiter charakterisiert, einzelne essenzielle Komponenten identifiziert und die zellulären Konsequenzen bei einem Ausfall dieses Systems untersucht. Anhand von Bindungsstudien mit rekombinanten Reparaturproteinen konnte gezeigt werden, dass das NER-Protein XPC an O6-MeG-Läsionen in der DNA bindet und für die Einleitung des Exzisionsvorganges essenziell ist. Dies wurde durch Reparaturkinetiken für O6-MeG an XPC-defizienten humanen Fibroblasten und an rekonstituierten Varianten und an primären hämatopoetischer Zellen aus XPC(-/-)-Mäusen nach einer ex vivo-Exposition mit Alkylanzien belegt. Ein funktioneller Ausfall führte zu einer erhöhten Sensitivität gegenüber alkylierenden Substanzen. Die Ergebnisse zeigen, dass die NER nicht alleine für die O6-MeG-Reparatur zuständig ist. Daher wurde die Beteiligung des Fanconi-Anämie(FA)-Systems analysiert, dem bisher nur eine Rolle bei der Prozessierung von DNA-Doppelstrang-Brüchen zugeschrieben wurde. Von den humanen Zelllinien mit Funktionsverlustmutationen für jeweils eines der FA-Proteine zeigten nur die Zellen mit Defekt im FancD2-Gen einen vollständigen Ausfall des alternativen Reparaturweges. Die essenzielle Rolle von FancD2 in diesem Prozess konnte durch einen Vergleich von primären Zellen und Gewebe aus FancD2(+/+) und FancD2(-/-) Mäusen bestätigt werden. Eine Interaktion von XPC und FancD2 konnte bisher nicht gezeigt werden, jedoch bilden XPC-defiziente Zellen nach einer Alkylierungsbehandllung keine FancD2-Foci an DNA-Schäden, wohingegen rekonstituierte Zellen dies können. Somit bleibt noch unklar, wie und in welcher Weise FancD2 und XPC nach einem Alkylierungsschaden miteinander reagieren oder koordiniert werden, um den DNA-Schaden zu beheben. Weitere Forschungen sind nötig um die Interaktion der beiden Proteine genauer zu definieren und um weitere Komponenten des alternativen O6-MeG-Reparatur-Mechanismuses zu beschreiben.O6-methylguanine can be caused by endogenous and exogenous alkylating substances or during tumor therapy used reagent. This damage can be highly mutagenic as well as cytotoxic for the cell. This study shows that not only the repair protein MGMT can repair this damage. Another novel repair mechanism recruits proteins from various other repair pathways to act together in the repair of the O6-methylguanine damages. This new repair pathway includes members of the nucleotide excision repair- as well as members of the Fanconi anemia pathway

Rapalink-1 Targets Glioblastoma Stem Cells and Acts Synergistically with Tumor Treating Fields to Reduce Resistance against Temozolomide.

Glioblastoma (GBM) is a lethal disease with limited clinical treatment options available. Recently, a new inhibitor targeting the prominent cancer signaling pathway mTOR was discovered (Rapalink-1), but its therapeutic potential on stem cell populations of GBM is unknown. We applied a collection of physiological relevant organoid-like stem cell models of GBM and studied the effect of RL1 exposure on various cellular features as well as on the expression of mTOR signaling targets and stem cell molecules. We also undertook combination treatments with this agent and clinical GBM treatments tumor treating fields (TTFields) and the standard-of-care drug temozolomide, TMZ. Low nanomolar (nM) RL1 treatment significantly reduced cell growth, proliferation, migration, and clonogenic potential of our stem cell models. It acted synergistically to reduce cell growth when applied in combination with TMZ and TTFields. We performed an in silico analysis from the molecular data of diverse patient samples to probe for a relationship between the expression of mTOR genes, and mesenchymal markers in different GBM cohorts. We supported the in silico results with correlative protein data retrieved from tumor specimens. Our study further validates mTOR signaling as a druggable target in GBM and supports RL1, representing a promising therapeutic target in brain oncology

A comparative pharmaco-metabolomic study of glutaminase inhibitors in glioma stem-like cells confirms biological effectiveness but reveals differences in target-specificity

