Dr Marie-Emilie Terret

Dennis Gomez D'

Dr Katia Cailliau

Dr Jean-François Bodart

Dr Matthieu Marin

Dr Ingrid Fliniaux

Yoshiko Uchimura

Guillaume Marchand

Dr Tony Lefebvre

Anne-Sophie Edouart

Dr Jérôme De Ruyck

Dr Julie Bouckaert

Keywords: Indenoisoquinoline, copper (II) complex, adenocarcinoma, topoisomerase s, DNA Damage Response (DDR), cell cycle, autophagy

REMERCIEMENTS

Mes remerciements vont aux Pr. Christophe D'Hulst et Dr. Yann Guerardel, précédent et actuel Directeurs de l'UGSF, pour avoir accepté mon inscription lors du M2 ainsi que de la thèse.

Je remercie Dr

Topoisomerases are the targets for inhibitors used in chemotherapy that induce DNA breaks accumulation leading to cancer cell death. A new copper (II) indenoisoquinoline complex was synthetized (WN197), and its activity and action mechanisms on cancer cells were determined. WN197 is cytotoxic below 0.5 µM, on MDA-MB-231 (triple-negative breast cancer), HeLa (cervical cancer), and HT-29 cells (colon cancer). At low doses, WN197 inhibits topoisomerase I. At higher doses, it inhibits topoisomerase IIα and IIβ and displays DNA intercalation properties. DNA damage, detected by the presence of γH2AX, is present in the three adenocarcinoma cell lines. The activation of the DNA Damage Response (DDR) occurs through the phosphorylation of ATM/ATR, Chk1/2 kinases, and an increase in p21 quantity, a p53 target. WN197 induces a G2 phase arrest characterized by the unphosphorylated form of histone H3, the accumulation of phosphorylated Cdk1, and an association of Cdc25C with 14.3.3. Cancer cells die by autophagy with Beclin-1 accumulation, LC3-II formation, p62 degradation, and RAPTOR phosphorylation in the mTOR complex. WN197 keeps its cytotoxic effect on multi-drug resistant cells, H69AR, with an IC50 below 0.7 µM, displays a higher IC50 on non-tumorigenic breast cells above 1 µM, and reveals no toxicity on Xenopus embryonic development. Finally, WN197, by inhibiting topoisomerase I at low concentration with high efficiency, is a promising agent for the development of future DNA damaging chemotherapies. Mots-clés : Indénoisoquinoline, complexe cuivre (II), adénocarcinome, topoisomérase s, DNA Damage Response (DDR), cycle cellulaire, apoptose, autophagie.

Les topoisomérases sont les cibles d'inhibiteurs en chimiothérapie induisant une accumulation de dommages à l'ADN, conduisant à la mort cellulaire. Un nouveau composé d'indénoisoquinoline cuivré a été synthétisé (WN197). Ses mécanismes d'action ont été déterminés sur trois lignées d'adénocarcinomes humains. WN197 est cytotoxique à des concentrations inférieures à 0,5 µM sur les lignées cancéreuses MDA-MB-231 (sein), HeLa (col de l'utérus) et . À ces doses, WN197 inhibe les topoisomérases I. À des doses plus élevées (> 1 µM), il inhibe les topoisomérases IIα et IIβ et possède des propriétés intercalantes. Les dommages à l'ADN sont détectés par la présence de γH2AX, et La phosphorylation d'ATM/ATR et des kinases Chk1/Chk2 et l'augmentation des quantités de p53 et p21 démontrent l'activation du DDR dans les trois lignées. WN197 provoque un arrêt des cellules en phase G2 caractérisé par le maintien de Cdk1 inactif dû à l'association de Cdc25C à 14.3.3, et par la forme non-phosphorylée de l'histone H3. Les cellules cancéreuses meurent par autophagie avec une accumulation de Beclin-1, la formation de LC3-II, la dégradation de p62 et la phosphorylation de RAPTOR dans le complexe mTOR. WN197 s'avère cytotoxique sur des cellules H69AR « multi-drug resistant », avec un IC50 inférieur à 0,7 µM, et possède un IC50 supérieur à 1 µM sur une lignée du sein non-tumorigénique, et ne se révèle pas toxique pour le développement embryonnaire du Xénope. En inhibant les topoisomérases I à faible concentration avec une forte efficacité, WN197 est un agent prometteur pour le développement de futures chimiothérapies ciblant l'ADN.

Parmi les inhibiteurs de topoisomérases I, le topotecan et l'irinotecan sont utilisés dans le traitement des cancers ovariens, de l'utérus et du côlon. La doxorubicine est un inhibiteur de topoisomérase II prescrits contre divers carcinomes, sarcomes et lymphomes. Néanmoins, les effets secondaires de ces molécules sont élevés (toxicité hématologique, vomissement, diarrhées, leucémies associées à la thérapie, cardiotoxicité), et elles provoquent des phénomènes de résistances des cellules cancéreuses, soit par augmentation de la production de transporteurs d'efflux (PGP, MRP, ABCG2…), soit par mutation des topoisomérases P a g e | 8 [START_REF] Pommier | DNA topoisomerase I inhibitors: chemistry, biology, and interfacial inhibition[END_REF]. Limiter au maximum les effets secondaires et contourner les phénomènes de résistances nécessitent la recherche permanente de nouvelles molécules toujours plus efficaces pour traiter ces cancers.

CONTEXTE DE RECHERCHE

Quatorze organométalliques dérivés de l'indénoisoquinoline (inhibiteurs de topoisomérase I) ont été développés par l'équipe du Pr. Pelinski (Unité de Catalyse et Chimie du Solide -Université de Lille). Leur toxicité a été testée dans notre laboratoire sur 5 lignées cellulaires cancéreuses via des tests MTS. La molécule appelée WN197 possédant les IC50 les plus faibles est une indénoisoquinoline à laquelle a été ajoutée une chaine latérale complexée à un atome de cuivre. Elle agit avec un IC50 de 0,144 µM sur la lignée MDA-MB-231 (cancer du sein triple négatif), de 0,644 µM sur la lignée MCF-7 (cancer du sein ER + , PR + , HER -), de 0,22 µM sur la lignée HeLa (cancer du col de l'utérus), de 0,358 µM sur la lignée HT-29 (cancer du côlon), et de 0,162 µM sur la lignée DU-145 (cancer de la prostate). Le ligand sans molécule de cuivre, WN170, est utilisé comme contrôle pour déterminer l'apport du cuivre au complexe.

Dans ma thèse, trois adénocarcinomes ont été choisis en raison de leur taux d'incidence très élevé : le cancer du col de l'utérus (lignée HeLa), le cancer du sein triple négatif (MDA-MB-231) et le cancer du côlon (HT-29). Le projet de thèse vise à comprendre les mécanismes d'action de WN197 sur ces trois lignées, avec successivement la détermination précise de l'induction des dommages à l'ADN et du type d'inhibition sur les topoisomérases (type I et/ou II ; type poison ou catalytique), des impacts sur le cycle cellulaire et les acteurs moléculaires ciblés, du type de mort cellulaire activée, des potentielles résistances associées et de la toxicité sur des lignées cellulaires non cancéreuses ainsi que sur le développement embryonnaire du Xénope.

RESULTATS I. Endommagement de l'ADN des lignées d'adénocarcinomes par le composé WN197

Les dommages à l'ADN induits par le composé WN197 ont été mis en évidence de manière indirecte par la présence du marqueur γH2AX en immunofluorescence et Western blot [START_REF] Kinner | Gamma-H2AX in recognition and signaling of DNA double-strand breaks in the context of chromatin[END_REF]. Les cellules témoins (DMSO, 0,5%) ne présentent pas ou peu de marquage indiquant une absence de cassures de l'ADN. La doxorubicine, un inhibiteur des topoisomérases II utilisé comme témoin des cassures de l'ADN, provoque une importante formation de γH2AX nucléaire, reflet de nombreuses cassures. Après traitement avec WN197 (0,5 µM) 30 min ou 24 h, de nombreux foci (accumulations de marquage) sont retrouvés,

III. Activation de la voie DDR des lignées d'adénocarcinomes

La voie DDR (DNA Damage Response) est une signalisation intracellulaire activée naturellement lorsque l'ADN subit des dommages pour dans un premier temps arrêter les cellules dans le cycle, ou induire la mort cellulaire si les dommages sont trop importants [START_REF] Hakem | DNA-damage repair; the good, the bad, and the ugly[END_REF]. Des effecteurs de cette voie ont été analysés par Western blot : ATR et ATM sont les premières kinases activées après cassures simple ou double brin(s) respectivement. Elles activent les kinases Chk1 et Chk2, elles-mêmes suivies de p53 et p21. La phosphorylation activatrice d'ATR (S428) a été observée sur les 3 lignées d'adénocarcinomes. Une nette augmentation de l'activation d'ATR est constatée pour les cellules traitées avec WN197 comparées aux cellules non traitées. La même augmentation se retrouve pour la phosphorylation activatrice d'ATM (S1981). Les deux kinases suivantes de la signalisation, Chk1 et Chk2, sont également activées (S317 et T68 respectivement), ainsi que la phosphorylation de p53 (S15) et la quantité de p21, confirmant que la voie DDR est activée.

IV. Effet de WN197 sur le cycle cellulaire des lignées d'adénocarcinomes

La répartition des cellules MDA-MB-231, HeLa et HT-29 dans le cycle cellulaire a été analysée par cytométrie en flux. Les cellules non traitées montrent une répartition classique de la population cellulaire dans le cycle. Après 24 h de traitement avec WN197 à 0,5 µM, en moyenne, une accumulation de 70,51% des cellules en phase G2/M est observée.

P a g e | 10

Pour les doses-réponses, les cellules ont été exposées à des doses croissantes de WN197 pendant 24 h puis analysées par cytométrie en flux. Une accumulation significative des cellules en phase G2/M est retrouvée à partir de 0,5 µM pour les MDA-MB-231, et pour les HeLa et HT-29 à 0,25 µM, mais avec une baisse d'accumulation à 1 µM. Ceci indiquerait un mode d'action différent du composé à de plus fortes doses comme observé avec les tests topoisomérases in vitro. Pour les cinétiques, les cellules ont été exposées à 0,5 µM de WN197 de 4 h à 48 h. Une accumulation significative des cellules en G2/M est observée dès 8 h pour les MDA-MB-231, 12 h pour les HT-29, et un peu plus tardivement pour les HeLa à partir de 16 h. Une diminution de l'accumulation est observée après 48 h.

La cytométrie ne permettant pas de distinguer si les cellules sont bloquées en phase G2 ou M, l'arrêt en phase G2 du cycle cellulaire a été précisé sur les trois lignées par une analyse en Western blot de l'état de phosphorylation et/ou de la quantité de certains acteurs majeurs de la régulation de la transition G2/M : la kinase Cdk1, la cycline B, la phosphatase Cdc25C et l'histone H3. La kinase Cdk1 couplée à la cycline B forme un complexe appelé MPF. Lors de la transition G2/M, Cdk1 est activée par les déphosphorylations de ses résidus thréonine 14 et tyrosine 15 (phosphorylations inhibitrices) réalisées par Cdc25C, et par une phosphorylation activatrice de la thréonine 161 par la CAK [START_REF] Hakem | DNA-damage repair; the good, the bad, and the ugly[END_REF]. Dans cet état, le MPF actif permet le passage des cellules de la phase G2 à la phase M du cycle cellulaire. La phosphorylation du résidu tyrosine 15 de Cdk1 augmente après traitement avec WN197 comparé au témoin sans traitement, ainsi que la quantité de cycline B, suggérant un défaut d'activation du MPF. Ce dernier serait la conséquence de l'inactivation de Cdc25C dans cette condition puisque les quantités de Cdc25C et de sa forme inactive phosphorylée sur le résidu sérine 216 (phosphorylation inhibitrice) en présence de WN197 étaient plus élevées que pour le témoin. Enfin, l'histone H3 phosphorylée sur la sérine 10, marqueur d'entrée en phase M du cycle cellulaire, n'est pas retrouvée après traitement avec WN197. Ces résultats démontrent que WN197 provoque un arrêt des cellules en phase G2 du cycle cellulaire plutôt qu'en phase M, en induisant une accumulation de Cdk1 inactive phosphorylée sur la tyrosine 15. Ce défaut d'activation de Cdk1 est accompagné d'une accumulation de cycline B et de l'absence de phosphorylation de l'histone H3 caractéristiques de la phase G2.

