Biology subjects

Bagci-Onder, T.

Publications and source records attributed to Bagci-Onder, T..

4 recordsLinked to original sources

bioRxiv2021

EPIKOL, a chromatin-focused CRISPR/Cas9-based screening platform, to identify cancer-specific epigenetic vulnerabilities

Dysregulation of the epigenome due to alterations in chromatin modifier proteins commonly contribute to malignant transformation. To discover new drug targets for more targeted and personalized therapies, functional interrogation of epigenetic modifiers is essential. We therefore generated an epigenome-wide CRISPR-Cas9 knock-out library (EPIKOL) that targets a wide-range of epigenetic modifiers and their cofactors. We conducted eight screens in two different cancer types and showed that EPIKOL performs with high efficiency in terms of sgRNA distribution, depletion of essential genes and steady behaviors of non-targeting sgRNAs. From this, we discovered novel epigenetic modifiers besides previously known ones that regulate triple-negative breast cancer and prostate cancer cell fitness. With further validation assays, we confirmed the growth-regulatory function of individual candidates, including SS18L2 and members of the NSL complex (KANSL2, KANSL3, KAT8) in triple negative breast cancer cells. Overall, we show that EPIKOL, a focused sgRNA library targeting approximately 800 genes, can reveal epigenetic modifiers that are essential for cancer cell fitness and serve as a tool to offer novel anti-cancer targets. With its thoroughly generated epigenome-wide gene list, and the relatively high number of sgRNAs per gene, EPIKOL offers a great advantage to study functional roles of epigenetic modifiers in a wide variety of research applications, such as screens on primary cells, patient-derived xenografts as well as in vivo models.

cancer biology↗

bioRxiv2021

Protein scaffold-based multimerization of soluble ACE2 efficiently blocks SARS-CoV-2 infection in vitro

Soluble ACE2 (sACE2) decoy receptors are promising agents to inhibit SARS-CoV-2, as their efficiency is less likely to be affected by common escape mutations in viral proteins. However, their success may be limited by their relatively poor potency. To address this challenge, we developed a large decoy library of sACE2 fusion proteins, generated with several protease inhibitors or multimerization tags. Among these decoys, multimeric sACE2 consisting of SunTag or MoonTag systems, which were originally utilized for signal amplification or gene activation systems, were extremely effective in neutralizing SARS-CoV-2 in pseudoviral systems and in clinical isolates. These novel sACE2 fusion proteins exhibited greater than 100-fold SARS-CoV-2 neutralization efficiency, compared to monomeric sACE2. SunTag or MoonTag in combination with a more potent version of sACE2, which has multiple point mutations for greater binding (v1), achieved near complete neutralization at a sub-nanomolar range, comparable with clinical monoclonal antibodies. Pseudoviruses bearing mutant versions of Spike, alpha, beta, gamma or delta variants, were also neutralized efficiently with SunTag or MoonTag fused sACE2(v1). Finally, therapeutic treatment of sACE2(v1)-MoonTag provided protection against SARS-CoV-2 infection in an in vivo mouse model. Overall, we suggest that the superior activity of the sACE2-SunTag or sACE2-MoonTag fusions is due to the greater occupancy of the multimeric sACE2 receptors on Spike protein as compared to monomeric sACE2. Therefore, these highly potent multimeric sACE2 decoy receptors may offer a promising treatment approach against SARS-CoV-2 infections. One Sentence SummaryMultimerization of sACE2 markedly enhanced the neutralization of SARS-CoV-2 by blocking multiple viral spike proteins simultaneously.

molecular biology↗

bioRxiv2020

The neutralization effect of Montelukast on SARS-CoV-2 is shown by multiscale in silico simulations and combined in vitro studies