Abstract: Cancer cells upregulate anabolic processes to maintain high rates of cellular turnover. Limiting the supply of macromolecular precursors by targeting enzymes involved in biosynthesis is a promising strategy in cancer therapy. Several tumors excessively metabolize glutamine to generate precursors for nonessential amino acids, nucleotides, and lipids, in a process called glutaminolysis. Here we show that pharmacological inhibition of glutaminase (GLS) eradicates glioblastoma stem-like cells (GSCs), a small cell subpopulation in glioblastoma (GBM) responsible for therapy resistance and tumor recurrence. Treatment with small molecule inhibitors compound 968 and CB839 effectively diminished cell growth and in vitro clonogenicity of GSC neurosphere cultures. However, our pharmaco-metabolic studies revealed that only CB839 inhibited GLS enzymatic activity thereby limiting the influx of glutamine derivates into the TCA cycle. Nevertheless, the effects of both inhibitors were highly GLS specific, since treatment sensitivity markedly correlated with GLS protein expression. Strikingly, we found GLS overexpressed in in vitro GSC models as compared with neural stem cells (NSC). Moreover, our study demonstrates the usefulness of in vitro pharmaco-metabolomics to score target specificity of compounds thereby refining drug development and risk assessment

A comparative pharmaco-metabolomic study of glutaminase inhibitors in glioma stem-like cells confirms biological effectiveness but reveals differences in target-specificity

Abstract: Cancer cells upregulate anabolic processes to maintain high rates of cellular turnover. Limiting the supply of macromolecular precursors by targeting enzymes involved in biosynthesis is a promising strategy in cancer therapy. Several tumors excessively metabolize glutamine to generate precursors for nonessential amino acids, nucleotides, and lipids, in a process called glutaminolysis. Here we show that pharmacological inhibition of glutaminase (GLS) eradicates glioblastoma stem-like cells (GSCs), a small cell subpopulation in glioblastoma (GBM) responsible for therapy resistance and tumor recurrence. Treatment with small molecule inhibitors compound 968 and CB839 effectively diminished cell growth and in vitro clonogenicity of GSC neurosphere cultures. However, our pharmaco-metabolic studies revealed that only CB839 inhibited GLS enzymatic activity thereby limiting the influx of glutamine derivates into the TCA cycle. Nevertheless, the effects of both inhibitors were highly GLS specific, since treatment sensitivity markedly correlated with GLS protein expression. Strikingly, we found GLS overexpressed in in vitro GSC models as compared with neural stem cells (NSC). Moreover, our study demonstrates the usefulness of in vitro pharmaco-metabolomics to score target specificity of compounds thereby refining drug development and risk assessment

Prognostic impact of vitamin B6 metabolism in lung cancer

Patients with non-small cell lung cancer (NSCLC) are routinely treated with cytotoxic agents such as cisplatin. Through a genome-wide siRNA-based screen, we identified vitamin B6 metabolism as a central regulator of cisplatin responses in vitro and in vivo. By aggravating a bioenergetic catastrophe that involves the depletion of intracellular glutathione, vitamin B6 exacerbates cisplatin-mediated DNA damage, thus sensitizing a large panel of cancer cell lines to apoptosis. Moreover, vitamin B6 sensitizes cancer cells to apoptosis induction by distinct types of physical and chemical stress, including multiple chemotherapeutics. This effect requires pyridoxal kinase (PDXK), the enzyme that generates the bioactive form of vitamin B6. In line with a general role of vitamin B6 in stress responses, low PDXK expression levels were found to be associated with poor disease outcome in two independent cohorts of patients with NSCLC. These results indicate that PDXK expression levels constitute a biomarker for risk stratification among patients with NSCLC.publishedVersio

Augmented Therapeutic Potential of Glutaminase Inhibitor CB839 in Glioblastoma Stem Cells Using Gold Nanoparticle Delivery

Gold nanoparticles (Au NPs) are studied as delivery systems to enhance the effect of the glutaminase1 inhibitor CB839, a promising drug candidate already in clinical trials for tumor treatments. Au NPs were synthesized using a bottom-up approach and covered with polymers able to bind CB839 as a Au-polymer-CB839 conjugate. The drug loading efficiency (DLE) was determined using high-performance liquid chromatography and characterization of the CB839-loaded NPs was done with various microscopic and spectroscopic methods. Despite the chemical inertness of CB839, Au NPs were efficient carriers with a DLE of up to 12%, depending on the polymer used. The therapeutic effect of CB839 with and without Au was assessed in vitro in 2D and 3D glioblastoma (GBM) cell models using different assays based on the colony formation ability of GBM stem cells (GSCs). To avoid readout disturbances from the Au metal, viability methods which do not require optical detection were hereby optimized. These showed that Au NP delivery increased the efficacy of CB839 in GSCs, compared to CB839 alone. Fluorescent microscopy proved successful NP penetration into the GSCs. With this first attempt to combine CB839 with Au nanotechnology, we hope to overcome delivery hurdles of this pharmacotherapy and increase bioavailability in target sites