Quand Cdc25C est inactivée par phosphorylation sur la S216, 14-3-3 reconnait et s'associe à la phosphatase, puis l'exporte hors du noyau. Pour confirmer les résultats précédents liant WN197 et l'inactivation de Cdc25C, des immunoprécipitations (IP) de Cdc25C ou 14-3-3 ont été réalisées sur les trois lignées. Sans traitement, les deux molécules ne sont pas retrouvées couplées (absence de Cdc25C après IP de 14-3-3, et absence de 14-3-3 après IP de Cdc25C). En revanche, après traitement avec le cisplatine (témoin positif) ou WN197, les deux molécules sont immunoprécipitées. Ces résultats confirment que Cdc25C est inhibée et couplée à 14-3-3 après un traitement des cellules avec WN197. P a g e | 11

V. Mort cellulaire induite par WN197 sur les lignées d'adénocarcinomes

Une des voies classiquement activées après des dommages à l'ADN est l'apoptose. Les formes clivées de la caspase 3 et de la PARP, marqueurs reconnus de ce type de mort cellulaire n'ont pas été retrouvées après un traitement avec WN197. De la même manière, le cytochrome c n'a pas été retrouvé dans le cytoplasme des cellules après traitement avec WN197, comparé au contrôle positif inducteur de l'apoptose, la doxorubicine. Les résultats ont été confirmés à l'aide de l'analyse annexine-V/iodure de propidium en cytométrie en flux. Un faible pourcentage de cellules est retrouvé en apoptose précoce et tardive avec une moyenne de 17% pour les MDA-MB-231, HeLa et HT-29. Ces résultats excluent donc une induction de l'apoptose par WN197. L'autophagie a ensuite été étudiée.

LC3, présent sous forme LC3-I dans les cellules, s'associe avec la phosphatidyléthanolamine pour former LC3-II lors du mécanisme d'autophagie (Kroemer et al., 2009). Ce changement de conformation a été analysé dans les 3 lignées d'adénocarcinomes après 24 h de traitement. Après traitement avec la rapamycine (inducteur d'autophagie) une augmentation de LC3-II est détectée, de même qu'après un traitement avec 0,5 µM de WN170 ou WN197. Un deuxième marqueur, p62, dégradé lors de l'autophagie a été observé par WB. Après traitement avec la rapamycine, une perte de p62 est constatée, tout comme pour les traitements avec WN170 et WN197. Le troisième marqueur étudié est la protéine Beclin-1, synthétisée lors de l'autophagie. Celle-ci est retrouvée de manière plus élevée pour les trois lignées cancéreuses traitées avec WN197.

Lors de l'autophagie, la protéine RAPTOR (regulatory-associated protein of mTOR) est phosphorylée sur la serine 792 dans le complexe mTOR [START_REF] Ma | mTORC1 pathway in DNA damage response[END_REF]. La présence de la forme phosphorylée de RAPTOR dans le complexe mTOR a été retrouvée par immunoprécipitation pour les trois lignées, après un traitement de 24 h avec la rapamycine ou WN197 (0,5 µM), contrairement au témoin et au contrôle négatif (doxorubicine, inducteur d'apoptose). L'activation de l'autophagie a finalement été vérifiée par un marquage fluorescent et une quantification des autophagosomes. Ces autophagosomes sont plus abondants dans les cellules traitées à la rapamycine et avec WN197 comparés au contrôle DMSO (0,5%, moyenne de 7) avec une moyenne de 186 pour la rapamycine et de 66 pour WN197 dans les lignées MDA-MB-231 et HeLa.

L'ensemble de ces résultats démontrent que WN197 induit la mort des cellules cancéreuses par autophagie. Table II: human somatic cells topoisomerases and their specificities. ……………………………………… page 38

Introduction

Table III: pattern of expression of Top2α/β in mice tissues according to [START_REF] Capranico | Different patterns of gene expression of topoisomerase II isoforms in differentiated tissues during murine development[END_REF]

……………………………………………………………………………………………………………………………………………….. page 40

Table IV: topoisomerases 1 and 2α/β tissues specificities, cell cycle fluctuation, and post-translational modifications. ………………………………………………………………………………………………………………………… page 42 Table V: main approved topoisomerases inhibitors as chemotherapy drugs.…………………………… page 48

Table VI: copper-complexes inhibitors of human topoisomerases, toxicity on cancer cell (IC50), and induced cell cycle arrests and/or cell deaths when determined (Molinaro et al., 2020). …… pages 51-53 P a g e | ) and an estimation of 28.9 million new cases in 2040. 52% of the new cancer cases affected men and 48% women. The cancers with the most incidence in men include lung (14.3% of total cancer number), prostate (14%) and colorectal cancers (10.6%). In woman, cancer incidence is the highest for breast cancer (24.5%), followed by colorectal (9.4%) lung (8.3%) and cervical (6.5%). These four cancers were also the deadliest in women (15.5%, 9.5%, 13.7% and 7.7% respectively), and lung, liver and colorectal cancers were the deadliest in men (21.5%, 10.4% and 9.3% respectively). Cancers originate from various tissues. Carcinoma cancers originate from epithelial cells, sarcomas from non-hematopoietic mesenchymal cells (bone, muscle, fat, cartilage, tendons, nerves), leukemias and lymphomas from hematopoietic cells, either from bone marrow cells that mature in the bloodstream, or that mature in the lymphatic system. Less P a g e | 22 common cancer includes blastoma, originating from precursor cells. Carcinomas are divided into various subgroups, determined by their histological aspects. Squamous cell carcinomas (SCC), also known as epidermoid carcinomas, arise from epitheliums covering both the outside of the body (skin) and the inside cavities. Adenocarcinomas (AC) arise from glandular cells and/or have glandular aspects. Mixtures of both types are called adenosquamous carcinomas (ASC). Anaplastic carcinomas are undifferentiated cells with excessive growth.

Breast cancer

Breast cancer is the most diagnosed cancer and the first cause of death by cancer among women with 58,500 new cases in France in 2018 and 12,100 deaths (with 1% of breast cancers in men) (INCa: France cancers panorama 2022). Most breast cancers arise from the gland duct (ductal carcinoma; 80%), but also from the lobules (lobular carcinoma) (Figure II) (Li et al., 2005). Carcinoma can be in situ (not spread) or invasive (spread to other tissues).

Figure II. Female breast anatomy.

Breast cancer cells can present receptors at their surface or not, classifying them hormono-dependent or -independent respectively. The three types of receptors present are the estrogen receptor (ER), the progesterone receptor (PR), and the human epidermal growth factor receptor 2 (HER2). Breast cancers can then be divided into three categories based on the receptors they express: luminal, HER2 + , or triple-negative [START_REF] Phipps | Reproductive and hormonal risk factors for postmenopausal luminal, HER-2-overexpressing, and triple-negative breast cancer[END_REF][START_REF] Waks | Breast Cancer Treatment: A Review[END_REF]. Luminal A tumor type represents cells expressing ERs and PRs but not HER2 (ER + , PR + , HER2 -), while luminal B type regroups cells expressing all three receptors (ER + , PR + , HER + ). HER2 + tumor type only expresses HER2 (ER -, PR -, HER2 + ). The last category does not express any of the receptors and is called triple-negative cancer (ER -, PR -, HER2 -). Receptorpositive cancer cells can be treated with drugs inhibiting the receptors (hormonotherapy), while triple-negative cannot, rendering them more difficult to treat. Breast cancer incidence has increased by 0.6% each year between 2010 and 2018 (in France), but the number of deaths has diminished by 1.6% per year. The net standard survival rate for 5 years, representing the P a g e | 23 people diagnosed and still alive 5 years later, is 87%. These cancers are detectable at early stages, and the widespread screening in the women's population explains the detection increase associated with the diminution of death. Yet, breast cancers aggressiveness depends on various factors including the expression of receptors (e.g. breast HER2 + cancers are highly aggressive [START_REF] Cossetti | Comparison of breast cancer recurrence and outcome patterns between patients treated from 1986 to 1992 and from 2004 to 2008[END_REF]), the lack of receptor (triple-negative breast cancers) [START_REF] Dent | Triple-negative breast cancer: clinical features and patterns of recurrence[END_REF], and the genetic aspect associated with the age of the patient [START_REF] Kuchenbaecker | Risks of Breast, Ovarian, and Contralateral Breast Cancer for BRCA1 and BRCA2 Mutation Carriers[END_REF]. Cervical cancers arise either from the squamous cells, causing a squamous cell carcinoma (SCC, about 70% of the cancer cases), or from the glandular cells, causing adenocarcinoma (AC, about 30% of the cancer cases) [START_REF] Vizcaino | International trends in incidence of cervical cancer: II. Squamous-cell carcinoma[END_REF][START_REF] Smith | The rising incidence of adenocarcinoma relative to squamous cell carcinoma of the uterine cervix in the United States--a 24-year population-based study[END_REF]. The cervical cancer incidence and the number of deaths decreased by 0.7% and 1.1% respectively every year between 2010 and 2018 (INCa: France cancers panorama 2022). The diminution can be explained by an increase of vaccination since the approval for the first vaccine in 2006 against the main infectious human papillomaviruses (HPV) (yet not all women and men eligible are vaccinated) [START_REF] Cohen | Cervical cancer[END_REF]. Moreover, pre-cancerous lesions of those cancers can be diagnosed by gynecologic screening. Nevertheless, the 5 years standardized survival rate for women diagnosed in 2010 is 63%, showing that those cancers remain difficult to treat. 1.1.1.3 Colorectal cancer With 23,200 new cases in men and 20,100 in women in 2018, colorectal cancer is the second most frequent cancer among women and the third among men in France (INCa: France P a g e | 24 cancers panorama 2022). It is also the third deadliest cancer among women and the second among men (9,200 deaths in men and 7,900 in women in France). Among colorectal cancers, an average of 60% is colon cancer, and 40% is rectum cancer. Because of the numerous glands of the colon, the majority of colon cancer are adenocarcinomas. They are either Lieberkühnian adenocarcinomas (carcinomas of the Lieberkühn glands, the most common) or mucinous adenocarcinomas (carcinomas of the epithelium tissue with abundant mucus secretion) [START_REF] Fleming | Colorectal carcinoma: Pathologic aspects[END_REF]. Colorectal cancers mainly arise from adenomatous polyps (benign tumor) [START_REF] Brissos | Possible DNA-interacting pathways for metal-based compounds exemplified with copper coordination compounds[END_REF]. A decrease of 1.4% number of cases per year is seen in men from 2010 to 2018, but no changes are reported for women. On the other side, the number of deaths has decreased for men and women, with 1.8% and 1.6% respectively. Those diminutions are explained by the increase of both immunologic tests to detect blood (hemoglobin) in feces, and high-risk factors in patients (polyps, genetics, inflammation) follow-up. However, the 5 years net survival rate after the established diagnosis is 63%, due to the high number of cancers detected at late stages and the lack of efficient treatments available.

Treatments

Several types of treatments are currently proposed to patients depending on the type and grade of cancer detected (Table I), but also the localization of the tumor, the age, and the health status given the medical and chirurgical antecedents, in addition to the side effects consequences of the treatments. The patient's choice is also considered, regarding his willingness to participate in therapeutic trials.

