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Glioblastoma is the most malignant primary brain tumor, and a cornerstone in its treatment is radiotherapy. However, tumor cells surviving after irradiation indicates treatment failure; therefore, better understanding of the mechanisms regulating radiotherapy response is of utmost importance. In this study, we generated clinically relevant irradiation-exposed models by applying fractionated radiotherapy over a long time and selecting irradiation-survivor (IR-Surv) glioblastoma cells. We examined the transcriptomic alterations, cell cycle and growth rate changes and responses to secondary radiotherapy and DNA damage response (DDR) modulators. Accordingly, IR-Surv cells exhibited slower growth and partly retained their ability to resist secondary irradiation. Concomitantly, IR-Surv cells upregulated the expression of DDR-related genes, such as CHK1, ATM, ATR, and MGMT, and had better DNA repair capacity. IR-Surv cells displayed downregulation of hypoxic signature and lower induction of hypoxia target genes, compared to naive glioblastoma cells. Moreover, Chk1 inhibition alone or in combination with irradiation significantly reduced cell viability in both naive and IR-Surv cells. However, IR-Surv cells' response to Chk1 inhibition markedly decreased under hypoxic conditions. Taken together, we demonstrate the utility of combining DDR inhibitors and irradiation as a successful approach for both naive and IR-Surv glioblastoma cells as long as cells are refrained from hypoxic conditions., Scientific and Technological Research Council of Turkey (TÜBİTAK)

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 enzyme (main protease) inhibition-based assay, surface plasmon resonance (SPR) spectroscopy, pseudovirus neutralization on HEK293T/hACE2+TMPRSS2, 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 the main protease enzyme inhibition and virus entry into the host cell (Spike/ACE2). The virus neutralization assay results showed that SARS-CoV-2 virus activity was delayed with Montelukast for 20 hours on the infected cells. 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., Graphical abstract, In the current drug repurposing study, we identified the leukotriene (D4) receptor antagonist Montelukast that simultaneously targets two important drug targets of SARS-CoV-2. Montelukast shows its effect both on the main protease and Spike/ACE2. The virus neutralization assay results showed that SARS-CoV-2 activity was delayed with Montelukast for 20 hours on the infected cells.

Discovery of point mutations in the genes encoding isocitrate dehydrogenases (IDH) in gliomas about a decade ago has challenged our view of the role of metabolism in tumor progression and provided a new stratification strategy for malignant gliomas. IDH enzymes catalyze the conversion of isocitrate to alpha-ketoglutarate (alpha-KG), an intermediate in the citric acid cycle. Specific mutations in the genes encoding IDHs cause neomorphic enzymatic activity that produces D-2-hydroxyglutarate (2-HG) and result in the inhibition of alpha-KG-dependent enzymes such as histone and DNA demethylases. Thus, chromatin structure and gene expression profiles in IDH-mutant gliomas appear to be different from those in IDH-wildtype gliomas. IDH mutations are highly common in lower grade gliomas (LGG) and secondary glioblastomas, and they are among the earliest genetic events driving tumorigenesis. Therefore, inhibition of mutant IDH enzymes in LGGs is widely accepted as an attractive therapeutic strategy. On the other hand, the metabolic consequences derived from IDH mutations lead to selective vulnerabilities within tumor cells, making them more sensitive to several therapeutic interventions. Therefore, instead of shutting down mutant IDH enzymes, exploiting the selective vulnerabilities caused by them might be another attractive and promising strategy. Here, we review therapeutic options and summarize current preclinical and clinical studies on IDH-mutant gliomas., Scientific and Technological Research Council of Turkey (TÜBİTAK)

