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IDH1 ve IDH2 genlerindeki mutasyonlar düşük dereceli gliomlar ve ikincil GBM tümörlerinde görülmekte olup, belirleyici epigenetik farklılıklara sebep olduğu bilinmektedir. Epigenetik hassasiyetlerini sorgulamak amacıyla, IDH mutant gliom hücrelerinde kromatin düzenleyici inhibitörlerinin bulunduğu kütüphane ile gerçekleştirdiğimiz kimyasal tarama sonucunda bu hücreleri yüksek oranda etkileyen 5azacytidine, Chaetocin, GSK-J4 ve Belionstat kimyasalları tanımlanmıştır. Belirlenen ajanların kombinasyon durumunda verimleri test edildiğinde, GSK-J4 ve Belinostat kombinasyonunun IDH mutant gliomlarda hücre canlılığını önemli ölçüde etkilediği görülmüştür. IDH1R132H mutasyonu hücrelerde ektopik olarak ifade edildiğinde IDH mutant hücrelerinin transkriptomunun ve hücresel stres kaynaklı yolakların değiştiğini, ve bu değişimlerin IDH mutant inhibitörü GSK864 ile geriye döndürülebileceğini gösterdik. GSK-J4 ve Belinostat kombinasyonunun IDH1R132 hücrelerinde etkisinin yüksek olduğunu, ancak yabanıl tip gliom hücrelerinde ve malignant olmayan fibroblast ve astrosit hücrelerini etkilemediği görülmüştür. GSK-J4 ve Belinostat kombinasyonu hücre ölümü ve apoptozun indüklenmesini içerir. RNA sekanslama analizleri glioma hücrelerinde artan stresi gösterir biçimde, GSK-J4 ve Belinostat verilen IDH-1 mutant hücrelerinde, inflamatuar ve katlanmamış protein cevabı yolaklarının aktive olduğunu göstermiştir. Özellikle, GSK-J4 indüklenen ATF4 aracılı entegre stres cevabı (ISR) ve Belinostat kaynaklı primer IDH1-mutant glioma hücrelerinde hücre döngüsünün durmasına neden olmuş, bunlara DDIT3/CHOP bağımlı apoptoz eşlik etmiştir. Buna ek olarak, sorumlu hedef histon demetilazını yok etmek için CRISPR/Cas9 aracılı genetik bir yaklaşım ile ve hem KDM6A hem de KDM6B'nin ablasyonunun, IDH1 mutant hücrelerinde GSK-J4'ün etkilerini fenokopi ettiğini gösterdik. Son olarak, GSK-J4 ve Belinostat kombinasyonu, farelerde ortotopik bir modelde tümör büyümesini ve sağkalımı artırmıştır. Bu sonuçlar IDH1-mutant gliomalara karşı potansiyel bir kombinasyon epigenetik tedavi önermektedir. Mutations in IDH1 and IDH2 genes are common in low grade gliomas and secondary GBM and are known to cause a distinct epigenetic landscape in these tumors. To interrogate the epigenetic vulnerabilities of IDH-mutant gliomas, we performed a chemical screen with inhibitors of chromatin modifiers and identified 5-azacytidine, Chaetocin, GSK-J4 and Belinostat as potent agents against primary IDH1-mutant cell lines. Testing the combinatorial efficacy of these agents, we demonstrated GSK-J4 and Belinostat combination as a very effective treatment for the IDH1-mutant glioma cells. Engineering established cell lines to ectopically express IDH1R132H, we showed that IDH1-mutant cells adopted a different transcriptome with changes in stress-related pathways that were reversible with the mutant IDH1 inhibitor, GSK864. The combination of GSK-J4 and Belinostat was highly effective on IDH1R132H cells, but not on wt glioma cells or non-malignant fibroblasts and astrocytes. The cell death induced by GSK-J4 and Belinostat combination involved the induction of cell cycle arrest and apoptosis. RNA sequencing analyses revealed activation of inflammatory and unfolded protein response pathways in IDH1-mutant cells upon treatment with GSK-J4 and Belinostat conferring increased stress to glioma cells. Specifically, GSK-J4 induced ATF4-mediated integrated stress response (ISR) and Belinostat induced cell cycle arrest in primary IDH1-mutant glioma cells, which were accompanied by DDIT3/CHOP-dependent upregulation of apoptosis. Moreover, to dissect out the responsible target histone demethylase, we undertook genetic approach and demonstrated that CRISPR/Cas9 mediated ablation of both KDM6A and KDM6B phenocopied the effects of GSK-J4 in IDH1-mutant cells. Finally, GSK-J4 and Belinostat combination significantly decreased tumor growth and increased survival in an orthotopic model in mice. Together, these results suggest a potential combination epigenetic therapy against IDH1-mutant gliomas. 116