Surgery

Surgery of the primary tumor and surrounding tissues is the main treatment but is not sufficient to eradicate cancers that have reached the metastasis stage. Sometimes surgery can be preceded by neoadjuvant chemotherapy to diminish the size of the tumor, or after surgery with adjuvant chemotherapy to kill any remaining cancer cells and to avoid cancer recidivism [START_REF] Lambert | Neoadjuvant Treatment Strategies in Resectable Pancreatic Cancer[END_REF][START_REF] Kerr | Adjuvant and neoadjuvant breast cancer treatments: A systematic review of their effects on mortality[END_REF].

Radiotherapy

Radiotherapy is also given after surgery, to kill cancer cells that may have escaped the surgery, or before to reduce the tumor. It can also be proceeded after an adjuvant chemotherapy to complete the treatment. Radiotherapy can be realized internally, as brachytherapy (also known as curie therapy in French), or externally at determined doses (Ahmad et al., 2012;[START_REF] De Ruysscher | Radiotherapy toxicity[END_REF]. Both technics can be combined. Brachytherapy requires the implantation of the material next to the tumor [START_REF] Chargari | Brachytherapy: An overview for clinicians[END_REF]. However, the ionizing radiations can also damage normal cells, leading to adverse effects [START_REF] De Ruysscher | Radiotherapy toxicity[END_REF] Table I. Breast, cervical, and colorectal cancers and their cell types, incidences, associated deaths, risk factors, and treatments. AC: Adenocarcinoma; SCC: Squamous cell carcinoma; WW: Worldwide. References: GLOBOCAN 2020 -WHO; [START_REF] Waks | Breast Cancer Treatment: A Review[END_REF][START_REF] Cohen | Cervical cancer[END_REF][START_REF] Ciombor | Recent therapeutic advances in the treatment of colorectal cancer[END_REF][START_REF] Biller | Diagnosis and Treatment of Metastatic Colorectal Cancer: A Review[END_REF].

P a g e | 26

Immunotherapy

Emerging therapies including immunotherapy can be proposed to patients, to help their immune system to fight cancer (monoclonal antibody inhibitors are discussed in targeted therapy §1. 1.2.5.2, page 30). The first type of immunotherapy uses immune checkpoint inhibitors. Normal cells express immune checkpoint receptors on their surface, like PD-L1 or B7. These immune checkpoint receptors are recognized by specific ligands expressed on immune T cell surfaces, like PD-1 and CTLA-4 [START_REF] Waldman | A guide to cancer immunotherapy: from T cell basic science to clinical practice[END_REF]. They allow an inhibitory signal to be sent to the immune system cells to avoid their killing. Cancer cells have developed a mechanism to escape the immune system by expressing these receptors [START_REF] Seidel | Anti-PD-1 and Anti-CTLA-4 Therapies in Cancer: Mechanisms of Action, Efficacy, and Limitations[END_REF]. Immune checkpoint inhibitors target these surface receptors to inhibit the escape signal, which induces cell destruction by the immune system [START_REF] Wei | Fundamental Mechanisms of Immune Checkpoint Blockade Therapy[END_REF] (Figure IV). Actual treatments are composed of anti-PD-L1 (ex: atezolizumab), anti-PD-1 (ex: nivolumab), anti-CTLA-4 (ipilimumab), and anti-LAG-3 (relatlimab, in combination with nivolumab) antibodies [START_REF] Hodi | Improved survival with ipilimumab in patients with metastatic melanoma[END_REF][START_REF] Seidel | Anti-PD-1 and Anti-CTLA-4 Therapies in Cancer: Mechanisms of Action, Efficacy, and Limitations[END_REF][START_REF] Wei | Fundamental Mechanisms of Immune Checkpoint Blockade Therapy[END_REF][START_REF] Paik | Nivolumab Plus Relatlimab: First Approval[END_REF]. Nonetheless, immune checkpoint inhibitors do not only target cancer cells, but also normal cells and are associated with auto-immune and infusion (allergic) reactions [START_REF] Seidel | Anti-PD-1 and Anti-CTLA-4 Therapies in Cancer: Mechanisms of Action, Efficacy, and Limitations[END_REF][START_REF] Marin-Acevedo | Immune Checkpoint Inhibitor Toxicities[END_REF].

The T-cell transfer therapy consists in the extraction and collection of patient T-cells (leukapheresis), called TIL (tumor-infiltrating lymphocytes). The most effective TIL against the patient tumor can be either collected and multiplied before a transfer into the patient's vain, called TIL therapy, or modified with the CAR (chimeric antigen receptor) gene (through a retrovirus) to produce the associated protein, called CAR T-cell therapy [START_REF] Wang | Current advances in T-cell-based cancer immunotherapy[END_REF][START_REF] Houot | T-cell-based Immunotherapy: Adoptive Cell Transfer and Checkpoint Inhibition[END_REF] (Figure IV). CARs recognize specific antigens on the cancer cell surface to improve the effectiveness of T-cells against the tumorigenic cells. The first CAR-T drug FDA approved was tisagenlecleucel in 2017 [START_REF] O'leary | FDA Approval Summary: Tisagenlecleucel for Treatment of Patients with Relapsed or Refractory B-cell Precursor Acute Lymphoblastic Leukemia[END_REF], but the CAR-T treatments are rarely used or in clinical trials [START_REF] Wang | Current advances in T-cell-based cancer immunotherapy[END_REF][START_REF] Houot | T-cell-based Immunotherapy: Adoptive Cell Transfer and Checkpoint Inhibition[END_REF]. Serious side effects can be induced by these treatments, such as cytokine release syndrome (fever, nausea, headache, rash, tachycardia, hypotension, dyspnea), and several deaths happened during clinical trials [START_REF] Wang | Current advances in T-cell-based cancer immunotherapy[END_REF]. CAR-NK (Natural Killer) could be an alternative to CAR-T. They are highly cytotoxic by favoring antibody-dependent cell-mediated cytotoxicity and by activation and persistence of NK cells via IL-15 cytokine (Nguyen et al., 2021). At the moment, CAR-NK therapy is only at the pre-clinical stage or recruiting for phase 1.

Therapeutic vaccination with the use of dendritic cells is another approach to immunotherapy. Dendritic cells are a type of antigen-presenting cells in the immune system. For immunotherapy, dendritic cells of the patient are extracted and incubated with antigens expressed at the tumor cells' surface before reinfusion into the patient [START_REF] Hammerstrom | Cancer immunotherapy: sipuleucel-T and beyond[END_REF]. The dendritic cells will present the antigens to the immune system and activate an Cytokine immunotherapy can also be used to stimulate the immune system against cancer cells (Figure IV). Cytokine can be directly infused into the body with IL-α or IL-2, or increased via the use of drugs that allow cytokine production. These drugs cause a release of interleukin 2 (IL-2) that increases the activity of the immune T-cells and natural killer, and also works as an anti-angiogenesis factor [START_REF] Berraondo | Cytokines in clinical cancer immunotherapy[END_REF]. Imiquimod acts by activating TLR7 that releases cytokines and is used against basal cell carcinoma [START_REF] Testerman | Cytokine induction by the immunomodulators imiquimod and S-27609[END_REF]. Flu-like symptoms are also related to these treatments, as long with serious side effects associated with the release of a large number of cytokines (severe allergic reactions, dyspnea, hypo and hypertension, blood clots, and organ damage). Cytokine release is also linked to troubles in behavior, and depression [START_REF] Pollak | Cytokine-induced changes in mood and behaviour: implications for 'depression due to a general medical condition', immunotherapy and antidepressive treatment[END_REF][START_REF] Kovacs | Psychological side effects of immune therapies: symptoms and pathomechanism[END_REF].

Hormonotherapy

Hormonotherapy is used against various hormono-dependent cancers (breast, prostate principally, but also endometrium, thyroid, neuroendocrine cancers), [START_REF] Van Weelden | Anti-estrogen Treatment in Endometrial Cancer: A Systematic Review[END_REF][START_REF] Grani | Thyroid hormone therapy in differentiated thyroid cancer[END_REF]. The principle is to inhibit the stimulating action of female hormones (progesterone or estrogens) or male hormones (testosterone). The non-drug treatments stop hormone production by the body, using chirurgical approaches such as ovariectomy (ovarian ablation), pulpectomy (the external membrane of testicular is conserved), orchiectomy (testicular ablation), or radiotherapy. The systemic treatments use drugs to inhibit the hormonal receptor expressed at the cancer cell surfaces, damage these receptors, or inhibit hormonal production (Figure V). For breast cancers, the most used hormonotherapy is the anti-estrogens treatment with tamoxifen [START_REF] Waks | Breast Cancer Treatment: A Review[END_REF]. It acts as a competitive ligand for the estrogen receptors, and belongs to the drug class of SERM (Selective Estrogen Receptor Modulator). The other category of anti-estrogen is classified into SERD (Selective Estrogen Receptor Degradation), and includes fulvestrant that induces the degradation of the receptor [START_REF] Iorfida | Fulvestrant in Combination with CDK4/6 Inhibitors for HER2-Metastatic Breast Cancers: Current Perspectives[END_REF]. LHRH (Luteinizing Hormone Releasing Hormone) analogues can also be used to inhibit the P a g e | 29 hormone production by the ovaries. LHRH, produced by the hypothalamus, is responsible for the stimulation of the hypophysis (/pituitary gland) that produces LH (luteinizing hormone) that consequently stimulates estrogens secretion by the ovaries. The hypophysis is hyperstimulated by LHRH analogues, resulting in an arrest of ovary stimulation [START_REF] Schally | Mode of Action of LHRH Analogs[END_REF]. LHRH analogues and antagonists are goserelin and leuprorelin. Finally, anti-aromatases are prescribed to menopaused women. They compete with aromatase, the enzyme responsible for the transformation of androgen (produced by the adrenal gland) into estrogens. Anti-aromatases used in hormonotherapy are letrozole and anastrazol [START_REF] Waks | Breast Cancer Treatment: A Review[END_REF]. Hormonotherapy lasts for three to five years and can be linked to recidivism after the arrest of the treatment [START_REF] Haque | Pathways to Endocrine Therapy Resistance in Breast Cancer[END_REF]. The main side effects of hormonotherapy are the menopause effects and a diminution in mineral bone density (Narayan et al., 2021).

Anti-androgens act by fixation to the testosterone receptors. Flutamide, bicalutamide, nilutamide and cyproterone acetate are mostly used [START_REF] Pronzato | Hormonotherapy of advanced prostate cancer[END_REF]. They are generally used before LHRH antagonists'/analogues' treatments. The most common LHRH analogues are leuprorelin, goserelin, buserelin, triptorelin and histrelin [START_REF] Shim | Effectiveness of three different luteinizing hormone-releasing hormone agonists in the chemical castration of patients with prostate cancer: Goserelin versus triptorelin versus leuprolide[END_REF]. The antagonist used is degarelix. The most common side effects are osteoporosis, gynecomastia, libido and mood troubles, and heat flashes [START_REF] Tucci | Hormonal treatment and quality of life of prostate cancer patients: new evidence[END_REF].

Chemotherapy

Chemotherapies are proceeded with diverse cytotoxic molecules, targeting a variety of cellular effectors: the mitotic spindle, extracellular receptors, cell cycle kinases, and/or DNA and its metabolism.