Current technologies for RNA-directed liquid diagnostics

There is unequivocal acceptance of the variety of enormous potential liquid nucleic acid-based diagnostics seems to offer. However, the existing controversies and the increased awareness of RNA-based techniques in society during the current global COVID-19 pandemic have made the readiness of liquid nucleic acid-based diagnostics for routine use a matter of concern. In this regard—and in the context of oncology—our review presented and discussed the status quo of RNA-based liquid diagnostics. We summarized the technical background of the available assays and benchmarked their applicability against each other. Herein, we compared the technology readiness level in the clinical context, economic aspects, implementation as part of routine point-of-care testing as well as performance power. Since the preventive care market is the most promising application sector, we also investigated whether the developments predominantly occur in the context of early disease detection or surveillance of therapy success. In addition, we provided a careful view on the current biotechnology investment activities in this sector to indicate the most attractive strategies for future economic success. Taken together, our review shall serve as a current reference, at the interplay of technology, clinical use and economic potential, to guide the interested readers in this rapid developing sector of precision medicine

A comparative pharmaco-metabolomic study of glutaminase inhibitors in glioma stem-like cells confirms biological effectiveness but reveals differences in target-specificity.

Cancer cells upregulate anabolic processes to maintain high rates of cellular turnover. Limiting the supply of macromolecular precursors by targeting enzymes involved in biosynthesis is a promising strategy in cancer therapy. Several tumors excessively metabolize glutamine to generate precursors for nonessential amino acids, nucleotides, and lipids, in a process called glutaminolysis. Here we show that pharmacological inhibition of glutaminase (GLS) eradicates glioblastoma stem-like cells (GSCs), a small cell subpopulation in glioblastoma (GBM) responsible for therapy resistance and tumor recurrence. Treatment with small molecule inhibitors compound 968 and CB839 effectively diminished cell growth and in vitro clonogenicity of GSC neurosphere cultures. However, our pharmaco-metabolic studies revealed that only CB839 inhibited GLS enzymatic activity thereby limiting the influx of glutamine derivates into the TCA cycle. Nevertheless, the effects of both inhibitors were highly GLS specific, since treatment sensitivity markedly correlated with GLS protein expression. Strikingly, we found GLS overexpressed in in vitro GSC models as compared with neural stem cells (NSC). Moreover, our study demonstrates the usefulness of in vitro pharmaco-metabolomics to score target specificity of compounds thereby refining drug development and risk assessment

CD133-Functionalized Gold Nanoparticles as a Carrier Platform for Telaglenastat (CB-839) against Tumor Stem Cells

The failure of a long-lasting curative therapeutic benefit of currently applied chemotherapies against malignant cancers is suggested to be caused by the ineffectiveness of such interventions on cancer stem cells (CSCs). CD133/AC133 is a cell surface protein previously shown to have potential to identify CSCs in various tumors, including brain tumors. Moreover, an increase in the rate of cellular metabolism of glutamine and glucose are contributors to the fast cellular proliferation of some high-grade malignancies. Inhibition of glutaminolysis by utilizing pharmacological inhibitors of the enzyme glutaminase 1 (GLS1) can be an effective anti-CSC strategy. In this study, the clinical-stage GLS1 inhibitor Telaglenastat (CB-839) was loaded into PEGylated gold nanoparticles equipped with the covalently conjugated CD133 aptamer (Au-PEG-CD133-CB-839) and exposed to a collection of CD133-positive brain tumor models in vitro. Our results show that Au-PEG-CD133-CB-839 significantly decreased the viability of CD133-postive cancer cells in a dose-dependent manner, which was higher as compared to the effects of treatment of the cells with the individual components of the assembled nanodrug. Interestingly, the treatment effect was observed in glioblastoma stem cells modeling different transcriptomic subtypes of the disease. The presented platform is the fundament for subsequent target specificity characterization and in vivo application