Yağ doku mühendisliği, yumuşak doku hasarlarının rejenerasyonu için umut verici biralandır. Kalın implantların iç katmanlarındaki hücreler besin ve oksijene ulaşamadığıiçin damarlaşma sağlanması ihtiyaç vardır. Diğer bir problem de biyouyumlu ve iyi mekanik özelliklere sahip hücre taşıyıcılar bulabilmektir. Bu çalışmada, doğal dokuylamekanik olarak uyumlu, hücre canlılığını ve farklılaşmasını destekleyen, kalın ve fonksiyonel bir damarlaşma sağlanmış yağ doku üretmeyi amaçladık. Bu amaçla, deselülerize edilmiş yağ doku (DAT) ve ipek fibroini farklı oranlarda karıştırılarak hidrojeller elde edilmiştir. Daha sonra, yağ doku kökenli kök hücrelerden farklılaştırılan öncül yağ doku hücreleri ve endotel-benzeri hücreler bu hidrojeller içine hapsedilmiştir. In vitro çalışmalar, 1:3 (v/v) DAT:Fibroin oranlı hidrojellerin hücre canlılığını desteklediğini göstermiştir. Aynı zamanda, hidrojel içine hapsedildikten yalnızca 3 gün sonra adipojenik yönde 1 hafta farklılaştırılan ASC'ler yağ damlacıkları toplamaya, endotel yönde 1 hafta farklılaştırılan ASC'ler ise kılcal damar-benzeri yapılar oluşturmaya başlamıştır. Bu karışım in vivo uygulamada da jelleştirilebilmiş ve deri altına yerleştirilmiştir. Erken analizlerde (1 haftalık) şiddetli bir immün reaksiyon veya parçalanma gözlemlenmemiştir. Geç analizlerde (3 haftalık) ise hidrojelin dıştan başlayarak parçalandığı ve kılcal damarların hidrojelin merkezine kadar ulaştığı gözlemlenmiştir. Hücre enkapsüle edilmiş hidrojellerin in vivo analizleri devam etmektedir. Mekanik testler, in vitro hücre kültür çalışmaları ve in vivo uygulamaların ön analiz sonuçlarına dayanarak, 1:3 DAT:Fib hidrojelleri yağ doku mühendisliği için uygun taşıyıcılar olarak önerilmiştir. Adipose tissue engineering is a promising field for regeneration of soft tissue defects.However, vascularization is needed since cells in the middle layer of thick implants cannot reach to nutrient and oxygen by diffusion. Finding a biocompatible scaffold with good mechanical properties is another problem in this field. In this study, we aimed to develop a thick functional vascularized adipose tissue which supports cell viability and functionality with similar mechanical properties with the adipose tissue. For this purpose, hydrogels were prepared by mixing human decellularized adipose tissue (DAT) and silk fibroin at different ratios. Then, both preadipocytes and endothelial-like cells pre-differentiated from rat adipose derived stem cells (ASCs) were encapsulated in hydrogels. In vitro analyses showed that hydrogels with 1:3 (v/v) DAT:Fibroin ratio support cell viability. ASCs pre-differentiated into adipogenic lineage for 1 week started to accumulate lipid vesicles, and ones pre-differentiated into endothelial lineage formed capillary-like structures inside hydrogel only after 3 days of encapsulation. This mixture was shown to be gelated in vivo too, and implanted subcutaneously. No severe immunological response or significant degradation of the hydrogels was observed in histopathological analysis 3 days postimplantation. Histology results for 3 weeks showed that hydrogel was degraded from outside, and capillaries reached to the center of hydrogel. In vivo analysis of cell encapsulated hydrogels are under study. Based on in vitro and in vivo results 1:3 - DAT:Fib hydrogels hold promise for adipose tissue engineering applications. 128

Dysregulation of the epigenome due to alterations in chromatin modifier proteins commonly contribute to malignant transformation. To interrogate the roles of epigenetic modifiers in cancer cells, we generated an epigenome-wide CRISPR-Cas9 knockout 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 and depletion of essential genes. We discovered novel epigenetic modifiers that regulate triple-negative breast cancer (TNBC) and prostate cancer cell fitness. We confirmed the growth-regulatory functions of individual candidates, including SS18L2 and members of the NSL complex (KANSL2, KANSL3, KAT8) in TNBC cells. Overall, we show that EPIKOL, a focused sgRNA library targeting similar to 800 genes, can reveal epigenetic modifiers that are essential for cancer cell fitness under in vitro and in vivo conditions and enable the identification of novel anti-cancer targets. Due to its comprehensive epigenome-wide targets and relatively high number of sgRNAs per gene, EPIKOL will facilitate studies examining functional roles of epigenetic modifiers in a wide range of contexts, such as screens in primary cells, patient-derived xenografts as well as in vivo models., Scientific and Technological Research Council of Turkey (TÜBİTAK); pLenti-CMVBlast-PIP-FUCCI