Mitotic spindle chemotherapy

The mitotic spindle is targeted by two main groups of drugs: alkaloids and taxanes, mainly originating from plants. Alkaloids prevent the assembly of the cell microtubules while the taxanes their disassembly, resulting in a cell cycle arrest, and cell death (Ballout et al., 2019). The first alkaloids used in chemotherapy were vincristine and vinblastine, extracted from the Catharanthus roseus (Madagascar periwinkle). Derivatives of these compounds were produced including vinorelbine, vindesine, and vinflunin. Original and derivative compounds are used to treat various carcinomas, sarcomas, lymphomas, and myelomas. Regarding taxanes, the first extracted drug was paclitaxel, from Taxus brevifolia, approved in 1993 (Ballout et al., 2019). Paclitaxel and its analogue docetaxel are used in chemotherapy alone or combined with other compounds mainly against carcinomas. The main side effects of these treatments are myelosuppression, and cardiovascular and gastrointestinal toxicities. Neurotoxicity can also occur as a chronic persistent side effect [START_REF] Ho | Presentation and management of docetaxel-related adverse effects in patients with breast cancer[END_REF]. P a g e | 30 1.1.2.5.2 Targeted therapy Another type of emerging therapy is targeted therapy. Targeted therapy can either be the use of small molecules or monoclonal antibodies acting at different cellular levels: on growth factors, cell receptors, or regulatory enzymes (Figure VI). The drugs can either inhibit cancer cells growth and division, inhibit cancer cell angiogenesis, help the immune system to attack cancer cells, or induce cancer cell apoptosis. The main targets are VEGFR, EGFR, tyrosine kinase enzymes, and proteasome, but many other cell pathways can be targeted [START_REF] Ma | Novel agents on the horizon for cancer therapy[END_REF]Tsimberidou, 2015;[START_REF] Zhong | Small molecules in targeted cancer therapy: advances, challenges, and future perspectives[END_REF]. In some chemotherapies, drugs are carried to cancer cells using nanocarriers like liposomes, polymeric nanoparticles, or antibodies [START_REF] Beltrán-Gracia | Nanomedicine review: clinical developments in liposomal applications[END_REF]. Cancer cell growth can be targeted by tyrosine-kinase inhibitors. Tyrosine kinases encompass a multitude of proteins belonging to several signal transduction pathways [START_REF] Pottier | Tyrosine Kinase Inhibitors in Cancer: Breakthrough and Challenges of Targeted Therapy[END_REF]. Imatinib (2001 approval) is an inhibitor of the Bcr-Abl oncoprotein (fusion protein from a translocation between chromosome 9 and 22, known as Philadelphia gene) targeting the tyrosine kinase domain by a competitive binding [START_REF] Rossari | Past, present, and future of Bcr-Abl inhibitors: from chemical development to clinical efficacy[END_REF]. As a consequence, the cell substrate cannot enter the kinase site for phosphorylation, resulting in an inactivation of the cancer proliferation signal. Imatinib also inhibits PDGFR (platelet-derived growth factor receptor), and c-kit (oncogene encoding for tyrosine-protein kinase KIT). The Bcr-Abl oncoprotein characterizes the chronic myeloid leukemia and is also found in various P a g e | 31 percentage in other leukemias [START_REF] Jabbour | Chronic myeloid leukemia: 2018 update on diagnosis, therapy and monitoring[END_REF]. Imatinib is prescribed against these cancers, while dasatinib, nilotinib, bosutinib, and ponatinib are used in first-or secondline treatment depending on the patient's mutations and responsiveness to the previous treatment.

Another target is the EGFR (epidermal growth factor receptor) overexpressed in a number of carcinomas [START_REF] Yarden | The ERBB network: at last, cancer therapy meets systems biology[END_REF]. The EGFR signaling pathway activation results in cell growth, proliferation, invasion, metastasis, and angiogenesis (Cruijsen et al., 2006;[START_REF] Sigismund | Emerging functions of the EGFR in cancer[END_REF]. EGFR inhibitors are both small molecules and monoclonal antibodies. Cetuximab is a chimeric monoclonal antibody approved by the FDA in 2009 against colon cancer [START_REF] García-Foncillas | Distinguishing Features of Cetuximab and Panitumumab in Colorectal Cancer and Other Solid Tumors[END_REF]. It is also administered to treat head and neck cancers. Other monoclonal antibodies are panitumumab and necitumumab. Small molecules inhibiting EGFR, like erlotinib (approval 2004), afatinib, and osimertinib are prescribed against metastatic NSCLC (non-small cell lung cancer), depending on the cancer cell mutations [START_REF] Tagliamento | Afatinib and Erlotinib in the treatment of squamous-cell lung cancer[END_REF][START_REF] Wu | Osimertinib in Resected EGFR-Mutated Non-Small-Cell Lung Cancer[END_REF]. EGFR2/HER2 (Human Epidermal growth factor Receptor 2) is involved in the regulation, proliferation, and survival of epidermal cells. HER2 is a known amplificated and overexpressed oncogene in 15-20% of breast cancers associated with a poor prognosis. HER2 inhibitors pertuzumab, trastuzumab, and lapatinib are approved for the treatment of advanced or metastatic breast cancers associated with HER2 overexpression [START_REF] Waks | Breast Cancer Treatment: A Review[END_REF]. Some agents do not target receptors but proteins from their signaling pathways. In the Ras/Raf pathway, BRAF is a target of vemurafenib and dabrafenib approved by the FDA for the treatment of metastatic melanoma characterized by the BRAF mutation. BRAF mutations are found in 62-72% of patients with metastatic melanomas [START_REF] Dong | BRAF oncogenic mutations correlate with progression rather than initiation of human melanoma[END_REF], making it an important effector for targeted therapy. MEK1 and MEK2 are targeted by trametinib, and also approved for the treatment of metastatic melanomas (Tsimberidou, 2015). mTOR from the PI3K pathway is also an interesting target for approved everolimus and temsirolimus to treat several carcinomas. Cancer cells angiogenesis is inhibited using VEGF (vascular endothelial growth factor) inhibitors such as bevacizumab (2004 approval), a monoclonal antibody that binds to VEGF and inhibits its interaction with the VEGF receptor (VEGFR) [START_REF] You | Chemotherapy plus bevacizumab as an optimal first-line therapeutic treatment for patients with right-sided metastatic colon cancer: a meta-analysis of first-line clinical trials[END_REF]. Bevacizumab is used to treat metastatic colorectal and NSCL cancers, and breast cancer [START_REF] Ferrara | Single or combined immune checkpoint inhibitors compared to first-line platinum-based chemotherapy with or without bevacizumab for people with advanced non-small cell lung cancer[END_REF][START_REF] Hey | A Systematic Review and Meta-Analysis of Bevacizumab in First-Line Metastatic Breast Cancer: Lessons for Research and Regulatory Enterprises[END_REF]. Ramucirumab is another antibody inhibitor used in advanced gastric cancer, but there are also small molecules targeting VEGFR like sorafenib, sunitinib, and pazopanib approved for advanced renal and hepatocellular cancers, and gastrointestinal stromal tumors [START_REF] Escudier | Sorafenib: key lessons from over 10 years of experience[END_REF][START_REF] Zhong | Small molecules in targeted cancer therapy: advances, challenges, and future perspectives[END_REF].

P a g e | 32

CDK4/CDK6, kinases involved in cell cycle progression, are targeted by palbociclib, ribociclib, and abemaciclib approved in 2017 for the treatment of advanced/metastasis breast cancers in combination with fulvestrant [START_REF] Waks | Breast Cancer Treatment: A Review[END_REF].

Bortezomib, carfilzomib, and ixazomib are proteasome inhibitors used to treat multiple myeloma [START_REF] Sherman | Proteasome Inhibitors: Harnessing Proteostasis to Combat Disease[END_REF]. They result in the downregulation of the NF-κB (nuclear factor kappa B) pathway, a major signaling pathway of cell survival in multiple myeloma (Annunziata et al., 2007). Moreover, the inhibition of NF-κB has been shown to restore chemosensitivity in pancreatic cancer [START_REF] Li | NF-κB in pancreatic cancer: Its key role in chemoresistance[END_REF].

PARP is a major enzyme involved in DNA single strand break reparation. PARP inhibitors are actively studied, and some of them are in clinical trials [START_REF] Zhong | Small molecules in targeted cancer therapy: advances, challenges, and future perspectives[END_REF]. The inhibition of PARP alone is not sufficient to induce cell death, but the association with another treatment or given to patient bearing mutations results in the increased toxicity of the associated drugs against cancer cells. This phenomenon is called the synthetic lethality: the loss of one gene alone is not lethal, while the concomitant inactivation of two genes leads to cell death (Huang et al., 2020). Olaparib has been approved in 2014 for patients with BRCA gene mutations in breast and ovarian cancers [START_REF] Mateo | A decade of clinical development of PARP inhibitors in perspective[END_REF], and metastatic prostate cancer. Talazoparib, FDA approved in 2018, is used against advanced breast cancer with BRCA mutations [START_REF] Eskiler | Talazoparib to treat BRCA-positive breast cancer[END_REF].

Targeted therapy is associated with classical side effects, like nausea, vomiting and fever, but also hypertension, myelosuppression, infusion reactions [START_REF] Waks | Breast Cancer Treatment: A Review[END_REF][START_REF] Zhong | Small molecules in targeted cancer therapy: advances, challenges, and future perspectives[END_REF], severe hepatotoxicity [START_REF] Wang | Comparison of Hepatotoxicity Associated With New BCR-ABL Tyrosine Kinase Inhibitors vs Imatinib Among Patients With Chronic Myeloid Leukemia: A Systematic Review and Meta-analysis[END_REF], and severe or fatal allergic reactions [START_REF] Keramida | Cardiovascular complications of metastatic colorectal cancer treatment[END_REF]. Sudden cardiac death has been associated with cetuximab treatments [START_REF] Keramida | Cardiovascular complications of metastatic colorectal cancer treatment[END_REF]. Additionally, treated cancer cells tend to develop resistance to these treatments [START_REF] Mateo | A decade of clinical development of PARP inhibitors in perspective[END_REF][START_REF] Zhong | Small molecules in targeted cancer therapy: advances, challenges, and future perspectives[END_REF].

Concerning the use of nanocarriers, few are FDA approved and most are in clinical trials [START_REF] Beltrán-Gracia | Nanomedicine review: clinical developments in liposomal applications[END_REF]. Liposomes are molecules containing two layers of phospholipids that self-form vesicles in water. The first liposomes were approved by the FDA in 1995 and in Europe in 1997 and contain doxorubicin [START_REF] Sanna | Targeted therapy using nanotechnology: focus on cancer[END_REF]. They are used to treat metastatic breast cancer, ovarian cancer, multiple myeloma, and Kaposi's sarcoma. Liposomes carrying daunorubicin were approved in 1996 against Kaposi's sarcoma. Liposomes increase the halflife circulation time and maximize drug accumulation in tumor tissues. Nevertheless, the use of liposomes does not allow to control the kinetic profile and the drug localization. Further research about dosage regimen needs to be carried out, as the lipids could affect the efficiency of the drug or its formulation. P a g e | 33 1.1.2.5.3 DNA targeting chemotherapies Molecules targeting DNA act in different ways. Alkylating agents interact with DNA and attach alkyl groups to guanines, resulting in cytotoxic DNA crosslinks. They are separated into six classes. The class of nitrogen mustards were the first alkylating agent used in chemotherapy, with cyclophosphamide being the most used [START_REF] Lehmann | Evolution of Nitrogen-Based Alkylating Anticancer Agents[END_REF]. This class also includes ethylenamine and methylenamine derivatives (altretamine, thiotepa), alkyl sulfonates (busulfan), nitrosoureas (carmustine, lomustine, bedamustine), triazenes (dacarbazine, procarbazine) and platinum derivative compounds used against a large range of cancers (Dasari & Tchounwou, 2014). Cisplatin was the first platinum compound approved in 1978, currently used alongside carboplatin and oxaliplatin. Cisplatin and carboplatin work as inactive prodrugs. Their planar conformation and their metabolite diaquaplatin render them active [START_REF] Chatelut | Pharmacologie des dérivés du platine : différences entre les trois composés et les facteurs de variabilité entre patients [Pharmacology of platinum compounds: differences between the three molecules and factors of interpatient variability[END_REF]. The main side effects associated with the use of alkylating agents are numerous and include immunosuppression, treatment-associated leukemia (t-AML), digestive troubles, hair loss, myelotoxicity, ototoxicity, renal toxicity, neuro and nephrotoxicity [START_REF] Ma | Utilizing Melatonin to Alleviate Side Effects of Chemotherapy: A Potentially Good Partner for Treating Cancer with Ageing[END_REF].

