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Acquired resistance to anti-cancer therapy is common and is a major clinical issue. Functional DNA repair pathways provide a common mechanism for drug resistance, but it can also result from mutations or reduced expression of the targeted protein. The overexpression or nuclear localisation of the multifunctional Y-box binding protein (YB-1) is considered as a prognostic marker for drug resistance in tumours. YB-1 has several interaction partners in cells; in this study, we have focused on its interaction with the human DNA repair enzyme NTH1 (hNTH1) and human DNA topoisomerase I (hTopoI), two enzymes that have been shown to be stimulated by YB-1. The abundance of the hNTH1/YB-1 complex was shown to increase in cisplatin-resistant tumour cells. Human TopoI is an essential enzyme involved in cellular regulation of DNA supercoiling and is the target of several anti-cancer agents. YB-1 enhances the activity of hTopoI and its sensitivity to hTopoI inhibitor, camptothecin in tumour cells. We have characterised the YB-1/hNTH1 and YB-1/hTopoI complexes in vitro and in vivo using Fluorescence Resonance Energy Transfer (FRET) measurements to identify and develop new strategies for the treatment of drug-resistant tumours. We also designed and optimised an original FRET-based biosensor to screen two medium-sized chemical libraries in order to find potential inhibitors of the hNTH1/YB-1 complex. Several “hits” were identified that significantly reduced the FRET level of our biosensor. For some of these compounds, we have reproduced these results starting from powders, have performed dose-response curves and have validated their actions as inhibitors of the hNTH1/YB-1 interface using alternative binding assays. Taken together, our results demonstrate that YB-1 directly interacts and stimulates a DNA repair and a DNA relaxing enzyme and targeting the YB-1/hNTH1 interface represents an interesting new strategy for the development of anti-cancer drugs.; La résistance acquise aux traitements anticancéreux représente un problème clinique majeur. Les voies de réparation de l'ADN fournissent un mécanisme de résistance, mais celle-ci peut aussi résulter de mutations ou d'une expression réduite de la protéine ciblée. La surexpression ou la localisation nucléaire de la Y-Box binding (YB-1) protéine est considérée comme un marqueur pronostique de chimiorésistance de la tumeur. YB-1 interagit avec plusieurs partenaires ; dans cette étude, nous nous sommes concentrés sur son interaction avec l'enzyme de réparation de l'ADN NTH1 (hNTH1) et l'ADN topoisomérase I (hTopoI), deux enzymes stimulées par YB-1. L'abondance du complexe hNTH1/YB-1 est accrue dans les cellules tumorales résistantes au cisplatine. La TopoI humaine est une enzyme essentielle impliquée dans la régulation cellulaire du surenroulement de l'ADN et est la cible de plusieurs agents anticancéreux. YB-1 augmente la sensibilité à l'inhibiteur de TopoI, la camptothécine, dans les tumeurs. Nous avons caractérisé les complexes YB-1/hNTH1 et YB-1/hTopoI in vitro et in vivo en utilisant des mesures de transfert d'énergie par résonance en fluorescence (ou FRET) pour identifier et développer de nouvelles stratégies pour le traitement de tumeurs chimio-résistantes. Nous avons développé et optimisé un biosenseur original basé sur le FRET pour cribler deux chimiothèques de taille moyenne afin d’identifier des inhibiteurs potentiels du complexe hNTH1/YB-1. Plusieurs «hits» ont été identifiés qui réduisent de façon significative le niveau de FRET de notre biosenseur. Pour certains de ces composés, nous avons reproduit ces résultats à partir de poudres, effectué des courbes dose-réponse et validé leurs actions en tant qu'inhibiteurs de l'interface hNTH1/YB-1 en utilisant d'autres tests d’interactions. Ensemble nos résultats démontrent que YB-1 interagit directement et stimule des enzymes de la réparation de l'ADN et du relaxation de l’ADN, et que cibler l’interface YB-1/hNTH1 représente une nouvelle stratégie intéressante pour le développement de traitements anticancéreux.