Soluble ACE2 (sACE2) decoys are promising agents to inhibit SARS-CoV-2, as their efficiency is unlikely to be affected by escape mutations. However, their success is limited by their relatively poor potency. To address this challenge, multimeric sACE2 consisting of SunTag or MoonTag systems is developed. These systems are extremely effective in neutralizing SARS-CoV-2 in pseudoviral systems and in clinical isolates, perform better than the dimeric or trimeric sACE2, and exhibit greater than 100-fold neutralization efficiency, compared to monomeric sACE2. SunTag or MoonTag fused to a more potent sACE2 (v1) achieves a sub-nanomolar IC50, comparable with clinical monoclonal antibodies. Pseudoviruses bearing mutations for variants of concern, including delta and omicron, are also neutralized efficiently with multimeric sACE2. Finally, therapeutic treatment of sACE2(v1)-MoonTag provides protection against SARS-CoV-2 infection in an in vivo mouse model. Therefore, highly potent multimeric sACE2 may offer a promising treatment approach against SARS-CoV-2 infections., Koç University Isbank Center for Infectious Diseases (KUISCID); Koç University Research Center for Transla-tional Medicine (KUTTAM)

Harakiri (HRK) is a BH3-only protein of the Bcl-2 family and regulates apoptosis by interfering with anti-apoptotic Bcl-2 and Bcl-xL proteins. While its function is mainly characterized in the nervous system, its role in tumors is ill-defined with few studies demonstrating HRK silencing in tumors. In this study, we investigated the role of HRK in the most aggressive primary brain tumor, glioblastoma multiforme (GBM). We showed that HRK is differentially expressed among established GBM cell lines and that HRK overexpression can induce apoptosis in GBM cells at different levels. This phenotype can be blocked by forced expression of Bcl-2 and Bcl-xL, suggesting the functional interaction of Bcl-2/ Bcl-xL and HRK in tumor cells. Moreover, HRK overexpression cooperates with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a known tumor-specific pro-apoptotic agent. Besides, secondary agents that augment TRAIL response, such as the histone deacetylase inhibitor MS-275, significantly increases HRK expression. In addition, GBM cell response to TRAIL and MS-275 can be partly abolished by HRK silencing. Finally, we showed that HRK induction suppresses tumor growth in orthotopic GBM models in vivo, leading to increased survival. Taken together, our results suggest that HRK expression is associated with GBM cell apoptosis and increasing HRK activity in GBM tumors might offer new therapeutic approaches., Scientific and Technological Research Council of Turkey (TÜBİTAK); Marie Curie FP7 Career Reintegration Grant; European Union (European Union); H2020; Unesco L'oreal Women in Science Grant; BAGEP

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)

Glioblastoma Multiforme (GBM) is the most common and aggressive primary brain tumor. Despite recent developments in surgery, chemo- and radio-therapy, a currently poor prognosis of GBM patients highlights an urgent need for novel treatment strategies. TRAIL (TNF Related Apoptosis Inducing Ligand) is a potent anti-cancer agent that can induce apoptosis selectively in cancer cells. GBM cells frequently develop resistance to TRAIL which renders clinical application of TRAIL therapeutics inefficient. In this study, we undertook a chemical screening approach using a library of epigenetic modifier drugs to identify compounds that could augment TRAIL response. We identified the fungal metabolite chaetocin, an inhibitor of histone methyl transferase SUV39H1, as a novel TRAIL sensitizer. Combining low subtoxic doses of chaetocin and TRAIL resulted in very potent and rapid apoptosis of GBM cells. Chaetocin also effectively sensitized GBM cells to further pro-apoptotic agents, such as FasL and BH3 mimetics. Chaetocin mediated apoptosis sensitization was achieved through ROS generation and consequent DNA damage induction that involved P53 activity. Chaetocin induced transcriptomic changes showed induction of antioxidant defense mechanisms and DNA damage response pathways. Heme Oxygenase 1 (HMOX1) was among the top upregulated genes, whose induction was ROS-dependent and HMOX1 depletion enhanced chaetocin mediated TRAIL sensitization. Finally, chaetocin and TRAIL combination treatment revealed efficacy in vivo. Taken together, our results provide a novel role for chaetocin as an apoptosis priming agent and its combination with pro-apoptotic therapies might offer new therapeutic approaches for GBMs., Scientific and Technological Research Council of Turkey (TÜBİTAK); European Union (European Union); Horizon 2020; Marie Curie FP7 Career Reintegration Grant; People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme; Innovative Medicines Initiative (EU/EFPIA) [ULTRA-DD]; Wellcome; Koç University Center for Translational Medicine (KUTTAM); Cancer Research UK; National Institute for Health Research (NIHR); AbbVie; Bayer Pharma AG; Boehringer Ingelheim; Canada Foundation for Innovation; Eshelman Institute for Innovation; Genome Canada; Johnson & Johnson USA, Janssen Biotech Inc.; Merck KGaA Darmstadt Germany; MSD; Novartis Pharma AG; Ontario Ministry of Economic Development and Innovation; Pfizer; Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP); Takeda Pharmaceutical Company Ltd.