Antimetabolites inhibit the DNA production by incorporating altered nucleotides, or by inhibiting the synthesis of new nucleotides. The incorporation of altered nucleotides or the absence of nucleotides incorporation lead to DNA damage and cell death, while the inhibition of nucleotides synthesis prevents cancer cell mitosis [START_REF] Valenzuela | Antimetabolite Treatment for Pancreatic Cancer[END_REF]. Anti-folate methotrexate is widely used to inhibit DHFR (dihydrofolate reductase), necessary for nucleotides synthesis [START_REF] Chan | Methotrexate--how does it really work?[END_REF]. 5-fluorouracile is an anti-pyrimidine, used in 60% of the chemotherapies, that acts as an uracil substitute [START_REF] Valenzuela | Antimetabolite Treatment for Pancreatic Cancer[END_REF]. Side effects possible are cardiotoxicity, mucositis, and palmar-plantar erythrodesia [START_REF] Fabian | Pyridoxine therapy for palmar-plantar erythrodysesthesia associated with continuous 5-fluorouracil infusion[END_REF]Alter et al., 2006;[START_REF] Ribeiro | Irinotecan-and 5-fluorouracil-induced intestinal mucositis: insights into pathogenesis and therapeutic perspectives[END_REF]. Fludarabine is an anti-purine used to treat chronic lymphoid leukemia [START_REF] Ricci | Fludarabine in the treatment of chronic lymphocytic leukemia: a review[END_REF].

Topoisomerase inhibitors impede the replication/transcription of the DNA and induce DNA damage that result in cell death (Pommier et al., 2010). They will be discussed in paragraph number 1.1.5, page 44.

DNA Damage Response, cell cycle, and cell death

DNA damage can be induced by a variety of endogenous or exogenous factors (O 'Connor, 2015). The DNA damage response (DDR) pathway has evolved to protect the cells' genomic integrity. DDR includes a large network of proteins starting with sensors, and followed by transducers, mediators, and effectors. The DDR control mechanisms sense DNA damage, coordinate DNA repair with cell cycle arrest, and ensure cell death when repair is not possible (Pearl et al., 2015;Molinaro et al., 2021) The damage sensors allow the detection of DNA breaks and serve to recruit various protein complexes depending on the type of break (Molinaro et al., 2021). Classically, a series of kinases, PI3KK (phosphatidylinositol-3-kinase-related kinase), ATM (ataxia-telangiectasia mutated), ATR (ATM-and Rad3-related), and DNA-PKcs (DNA-dependent protein kinase, catalytic subunit) are activated (Schlam-Babayov et al., 2021). These kinases phosphorylate multiple substrates, firstly to allow a cell cycle arrest and then DNA repair. Notably, PI3KKs phosphorylate H2AX (γH2AX) to allow the early recruitment of repair effectors to the damaged DNA site, and Chk1/Chk2 to arrest the cell cycle (Figure VII) (Molinaro et al., 2021).

The cell cycle is composed of checkpoints to avoid multiplication of incorrect DNA. These checkpoints rely on several Cdk/Cyclin complexes that display phase-specific activity. The G2/M checkpoint is regulated by the Cyclin B/Cdk1 complex, called MPF (mitosis promoting factor). To allow the transition through the M phase, Cdk1 is activated through dephosphorylations of its threonine 14 and tyrosine 15 residues by Cdc25C, and by a threonine 161 phosphorylation by CAK (Cdk7/Cyclin H/Mat1 complex) [START_REF] Hakem | DNA-damage repair; the good, the bad, and the ugly[END_REF]. After DNA damage, Chk1 and Chk2 inhibit Cdc25C by phosphorylation on serine 216, recognized by the 14-3-3 chaperon which exports Cdc25C to the cytoplasm. The MPF complex can not be activated and the cells are arrested in the G2 phase. Phosphorylated Chk1 and Chk2 can add a ubiquitination signal to Cdc25A for proteasomal degradation, resulting in a G1/S arrest due to the lack Cdk2/Cyclin A/E complexes activation (Donzelli & Draetta, 2003).

In parallel to the cell cycle arrest, DNA damage can be repaired by various mechanisms, depending on the type of damage (single-or double-strand breaks, adducts, interstrand crosslinks…) (Molinaro et al., 2021). The different types of damage are detected by different sensors that determine the type of repair. ROS-induced DNA damage are repaired by NER (nucleotide excision repair) or HR (homologous recombination). DNA damage induced by alkylating agents are repaired by NER, BER (base excision repair), MMR (mismatch repair), TLS (translesion synthesis process), HR, FA proteins (Fanconi anemia) or NER. X-rays, IR, and UV induced damage are repaired by TLS or HR, and the DNA damage induced by topoisomerase inhibitors are repaired by HR or NHEJ (non-homologous excision repair), with the proteins TDP1 and TDP2 (tyrosyl-DNA phosphodiesterases 1 and 2), eukaryotic enzymes involved in the aberrant topoisomerase activity reparation. TDP1 hydrolyzes phosphotyrosyl peptides emanating from the 3' DNA ends from trapped Top1, and TDP2 has the same role for Top2induced 5'-phosphotyrosyl residues [START_REF] Pommier | Tyrosyl-DNA-phosphodiesterases (TDP1 and TDP2)[END_REF][START_REF] Kawale | Tyrosyl-DNA phosphodiesterases: rescuing the genome from the risks of relaxation[END_REF].

If the DNA damage are too extended or unrepairable, the DDR pathway leads to a cell death. The most common cell death induced is apoptosis, mediated by p53 (Figure VII). p53 and its target p21 are activated by ATM/ATR, Chk1 and Chk2. The p53 level oscillates and the oscillation numbers directly relate to the DSB (double-strand break) quantities and to the cell death outcome that also depends on the genetic context of the damaged cells (Reyes et al.,P a g e | 36 2018). When p53 is mutated in high-grade serous carcinomas, its reliance on the G2/M checkpoint is increased (Ngoi et al., 2020). Otherwise, p53 (phosphorylated on S15 or S20) activates the transcription of various apoptosis genes, the pro-apoptotic BCL-2 members (PUMA, NOXA, BAX, BAK…), and inhibits the transcription of the anti-apoptotic BCL-2 members (BCL-2, BCL-XL…). In normal cells, cytochrome c is located in the mitochondria intermembrane space where it functions as an electron shuttle in the respiratory chain [START_REF] Garrido | Mechanisms of cytochrome c release from mitochondria[END_REF]. Upon apoptotic stimulus, BAX and BAK oligomerization at the mitochondria membrane forms pores and triggers membrane permeabilization, resulting in the cytochrome c release from the mitochondria to the cytosol and the activation of Apaf-1 (apoptosis protease activating factor 1). Apaf-1 and cytochrome c together form the apoptosome, and in combination with caspase 9, allow the cleavage of the procaspase 3. The activated caspases catalyze the cleavage of other key proteins, such as PARP (poly ADP ribose polymerase), a DNA repair enzyme, and procaspases 6 and 7, leading to cell death [START_REF] Oda | p53AIP1, a potential mediator of p53-dependent apoptosis, and its regulation by ser-46-phosphorylated p53[END_REF]Matt and Hofmann, 2016).

Autophagy can also be triggered after DNA damage. Autophagy is mediated by ATM/ATR and nuclear p53, which activates the transcription of PTEN (phosphatase and tensin homolog) and AMPK, to inhibit PI3K/AKT and by extension the autophagy inhibitor mTORC1 (Lorin et al., 2013) (Figure VII). At the same time, p53 activates DAPK1 to allow the activation of LC3, and the synthesis of Beclin-1 [START_REF] Zalckvar | Phosphorylation of Beclin 1 by DAP-kinase promotes autophagy by weakening its interactions with Bcl-2 and Bcl-XL[END_REF]. LC3-I is complexed with phosphatidyl-ethanolamine (forming LC3-II) at the membrane of the phagophore by ATG12-5-16. p62 associated with a ubiquitination signal ligates LC3-II on the inside of the membrane [START_REF] Liu | p62 links the autophagy pathway and the ubiqutin-proteasome system upon ubiquitinated protein degradation[END_REF] to allow the formation of autophagosomes that fuse with lysosomes, generating autolysosomes that are further degraded.

Topoisomerases

Discovered by J.C. Wang in 1971 while working on Escherichia coli, topoisomerases are DNA interacting enzymes that regulate DNA topology, by increasing or decreasing the unwinding to allow replication, transcription, and chromosome segregation. These enzymes bind to DNA, form a cleavage complex (DNA-topoisomerase), generate transitory breaks, relax and religate the DNA before unbinding [START_REF] Champoux | DNA topoisomerases: structure, function, and mechanism[END_REF]Pommier, 2013;[START_REF] Pommier | Human topoisomerases and their roles in genome stability and organization[END_REF] II). Type IA regroups Top3α and Top3β. Their mechanisms of action were recently determined. Top3α acts during replication and removes negative supercoiling (Pommier et al., 2016). Top3β acts during transcription and translation by removing R-loops on CpG sites in active promoters. Top3β KO is non-lethal [START_REF] Joo | Topoisomerase 3β knockout mice show transcriptional and behavioural impairments associated with neurogenesis and synaptic plasticity[END_REF]. Both Top3 enzymes require Mg 2+ metal cofactor for their activity [START_REF] Sissi | Effects of magnesium and related divalent metal ions in topoisomerase structure and function[END_REF] but are ATP-independent, and act as monomers, cleaving one strand of the DNA at the 5' end. Type IB includes nuclear Top1 and mitochondrial Top1mt. Top1 allows replication and transcription, while Top1mt allows specific mitochondrial DNA replication. They both remove positive and negative supercoiling and support fork movement during replication. They also act as monomers but do not require Mg 2+ nor ATP for their activity. They cleave one strand of the DNA at the 3' end involving sequential transesterifications. Type IIA is composed of Top2α and Top2β. Top2α allows DNA replication and Top2β DNA transcription. Differently, Top2 acts as a heterodimer, cleaves both strands of the DNA at the 5' end, and requires both Mg 2+ and ATP. Top2α relaxes positive supercoils faster than negative ones (> 10-fold), while Top2β relaxes both positive and negative P a g e | 38 supercoils in the same way [START_REF] Mcclendon | Human topoisomerase IIalpha rapidly relaxes positively supercoiled DNA: implications for enzyme action ahead of replication forks[END_REF]. Top1, Top2, and Top3α are essential as their KOs are lethal (Table II) [START_REF] Morham | Targeted disruption of the mouse topoisomerase I gene by camptothecin selection[END_REF][START_REF] Li | Mammalian DNA topoisomerase IIIalpha is essential in early embryogenesis[END_REF]Akimitsu et al., 2003). Only one topoisomerase is expressed in germinal cells, Spo11, a type II topoisomerase acting as a dimer and required for meiosis recombination [START_REF] Vrielynck | A DNA topoisomerase VI-like complex initiates meiotic recombination[END_REF]. Table II. Human somatic cells topoisomerases and their specificities. DSB: double-strand break; SSB: single-strand break.

P a g e | 39

Topoisomerase structures

Top1 is composed of 765 amino acids and contains four domains: a N-terminal domain, a DNA-binding region (core domain), a catalytic domain, and a C-terminal domain containing the catalytic tyrosine (Figure X, XI) [START_REF] Redinbo | Crystal structures of human topoisomerase I in covalent and noncovalent complexes with DNA[END_REF][START_REF] Stewart | A model for the mechanism of human topoisomerase I[END_REF][START_REF] Mckinnon | Topoisomerases and the regulation of neural function[END_REF]. Top1mt is composed of 601 amino acids with the same domains as Top1, with whom they share 71% of identity, reflecting high conservation between Top1 and Top1mt.

Top2α and Top2β share a 69% identity. They both share the same domains: ATPase and ATP-binding regions in the N-terminal, a core with TOPRIM domain and DNA-binding regions, and a low-complexity region in the C-terminus. Top2α and Top2β are composed of 1,531 and 1,626 amino acids, respectively [START_REF] Berger | Structure and mechanism of DNA topoisomerase II[END_REF]Aravind et al., 1998;[START_REF] Mckinnon | Topoisomerases and the regulation of neural function[END_REF].