Acquired resistance to anti-cancer therapy is common and is a major clinical issue. Functional DNA repair pathways provide a common mechanism for drug resistance, but it can also result from mutations or reduced expression of the targeted protein. The overexpression or nuclear localisation of the multifunctional Y-box binding protein (YB-1) is considered as a prognostic marker for drug resistance in tumours. YB-1 has several interaction partners in cells; in this study, we have focused on its interaction with the human DNA repair enzyme NTH1 (hNTH1) and human DNA topoisomerase I (hTopoI), two enzymes that have been shown to be stimulated by YB-1. The abundance of the hNTH1/YB-1 complex was shown to increase in cisplatin-resistant tumour cells. Human TopoI is an essential enzyme involved in cellular regulation of DNA supercoiling and is the target of several anti-cancer agents. YB-1 enhances the activity of hTopoI and its sensitivity to hTopoI inhibitor, camptothecin in tumour cells. We have characterised the YB-1/hNTH1 and YB-1/hTopoI complexes in vitro and in vivo using Fluorescence Resonance Energy Transfer (FRET) measurements to identify and develop new strategies for the treatment of drug-resistant tumours. We also designed and optimised an original FRET-based biosensor to screen two medium-sized chemical libraries in order to find potential inhibitors of the hNTH1/YB-1 complex. Several "hits" were identified that significantly reduced the FRET level of our biosensor. For some of these compounds, we have reproduced these results starting from powders, have performed dose-response curves and have validated their actions as inhibitors of the hNTH1/YB-1 interface using alternative binding assays. Taken together, our results demonstrate that YB-1 directly interacts and stimulates a DNA repair and a DNA relaxing enzyme and targeting the YB-1/hNTH1 interface represents an interesting new strategy for the development of anti-cancer drugs., Anti-kanser tedavisine karşı kazanılan direnç yaygın ve önemli bir klinik sorundur. Fonksiyonel DNA onarım yolları, ilaç direnci için yaygın bir mekanizma sağlamaktadır, ancak aynı zamanda, hedeflenen proteinin mutasyonlarından veya ekspresyonunun azalmasından da kaynaklanabilmektedir. Çok işlevli Y-box bağlanma proteinin (YB-1) aşırı ekspresyonu veya nükleer lokalizasyonu, tümörlerde ilaç direnci için bir prognostik belirteç olarak kabul edilmektedir. YB-1'in hücrelerde birkaç etkileşim partneri vardır; bu çalışmada, YB-1 ile aktiviteleri uyarıldığı gösterilen insan DNA onarım enzimi NTH1 (hNTH1) ve insan DNA topoizomeraz I (hTopoI) ile olan etkileşimleri üzerine odaklandık. Cisplatin-dirençli tümör hücrelerinde hNTH1/YB-1 kompleks oluşumunun arttığı gösterilmiştir. İnsan TopoI, DNA süpersarmallarının hücresel regülasyonunda yer alan ve birkaç anti-kanser ajanının hedefi olan önemli bir enzimdir. YB-1, hTopoI aktivitesini ve tümör hücrelerinin hTopoI inhibitörü Campthotecin'e olan duyarlılığını artırmaktadır. Bu çalışmada YB-1/hNTH1 ve YB-1/hTopoI komplekslerini in vitro ve in vivo olarak, ilaca dirençli tümörlerin tedavisi için yeni stratejiler tanımlamak ve geliştirmek için Fluoresans Rezonans Enerji Transferi (FRET) ölçümlerini kullanarak karakterize ettik. Ayrıca, hNTH1/YB-1 kompleksinin potansiyel inhibitörlerini bulmak için iki orta ölçekli kimyasal kütüphaneyi taramak üzere orijinal bir FRET-bazlı biyosensör tasarladık ve optimize ettik. Biyosensörümüzün FRET seviyesini önemli ölçüde azaltan birkaç "hit" tespit ettik. Bu bileşiklerin bazıları için, toz bileşiklerden başlayarak bu sonuçları tekrar ürettik, doz-cevap eğrileri gerçekleştirdik ve alternatif bağlama deneyleri kullanarak hNTH1/YB-1 arayüzünün inhibitörleri olarak onayladık. Birlikte ele alındığında, sonuçlarımız YB-1'in DNA onarımı ve DNA relakse edici enzimleriyle doğrudan etkileşime girip uyardığını ve YB-1/hNTH1 arayüzünü hedef almanın, anti-kanser ilaçlarının geliştirilmesi için ilginç yeni bir stratejiyi temsil ettiğini göstermektedir.