Small molecule inhibitors have previously been investigated in different studies as possible therapeutics in the treatment of SARS-CoV-2. In the current drug repurposing study, we identified the leukotriene (D4) receptor antagonist Montelukast as a novel agent that simultaneously targets two important drug targets of SARS-CoV-2. We initially demonstrated the dual inhibition profile of Montelukast through multiscale molecular modeling studies. Next, we characterized its effect on both targets by different in vitro experiments including the Fluorescent Resonance Energy Transfer (FRET)-based main protease enzyme inhibition assay, surface plasmon resonance (SPR) spectroscopy, pseudovirus neutralization on HEK293T / hACE2, and virus neutralization assay using xCELLigence MP real time cell analyzer. Our integrated in silico and in vitro results confirmed the dual potential effect of the Montelukast both on virus entry into the host cell (Spike/ACE2) and on the main protease enzyme inhibition. The virus neutralization assay results showed that while no cytotoxicity of the Montelukast was observed at 12 M concentration, the cell index time 50 (CIT50) value was delayed for 12 hours. Moreover, it was also shown that Favipiravir, a well-known antiviral used in COVID-19 therapy, should be used by 16-fold higher concentrations than Montelukast in order to have the same effect of Montelukast. The rapid use of new small molecules in the pandemic is very important today. Montelukast, whose pharmacokinetic and pharmacodynamic properties are very well characterized and has been widely used in the treatment of asthma since 1998, should urgently be completed in clinical phase studies and if its effect is proven in clinical phase studies, it should be used against COVID-19.

biophysics↗

bioRxiv2020

Combined inhibition of KDM6A/B and HDACs exacerbates integrated stress response and mediates therapeutic effects in IDH1-mutant glioma

BackgroundIDH1/2-mutant gliomas are primary brain tumors for which curative treatments are lacking. Mutant IDH-dependent 2-hydroxyglutarate (2-HG) accumulation leads to DNA and histone hypermethylation. Based on this distinct phenotype, we interrogated epigenetic dependencies of IDH-mutant glioma that can be targeted therapeutically. MethodsWe conducted a chemical screen targeting chromatin modifiers in patient derived IDH1-mutant GBM cells. We investigated mechanisms of action of compound hits and their combinations through cell-based functional assays, live-cell imaging, Western blot, CRISPR knockout, RNA-seq and ChIP experiments. The therapeutic concept was validated in vivo using chemical inhibitors GSK-J4 and Belinostat in an orthotopic GBM model. ResultsWe identified the H3K27me3 demethylase (KDM6) inhibitor GSK-J4 and histone deacetylase inhibitor Belinostat as potent, genotype-selective agents against IDH1-mutant glioma. RNA-sequencing on paired wild-type and IDH1R132H cells revealed inhibition of cholesterol biosynthesis and activation of cellular stress in IDH1R132H cells, which were reversible with a mutant IDH1 inhibitor. GSK-J4 caused further repression of cholesterol biosynthesis pathway genes through H3K27me3 deposition and exacerbated the ATF4-mediated integrated stress response. Belinostat inhibited anti-apoptotic pathways through activation of TGF-{beta} signaling and induced cell cycle arrest. Together, the GSK-J4 and Belinostat combination activated DDIT3/CHOP-dependent apoptosis in IDH1-mutant cells and extended survival in an IDH1-mutant orthotopic model in vivo. ConclusionsThese results provide a possible therapeutic approach that exploits epigenetic vulnerabilities of IDH-mutant gliomas. Key points- Combination of GSK-J4 and Belinostat selectively targets IDH1-mutant cells. - GSK-J4 downregulates cholesterol biosynthesis and activates an ATF4-mediated stress response. - Belinostat activates the TGF{beta} pathway, induces G2/M arrest and inhibits anti-apoptotic pathways. Importance of the studyIDH1/2 genes are frequently mutated in low grade glioma and secondary glioblastoma. These tumors exhibit a distinct epigenomic signature with increased DNA and histone methylation; therefore, identifying and exploiting their epigenetic vulnerabilities may lead to effective therapies. We discovered that targeting of KDM6A/6B together with HDACs provides a promising therapeutic approach for IDH1-mutant glioma.

cancer biology↗