Regarding Top3α and Top3β, only 37% of the proteins are identical, with 1,001 amino acids for the first one and 862 for the second. Top3 is composed by four domains: the first domain (N-terminal) comprises the TOPRIM domain, responsible for Mg 2+ binding, domains 2 and 4 (C-terminal) are winged-helix domains that form a single-strand DNA-binding groove, and the third domain is a β-barrel. Domains 1 and 3 form the enzyme active site [START_REF] Garnier | Type IA DNA Topoisomerases: A Universal Core and Multiple Activities[END_REF].

Spo11, initiator of meiotic recombination by double-strand breaks in gametes, is the smallest topoisomerase with 396 amino acids. Spo11 has a C-terminal domain containing the TOPRIM domain, and a 5Y-CAP domain (catabolite gene activator protein) including the catalytic tyrosines [START_REF] Baudat | SPO11: une activité de coupure de l'ADN indispensable à la méiose [SPO11: an activity that promotes DNA breaks required for meiosis[END_REF].

Topoisomerases expression patterns and modifications

Interestingly, Top2α and β isoforms present different patterns of expression depending on the type of tissues. Northern blot analysis of expression pattern in mice tissues showed that the expression of Top2α is high in proliferating tissues, such as bone marrow and spleen, and undetectable in the other tissues, while Top2β expression is higher in quiescent cells, such as heart and lung with no expression of Top2α [START_REF] Capranico | Different patterns of gene expression of topoisomerase II isoforms in differentiated tissues during murine development[END_REF] (Table III). Some tissues do not express any Top2, like muscle and pancreas. On the opposite, Top1 has no tissue specificity and is expressed in every tissue, mainly at a high level except for adipose tissue, heart, smooth and skeletal muscles (Human Protein Atlas).

Table III. Pattern of expression of Top2α/β in mice tissues according to [START_REF] Capranico | Different patterns of gene expression of topoisomerase II isoforms in differentiated tissues during murine development[END_REF] Top1 protein level does not fluctuate during the cell cycle [START_REF] Heck | Differential expression of DNA topoisomerases I and II during the eukaryotic cell cycle[END_REF], but Top1 endorses post-translational modifications that are involved in the regulation of the enzyme activity. c-Abl phosphorylates Top1 at Y268 in the core subdomain II, which upregulates Top1 activity, as shown in vitro and in cellulo [START_REF] Yu | Phosphorylation of DNA topoisomerase I by the c-Abl tyrosine kinase confers camptothecin sensitivity[END_REF] (Table IV). This phosphorylation by c-Abl was induced after cells' ionizing radiation. c-Abl-deficient cells are lacking the accumulation of the classical protein-linked DNA breaks, leading to camptothecin resistance (a Top1 inhibitor). These results shown that the phosphorylation of Top1 is important for its activity. Moreover, in vitro and in vivo studies have shown that Top1 is phosphorylated by casein kinase II on a serine residue, which stimulates its activity [START_REF] Durban | Topoisomerase I phosphorylation in vitro and in rapidly growing Novikoff hepatoma cells[END_REF]. Additionally, an increase in the O-GlcNAcylation of Top1 results in the augmentation of Top1 activity, and vice versa in the mouse kidney [START_REF] Noach | Modification of topoisomerase I activity by glucose and by O-GlcNAcylation of the enzyme protein[END_REF].

Unlikely, Top2α stability and level undergo a cell-cycle dependent fluctuation (Table IV). The expression of Top2α starts at the beginning of the S phase and continues through G2 and M phases, and shows a pike of expression between the G2 and M phase (Lee and Berger, 2019). Top2α is not expressed during the G1 phase. On the contrary, Top2β level does not fluctuate during the cell cycle [START_REF] Woessner | Proliferation-and cell cycle-dependent differences in expression of the 170 kilodalton and 180 kilodalton forms of topoisomerase II in NIH-3T3 cells[END_REF]. Levels of expression and posttranslational modifications are related to Top2α activity during the cell cycle. Indeed, during the DNA replication in S phase, Top2α decatenates newly-replicated sister chromosomes and relaxes supercoils that accumulate ahead of replication forks (Lee and Berger, 2019). During the G2 phase, Top2α plays a role in the decatenation equilibrium. During prophase, Top2α and condensin work together for chromosome condensation. Top2α localizes to centromere in metaphase, facilitated by its SUMOylation, but DNA is protected from Top2 activity by the cohesin protein. During anaphase, the release of cohesion by separase allows decatenation of the sister chromatids by Top2 for chromosome segregation. During this phase, Top2α starts to be ubiquitinylated by APC/Cdh1 and is degraded by the proteasome (Lee and Berger, 2019). Additionally, Top2α phosphorylation increases during the G2 and M phases due to a multitude of kinases (CK1/2, PKC, CamKII, Cdc7, PLK1), which have been demonstrated to enhance the enzyme activity [START_REF] Gasser | Topoisomerase II: its functions and phosphorylation[END_REF]Lee and Berger, 2019). Finally, Top2α acetylation on its catalytic domain was recently demonstrated, and supposed to modulate the enzyme's catalytic activity [START_REF] Bedez | Post-translational modifications in DNA topoisomerase 2α highlight the role of a eukaryotespecific residue in the ATPase domain[END_REF].

P a g e | 42

Topoisomerases

Tissue specificity

Cell cycle fluctuation Post-translational modifications

Top1

No No

• Phosphorylation increases its activity

• O-GlcNAcylation increases its activity

Top2α

Yes (see Table III) • Absence in G1

• Expression starting at S phase • Pike of expression between G2 and M phase

• Phosphorylation increases its activity (G2/M phases)

• SUMOylation upon mitotic entry until anaphase (centromere localization)

• Acetylation (catalytic activity)

• Ubiquitination (anaphase)

Top2β

Yes (see Table III)

No No data

Table IV. Topoisomerases 1 and 2α/β tissue specificities, cell cycle fluctuation and posttranslational modifications.

Topoisomerase-associated diseases

Dysregulations of Top1 activity are associated with several human diseases, such as spinocerebellar ataxia disorders and autoimmune scleroderma (Li and Liu, 2016). Besides, in scleroderma, which autoimmune antibodies cause hardening of the skin and connective tissues, a high level of Top1 autoantibodies are found (Li and Liu, 2016), associated with a decrease in Top1 catalytic activity alongside an increase of its SUMOylation [START_REF] Zhou | Decreased catalytic function with altered sumoylation of DNA topoisomerase I in the nuclei of scleroderma fibroblasts[END_REF]. Top1 SUMOylation in scleroderma could lead to DNA damage and genome instability. In addition, topoisomerase mutations cause genomic instability in neurons associated with the early onset of neurodegeneration [START_REF] Fragola | Deletion of Topoisomerase 1 in excitatory neurons causes genomic instability and early onset neurodegeneration[END_REF]. The inhibition of Top1 not only counteracts tumorigenesis, but also some autism spectrum disorders by inhibiting a specific RNA transcript (UBE3A-ATS in Angelman syndrome) (Li and Liu, 2016), suppresses inflammatory genes, and protects from death by inflammation after a pathogen infection [START_REF] Rialdi | Topoisomerase 1 inhibition suppresses inflammatory genes and protects from death by inflammation[END_REF].

The involvement of topoisomerases in transcriptional regulation has become more in focus when several topoisomerases mutation, depletion or inactivation have been shown to be involved in neuronal diseases like schizophrenia and autism. In familial cases of schizophrenia, the TOP3B gene is directly inactivated [START_REF] Stoll | Deletion of TOP3β, a component of FMRP-containing mRNPs, contributes to neurodevelopmental disorders[END_REF], suggesting the importance of Top3β in neuronal integrity. In the same way, Top3β has been shown to interact

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with RNAs and has an RNA topoisomerase activity [START_REF] Stoll | Deletion of TOP3β, a component of FMRP-containing mRNPs, contributes to neurodevelopmental disorders[END_REF][START_REF] Xu | Top3β is an RNA topoisomerase that works with fragile X syndrome protein to promote synapse formation[END_REF]. It was demonstrated that Top3β can bind to mRNA encoded by genes linked to schizophrenia and autism. Autism causes a fragile X, resulting in the loss of the gene encoding for FMRP (fragile X mental retardation protein). Normally, FMRP functions in synapses to inhibit translation stimulated by metabotropic glutamate receptors [START_REF] Bhakar | The pathophysiology of fragile X (and what it teaches us about synapses)[END_REF]. Top3β and FMRP can both interact with TDRD3 (tudor domain-containing protein 3), which acts as a scaffold to integrate Top3β and FMRP to transcribed genes through the binding of TDRD3 to histones [START_REF] Yang | TDRD3 is an effector molecule for argininemethylated histone marks[END_REF][START_REF] Luftig | Viruses and the DNA Damage Response: Activation and Antagonism[END_REF]. This suggests that the Top3β-TDRD3-FMRP complex ensures a correct mRNA translation, and that the loss of one of its components, like FMRP in autism, could lead to its inactivation, resulting in diseases.

Top2β activity is essential for neural development and transcription (Madabhushi, 2018). Recently, a missense TOP2B variant has been identified in two individuals with global developmental delay and autism spectrum disorder [START_REF] Lam | Global developmental delay and intellectual disability associated with a de novo TOP2B mutation[END_REF][START_REF] Hiraide | A de novo TOP2B variant associated with global developmental delay and autism spectrum disorder[END_REF].

Topoisomerase inhibitors

Inhibition of topoisomerases is used as prime therapy to kill fast-growing cancer cells. Topoisomerase targeting compounds can act as poisons or catalytic inhibitors (Larsen et al., 2003;Nitiss, 2009a;Pommier, 2013) (Molinaro et al., 2021;[START_REF] Pommier | DNA topoisomerase I inhibitors: chemistry, biology, and interfacial inhibition[END_REF]Pommier, , 2013;;Pommier et al. 2010;Young, 2015). ➔ Catalytic inhibitors act in three different ways: as DNA intercalators, avoiding the complexation of the topoisomerase within the site of interaction with DNA; as ATP competitor, rendering Top2 unable to cut DNA; or finally, as ATP hydrolysis inhibitor, impeding Top2 detachment from the DNA (Figure XIII) (Larsen et al., 2003;Nitiss, 2009). Top1 inhibitors reversibly bind to Top1 cleavage complexes, so the drugs do not directly damage DNA. However, the collision between the replication fork or the transcription machinery and the cleavage complex (Top1-DNA-inhibitor) causes irreversible Top1 covalent complexes associated with double-strand breaks [START_REF] Pommier | The indenoisoquinoline noncamptothecin topoisomerase I inhibitors: update and perspectives[END_REF].