Androgen deprivation therapy (ADT) is the standard care for prostate cancer (PCa) patients who fail surgery or radiotherapy. While initially effective, the cancer almost always recurs as a more aggressive castration resistant prostate cancer (CRPC). Previous studies have demonstrated that chromatin modifying enzymes can play a critical role in the conversion to CRPC. However, only a handful of these potential pharmacological targets have been tested. Therefore, in this study, we conducted a focused shRNA screen of chromatin modifying enzymes previously shown to be involved in cellular differentiation. We found that altering the balance between histone methylation and demethylation impacted growth and proliferation. Of all genes tested, KDM3B, a histone H3K9 demethylase, was found to have the most antiproliferative effect. These results were phenocopied with a KDM3B CRISPR/Cas9 knockout. When tested in several PCa cell lines, the decrease in proliferation was remarkably specific to androgen-independent cells. Genetic rescue experiments showed that only the enzymatically active KDM3B could recover the phenotype. Surprisingly, despite the decreased proliferation of androgen-independent cell no alterations in the cell cycle distribution were observed following KDM3B knockdown. Whole transcriptome analyses revealed changes in the gene expression profile following loss of KDM3B, including downregulation of metabolic enzymes such as ARG2 and RDH11. Metabolomic analysis of KDM3B knockout showed a decrease in several critical amino acids. Overall, our work reveals, for the first time, the specificity and the dependence of KDM3B in CRPC proliferation., Scientific and Technological Research Council of Turkey (TÜBİTAK); Royal Society Newton Advanced Fellowship; Wellcome Trust Core Award; Royal Society Dorothy Hodgkin Fellowship; Cancer Research UK Programme

Type 1 diabetes (T1D) is an autoimmune disease characterized by destruction of pancreatic β cells. One of the promising therapeutic approaches in T1D is the transplantation of islets; however, it has serious limitations. To address these limitations, immunotherapeutic strategies have focused on restoring immunologic tolerance, preventing transplanted cell destruction by patients’ own immune system. Macrophage-derived chemokines such as chemokine-ligand-22 (CCL22) can be utilized for regulatory T cell (Treg) recruitment and graft tolerance. Stellate cells (SCs) have various immunomodulatory functions: recruitment of Tregs and induction of T-cell apoptosis. Here, we designed a unique immune-privileged microenvironment around implantable islets through overexpression of CCL22 proteins by SCs. We prepared pseudoislets with insulin-secreting mouse insulinoma-6 (MIN6) cells and human SCs as a model to mimic naive islet morphology. Our results demonstrated that transduced SCs can secrete CCL22 and recruit Tregs toward ​the implantation site in vivo. This study is promising to provide a fundamental understanding of SC-islet interaction and ligand synthesis and transport from SCs at the graft site for ensuring local immune tolerance. Our results also establish a new paradigm for creating tolerable grafts for other chronic diseases such as diabetes, anemia, and central nervous system (CNS) diseases, and advance the science of graft tolerance., Scientific and Technological Research Council of Turkey (TÜBİTAK)

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can selectively kill tumor cells. TRAIL resistance in cancers is associated with aberrant expression of the key components of the apoptotic program. However, how these components are regulated at the epigenetic level is not understood. In this study, we investigated novel epigenetic mechanisms regulating TRAIL response in glioblastoma multiforme (GBM) cells by a short-hairpin RNA loss-of-function screen. We interrogated 48 genes in DNA and histone modification pathways and identified KDM2B, an H3K36-specific demethylase, as a novel regulator of TRAIL response. Accordingly, silencing of KDM2B significantly enhanced TRAIL sensitivity, the activation of caspase-8, -3 and -7 and PARP cleavage. KDM2B knockdown also accelerated the apoptosis, as revealed by live-cell imaging experiments. To decipher the downstream molecular pathways regulated by KDM2B, levels of apoptosis-related genes were examined by RNA-sequencing upon KDM2B loss, which revealed derepression of proapoptotic genes Harakiri (HRK), caspase-7 and death receptor 4 (DR4) and repression of antiapoptotic genes. The apoptosis phenotype was partly dependent on HRK upregulation, as HRK knockdown significantly abrogated the sensitization. KDM2B-silenced tumors exhibited slower growth in vivo. Taken together, our findings suggest a novel mechanism, where the key apoptosis components are under epigenetic control of KDM2B in GBM cells., Republic of Turkey Ministry of Development Research; Marie Curie