Two Top1 inhibitors are used in chemotherapy to treat ovarian, cervical, SCLC, and colorectal cancers: topotecan and irinotecan (Table V) (Thomas and Pommier, 2019). They are derivatives of camptothecin, an alkaloid isolated from the Camptotheca acuminata tree in 1966 (Wall et al., 1966). Camptothecin has a low solubility, is unstable (its open ring form is unable to inhibit Top1 when inactivated by lactone hydrolysis at physiologic pH), and induces P a g e | 46 high toxicity (Thomas and Pommier, 2019), explaining why topotecan and irinotecan were developed in 1996 and 1998 respectively. Topotecan and irinotecan act as Top1 poisons. They bind to the DNA-Top1 cleavage complexes, avoiding DNA religation, leading to DNA breaks accumulation in the cell and therefore apoptotic cell death. Topotecan was firstly approved to treat ovarian cancer in 1996, then lately to treat cervical and SCLC in 2006 and 2007 respectively. Irinotecan was fully approved in 1998 to treat colorectal cancer. Side effects associated with topotecan and irinotecan are a severe myelosuppression, digestive troubles, and asthenia (persisting fatigue) (Thomas and Pommier, 2019). The myelosuppression in patients treated with topotecan is associated with sepsis, an inflammatory dysregulation associated with an infection that can lead to death (Pawel et al., 2001). Specifically, the neutropenia induced by topotecan can lead to fatale neutropenic colitis (colon infection) [START_REF] Takaoka | Neutropenic Colitis during Standard Dose Combination Chemotherapy with Nedaplatin and Irinotecan for Testicular Cancer[END_REF]. Topotecan is also associated with interstitial pulmonary pathologies (pulmonary inflammations) [START_REF] Shiozawa | Risk factors for severe adverse effects and treatment-related deaths in Japanese patients treated with irinotecan-based chemotherapy: a postmarketing survey[END_REF][START_REF] Meng | A Rare Case of Irinotecan-induced Interstitial Pulmonary Disease and Diffuse Alveolar Hemorrhage in a Patient with Pancreatic Cancer[END_REF], with rare cases being fatal. Belotecan is another camptothecin derivative, only approved in South Korea to treat ovarian and SCL cancers since 2003 [START_REF] Hur | Synergism of AZD6738, an ATR Inhibitor, in Combination with Belotecan, a Camptothecin Analogue, in Chemotherapy-Resistant Ovarian Cancer[END_REF]. More recently, new antibody-drug conjugates have been approved. Sacituzumab govitecan was fully approved in 2021 to treat triple-negative breast cancers. Sacituzumab is a monoclonal antibody design to bind Trop-2, a protein overexpressed on several solid cancers, responsible for cancer progression. Sacituzumab is linked to the small molecule govitecan (SN-38), which is the active metabolite of irinotecan, targeting Top1 [START_REF] Syed | Sacituzumab Govitecan: First Approval[END_REF]. The binding of sacituzumab to cancer cells allows the uptake of govitecan to induce cell death, avoiding SN-38 toxicity to normal cells. Another antibody-drug conjugate is trastuzumab deruxtecan, accepted in 2019 in USA and 2021 in the European Union against unresectable or metastasis HER2 + breast cancer [START_REF] Keam | Trastuzumab Deruxtecan: First Approval[END_REF]. Deruxtecan is a derivative of the exatecan camptothecin analog and trastuzumab is an antibody directed against HER2 [START_REF] De Langen | Trastuzumab and paclitaxel in patients with EGFR mutated NSCLC that express HER2 after progression on EGFR TKI treatment[END_REF]. However, the same side effects as the other camptothecin derivatives are observed: digestive trouble, fatigue, alopecia, neutropenia, leukopenia, and thrombopenia [START_REF] Bartsch | Trastuzumab-deruxtecan: an investigational agent for the treatment of HER2-positive breast cancer[END_REF].

Diverse Top2 inhibitors are used in chemotherapy (Table V). Most of them are not specific to one Top2 but target both Top2α and Top2β. Some of them belong to the anthracycline family and were extracted from the bacteria genus Streptomyces. Doxorubicin was extracted in 1971, and is currently prescribed against carcinomas, sarcomas, leukemias, and lymphomas, alongside daunorubicin (1979), epirubicin (1984), and idarubicin (1990) (Pommier, 2013). Anthracyclines act as poisons at low doses and as catalytic inhibitors at higher doses due to DNA intercalation property (Pommier et al., 2010;Pommier 2013). Nevertheless, associated side effects are high with hematological toxicity, vomiting, diarrhea, treatment-associated leukemias, and cardiotoxicity (Pommier 2013). The mechanisms responsible for doxorubicin's cardiotoxicity remain unclear. One possibility is that doxorubin targets Top2β, the topoisomerase highly expressed in cardiomyocytes, with absence of Top2α and a low level of Top1 [START_REF] Capranico | Different patterns of gene expression of topoisomerase II isoforms in differentiated tissues during murine development[END_REF][START_REF] Zhang | Identification of the molecular basis of doxorubicin-induced cardiotoxicity[END_REF]. Another possibility is the increased selective uptake of doxorubicin by cardiac tissue due to its daunosamine aminosugar moiety and the production of ROS (Evison et al., 2016). This cardiotoxicity prevents the treatment of patients with cardiac diseases or background, who must be given another treatment. Mitoxantrone is a doxorubicin analog developed in the 1980s [START_REF] Fox | Mechanism of action of mitoxantrone[END_REF]. It was approved for the treatment of AML in 1987, hormone-refractory prostate cancer in 1996, breast cancer, andNHL (Non-Hodgkin Lymphoma). In comparison to doxorubicin, mitoxantrone shows less cardiotoxicity (Evison et al., 2016). Mitoxantrone targets both Top2α and β, but Top2β seems to be the most important target as an increase in Top2β -/-cells survival is observed under mitoxantrone treatment [START_REF] Errington | Murine transgenic cells lacking DNA topoisomerase IIbeta are resistant to acridines and mitoxantrone: analysis of cytotoxicity and cleavable complex formation[END_REF].

Another category of Top2 inhibitor is the podophyllotoxins, extracted from the rhizomes of the genus Podophyllum in 1966 (Pommier, 2013). Etoposide was developed in 1966 and FDA approved in 1983. It acts as a poison and is prescribed against lung, testicular and ovarian cancers, neuroblastoma, leukemia and lymphoma. It can also induce t-AML (Pommier et al., 2010;Pommier 2013). Teniposide (1992 FDA first approval) is another podophyllotoxin used to treat ALL (acute lymphocytic leukemia), Hodgkin's lymphoma, and brain tumors [START_REF] Zia | Chemotherapeutic Drugs and Plasma Proteins: Exploring New Dimensions[END_REF]Schor, 2019).

Regarding catalytic inhibitors, dexrazoxane is used as a complementary treatment, and as a cardioprotector of anthracycline treatments. It stabilizes the closed conformation of the enzyme around DNA by blocking its ATPase activity (Larsen et al., 2003). It is a derivative of EDTA that chelates iron and decreases the formation of superoxide radicals, partly responsible for anthracycline cardiotoxicity. Novobiocin, another catalytic inhibitor, binds to Top2 ATP sites before or after Top2 fixation to DNA (Larsen et al., 2003), but was not approved for treatment due to lack of efficiency.

In addition to toxicity, another problem related to the use of topoisomerase inhibitors is the rise of resistance mechanisms of cancer cells. Resistance occurs in diverse ways, one of them being the increased production of efflux transporters production [START_REF] Pommier | DNA topoisomerase I inhibitors: chemistry, biology, and interfacial inhibition[END_REF](Pommier, , 2013;;Pommier et al., 2010). Efflux transporters are part of the MDR (multi-drug resistance) mechanisms, and involve specific members of the ABC transporter family. The ABC transporters are the largest family of transmembrane proteins divided into seven subfamilies (ABC-A to G). They include a majority of PGPs (P-glycoprotein/ABCB1), MRPs (multi-drug resistance-associated protein/ABCC1), and ABCG2s (ATP-binding cassette super-family G member 2). In cancer cells treated with a chemotherapeutic drug for a long period of time, transporters synthesis increases to expulse the treatment. Various MRPs are expressed differentially in function of the administered treatment [START_REF] Sodani | Multidrug resistance associated proteins in multidrug resistance[END_REF]. It has been shown that MRP1,2,4 are responsible for camptothecins resistance, MRP1,2 and 6 for P a g e | 48

anthracyclines resistance, and MRP1,2,3 and 6 for podophyllotoxins resistance [START_REF] Tiwari | Revisiting the ABCs of multidrug resistance in cancer chemotherapy[END_REF][START_REF] Sodani | Multidrug resistance associated proteins in multidrug resistance[END_REF]. Additional resistance mechanisms are: the mutation of topoisomerases, that avoids the recognition of the enzyme by the inhibitor; a reduction in the topoisomerase level; a change in the topoisomerase subcellular localization; and Top2 phosphorylation (Nitiss 2009a,b). The reduction in the Top2α expression level responsible for cancer resistance is a consequence of intronic polyadenylation [START_REF] Elton | Intronic Polyadenylation in Acquired Cancer Drug Resistance Circumvented by Utilizing CRISPR/Cas9 with Homology-Directed Repair: The Tale of Human DNA Topoisomerase IIα[END_REF]. It results in truncated Top2α with altered biological activities. Recently, the CRISPR/Cas9 technology has been used to circumvent a specific topoisomerase inhibitor resistance in leukemia cells [START_REF] Hernandez | CRISPR/Cas9 Genome Editing of the Human Topoisomerase IIα Intron 19 5' Splice Site Circumvents Etoposide Resistance in Human Leukemia K562 Cells[END_REF][START_REF] Hernandez | Use of CRISPR/Cas9 with homology-directed repair to silence the human topoisomerase IIα intron-19 5' splice site: Generation of etoposide resistance in human leukemia K562 cells[END_REF]. The technology allows the removal of an intron responsible for the truncated version of Top2α and restores the toxicity of Top2α-targeting drugs such as etoposide.

Due to their high proliferative activity, some cancers overexpress topoisomerases to allow a fast cell multiplication [START_REF] Gouveris | Topoisomerase I and II Expression in Recurrent Colorectal Cancer Cells: A Dubious Matter[END_REF]Pommier, 2013). Upon analysis of 24,262 patient solid tumors (26 types), 51% of the tumors showed overexpression of Top1 (Heestand P a g e | 49 et al., 2017). From 230 breast cancer samples, Top2α expression was found in 0.6 to 39.4% of the carcinomas' cells (mean 10.6%±7.9%), whereas expression was undetectable in nonmalignant breast epithelium [START_REF] Järvinen | Expression of topoisomerase IIalpha is associated with rapid cell proliferation, aneuploidy, and c-erbB2 overexpression in breast cancer[END_REF]. More specifically, the Top2α gene is located close to the HER2 oncogene and is amplified or deleted in almost 90% of the HER2 + breast tumors [START_REF] Järvinen | HER-2/neu and topoisomerase IIalpha in breast cancer[END_REF]. Chemotherapies targeting topoisomerases are more efficient in cancers overexpressing topoisomerases [START_REF] Gouveris | Topoisomerase I and II Expression in Recurrent Colorectal Cancer Cells: A Dubious Matter[END_REF][START_REF] Pommier | DNA topoisomerase I inhibitors: chemistry, biology, and interfacial inhibition[END_REF]Pommier, , 2013)). For example, patients with locally advanced or metastatic breast cancers had a better anthracycline response when they had Top2α amplification or overexpression [START_REF] Coon | Amplification and overexpression of topoisomerase IIalpha predict response to anthracycline-based therapy in locally advanced breast cancer[END_REF][START_REF] Cardoso | Correlation between complete response to anthracycline-based chemotherapy and topoisomerase II-alpha gene amplification and protein overexpression in locally advanced/metastatic breast cancer[END_REF]Arriola et al., 2007).

Biomarkers to predict clinical efficacy are under investigation to allow the selection of patients that could benefit from Top1 inhibitors chemotherapy the most. γH2AX (Furuta et al., 2003;[START_REF] Rao | Batracylin (NSC 320846), a dual inhibitor of DNA topoisomerases I and II induces histone gamma-H2AX as a biomarker of DNA damage[END_REF], and schlafen-11 (Murai et al., 2019;[START_REF] Coleman | The role of Schlafen 11 (SLFN11) as a predictive biomarker for targeting the DNA damage response[END_REF][START_REF] Bednarikova | Can Schlafen 11 Help to Stratify Ovarian Cancer Patients Treated with DNA-Damaging Agents?[END_REF] are interesting candidate markers. A gene signature developed for sensitivity and resistance to topotecan and other chemotherapeutic drugs is also underway to determine the value in patient identification [START_REF] Potti | Genomic signatures to guide the use of chemotherapeutics[END_REF]. Moreover, some genes hypermethylation involved in gene silencing are linked to drug sensitivity. Methylation of WRN (Werner Syndrome), CHFR (Checkpoint with Forkhead and Ring finger domains), and SULF2 (sulfatase 2) gene promoters has been linked to a better response to irinotecan in colorectal, cervical, gastric and NSCL cancers [START_REF] Tessema | SULF2 methylation is prognostic for lung cancer survival and increases sensitivity to topoisomerase-I inhibitors via induction of ISG15[END_REF][START_REF] Masuda | Association of epigenetic inactivation of the WRN gene with anticancer drug sensitivity in cervical cancer cells[END_REF][START_REF] Wang | Correlation between the methylation of SULF2 and WRN promoter and the irinotecan chemosensitivity in gastric cancer[END_REF][START_REF] Cha | Association of CHFR Promoter Methylation with Treatment Outcomes of Irinotecan-Based Chemotherapy in Metastatic Colorectal Cancer[END_REF]. WRN is a helicase that plays critical roles in DNA replication, recombination, repair, and telomere maintenance; CHFR is an E3 ubiquitin protein required for maintenance of the antephase checkpoint that regulates cell cycle entry into mitosis; and SULF2 is a heparan sulfate proteoglycan involved in cell signaling.

Altogether, despite the powerful effects of topoisomerase inhibitors to efficiently direct cancer cells to death, the limitations due to the side effects and the necessity to circumvent cancer cell resistance mechanisms require a constant development of new molecules.

Organometallics and Copper-complexes

The first discovery of an antitumoral metal-based agent arose in the 1960s with platinum, which opened a new era of chemotherapeutic drugs (Rosenburg, 1969). As described previously ( §1. 1.2.5.3, page 33), cisplatin is an alkylating agent widely used in chemotherapy to treat a variety of cancer, as long as its derivatives from second (carboplatin) and third generations (oxaliplatin). In cisplatin, the platinum (II) is located at the center of a squared planar structure and is coordinated with two chlorides and two ammonia molecules in a cis configuration.

P a g e | 50

In an attempt to circumvent the major problems associated with classical chemotherapeutics drugs including the severe side effects, a possible approach consists to design and develop new therapeutic metal-based anticancer drugs. Transition metals from the D-block of the periodic table (groups 3 to 12), such as essential trace metals, including copper and iron, can be used for the implementation of metal-based complexes in anticancer therapies. In this context, new organometallics containing iron have been developed and studied from preexisting compounds, including ferrocene species (originally derived from tamoxifen) with antiproliferative potential used due to their stable aromatic nature of ferrocene, associated with low toxicity and cost (Jaouen et al., 2015). The metal-based agent ferroquine, composed of the addition of an iron atom to the chloroquine core, was firstly used as an antimalarial drug and are now unveiled to be a promising agent to treat cancer (Kondratskyi et al., 2017). Amongst metals use in the chemotherapeutic strategy, copper was shown to be an interesting candidate. Physiologically, copper has a central role in various cellular processes, being an essential micronutrient, an important cofactor of metalloenzymes involved in mitochondrial metabolism (cytochrome c oxidase) and cellular radical detoxification against reactive oxygen species (ROS) (superoxide dismutase) (Hordyjewska et al., 2014). Copper is essential for angiogenesis, proliferation, and migration of endothelial cells (Hu, 1998;[START_REF] Urso | Behind the link between copper and angiogenesis: Established mechanisms and an overview on the role of vascular copper transport systems[END_REF].

A number of copper-complexes have been synthesized through the years, among them promising topoisomerase inhibitors (Molinaro et al., 2020), (Table VI). Many compounds have been newly designed and synthesized to enhance preexisting molecules. They have been added with diverse metals to improve their cytotoxicity against cancer cells. The copper complexes are mainly formed with a Cu(II) atom, a stable form of copper. Copper modifies the backbone of the complexed ligand and grants better DNA affinity (Krasnovskaya et al., 2020;Brissos et al., 2015). These compounds can either target Top1, such as the oxindolimine-Cu(II) or plumbagin-Cu(II) (Castelli et al., 2018;Chen et al., 2009), Top2α, such as thiazole-TSC-Cu-(II) or quinoline-TSC-Cu(II) (Lisic et al., 2018;Bisceglie et al., 2015), or are dual Top2α and β inhibitors such as bis-pyrazolyl-Cu(I) compound (Khan et al., 2017). Copper-complexes act as poison or catalytic inhibitors but are often associated with ROS production, which is the case for most metal-based drugs (Devi et al., 2018;[START_REF] Gulcin | Metal Ions, Metal Chelators and Metal Chelating Assay as Antioxidant Method[END_REF]. ROS are radical and non-radical oxygen species formed by a partial reduction of the oxygen (e.g. superoxide anion (O2-), hydrogen peroxide (H2O2), or hydroxyl radical (HO•)). ROS production arises from mitochondria oxidative metabolism at a low and stationary level and have a role in cell signaling and homeostasis, but also in physiological functions such as aging, inflammation, and immune responses [START_REF] Mirzaei | Elucidating Role of Reactive Oxygen Species (ROS) in Cisplatin Chemotherapy: A Focus on Molecular Pathways and Possible Therapeutic Strategies[END_REF]. When accumulated at a high level by exogenous factors like chemotherapeutic drugs, ROS induce DNA damage which increases the toxicity against cancer cells, but also towards normal cells.

P a g e | 51

As a consequence of their induced activation of the DDR, copper-complexes arrest cancer cells in different phases of the cell cycle and trigger cell death, mainly apoptosis, and in some cases autophagy (Molinaro et al., 2020). For example, pyrophosphate and tetrazolopyrimidine diimine copper complexes act with low IC50 under 1 µM (0.64 µM on an ovarian and 0.12 µM on a lung cancer lineage, respectively), with an induced apoptosis for tetrazolopyrimidine diimine (Ikotun et al., 2009;Haleel et al., 2016). The Cu(II)(GTSCHCl) complex acts with an IC50 of 1.45 µM on triple-negative breast cancer cell, 1.23 µM on colon cancer cell, and induces a G2/M arrest as long as apoptosis (Palanimuthu et al., 2013). Copper complexes inhibitors of topoisomerase have been published in our review included page 187. All these results obtained with copper complexes encourage the expansion of this highly active anticancer type of drug targeting the topoisomerase family. Copper-complexes inhibitors of human topoisomerases, toxicity on cancer cells (IC50), and induced cell cycle arrests and/or cell deaths when determined (Molinaro et al., 2020).

RESEARCH CONTEXT, INDENOISOQUINOLINE COPPER-COMPLEXES

Indenoisoquinolines

A new Top1 inhibitor family of molecules, the indenoisoquinolines, was developed by Pommier and Cushman from 1998 at the National Cancer Institute (NCI) using a COMPARE algorithm to predict biological targets from data generated by the screen of 60 human cancer cell lines (NCI-60) [START_REF] N Nagarajan | Synthesis and evaluation of indenoisoquinoline topoisomerase I inhibitors substituted with nitrogen heterocycles[END_REF][START_REF] Pommier | The indenoisoquinoline noncamptothecin topoisomerase I inhibitors: update and perspectives[END_REF]. The indenoisoquinoline backbone is composed of four carbon cycles, including two aromatic cycles, one azote, and two oxygens (Figure XIV). Recently, modulation of the chemical nature of substituents on the indenoisoquinoline has resulted in additional biological targets with convincing evidence for the use of indenoisoquinolines for the treatment of many various diseases [START_REF] Cushman | Design and Synthesis of Indenoisoquinolines Targeting Topoisomerase I and Other Biological Macromolecules for Cancer Chemotherapy[END_REF].

P a g e | 54

These compounds are good protagonists for chemotherapy as they are chemically stable in the absence of e-ring compared to camptothecins (inactivated at physiologic pH) (Pommier et Cushman, 2009). In addition, the DNA-protein crosslinks (DPCs) induced by the indenoisoquinoline via the inhibition of Top1 are persistent at least 1 h after drug removal, while the DPCs are reversed less than 15 min after camptothecin or topotecan removal (Antony et al., 2007). Therefore, indenoisoquinoline chemotherapy requires shorter infusion times because the Top1 inhibitor complex is less likely to dissociate. Moreover, indenoisoquinolines are less or not substrates for PGP and ABCG2 efflux transporters responsible for MDR, in comparison to topotecan and irinotecan, and can circumvent established chemoresistance (Antony et al., 2007).

Co-crystal structures of indenoisoquinolines with Top1-DNA elucidated the structure of the ternary complex showing that they interact with DNA in a hydrophobic stacking interaction, and with Top1 by a network of hydrogen bonds, a common feature of interfacial inhibition [START_REF] Marchand | A novel norindenoisoquinoline structure reveals a common interfacial inhibitor paradigm for ternary trapping of the topoisomerase I-DNA covalent complex[END_REF][START_REF] Pommier | Topoisomerase I inhibitors: camptothecins and beyond[END_REF].

Three indenoisoquinolines have been selected for clinical trials: indotecan (LMP-400), indimitecan (LMP-776), and LMP-744, and have completed or are in phase 1 to treat lymphoma and advanced solid tumors [START_REF] Kummar | Clinical and pharmacologic evaluation of two dosing schedules of indotecan (LMP400), a novel indenoisoquinoline, in patients with advanced solid tumors[END_REF]Pommier et al., 2018). They have good pharmacokinetics proprieties, including a good cell absorption, body diffusion, and excretion characteristics [START_REF] Muzzio | Plasma pharmacokinetics of the indenoisoquinoline topoisomerase I inhibitor, NSC 743400, in rats and dogs[END_REF][START_REF] Kummar | Clinical and pharmacologic evaluation of two dosing schedules of indotecan (LMP400), a novel indenoisoquinoline, in patients with advanced solid tumors[END_REF]. Myelosuppression has been linked to these treatments, without significant gastrointestinal problems [START_REF] Kummar | Clinical and pharmacologic evaluation of two dosing schedules of indotecan (LMP400), a novel indenoisoquinoline, in patients with advanced solid tumors[END_REF].

To enhance their efficiency towards cancer cells, new organometallic compounds derivative of indenoisoquinoline were synthetized by Pr. L. Pelinksi's team (Unité de Catalyse et Chimie du Solide -Lille University) (Wambang et al. 2016). Associated with the backbone of indenoisoquinoline, a carbonated chain was added linked to a metal from the D bloc of the periodic table such as: copper, zinc and ruthenium (Figure XIV).

Metal complexes of indenoisoquinoline

Fourteen complexes were synthesized by Pr. L. Pelinski team, and their cytotoxicity were determined using MTS on five cancer cell lines, MDA-MB-231 (triple-negative breast cancer), MCF-7 (hormone-independent breast cancer), HeLa (cervix cancer), HT-29 (colon cancer), and DU-145 (prostate cancer) (Figure XV). The copper complexes came out to be efficient organometallic complexes, with the lowest IC50 on the five cell lines (mean of 0.455 µM for the copper complexes; mean of 0.556 µM for the zinc complexes; mean of 149.239 µM for the ruthenium complexes). Another piece of evidence that came out of the MTS tests was the high efficiency of a four-carbon spacer compared to shorter spacers. An additional interesting feature demonstrated an increased efficiency provided by two symmetrical molecules of indenoisoquinoline surrounding the copper atom (Table VII) [START_REF] Wambang | Nouveaux Complexes Organométalliques d'Indéno[1,2-c]isoquinoléines Anticancéreux : Synthèse, Caractérisation et Evaluation Biologique en tant que Ligands de l'ADN et Inhibiteurs de Topoisomérase[END_REF][START_REF] Wambang | Nouveaux Complexes Organométalliques d'Indéno[1,2-c]isoquinoléines Anticancéreux : Synthèse, Caractérisation et Evaluation Biologique en tant que Ligands de l'ADN et Inhibiteurs de Topoisomérase[END_REF]. P a g e | 56 The compound with the lowest IC50, named WN197 (Cu-2 on the figure above), shows IC50 of 0.144 µM on MDA-MB-231, 0.644 µM on MCF-7, 0.220 µM on HeLa, 0.358 µM on HT-29 and 0.162 µM on DU-145. The WN197 IC50 mean was significantly higher compared to all other compounds except for L3 and Ru-2 (Table VII). WN197 is composed of a copper-core surrounded by two indenoisoquinolines connected with a four carbons chains. WN170 (L2 on the figure above) was studied as a control to understand the benefit of the copper addition. WN170 is composed of one indenoisoquinoline with a four carbons chain bound to the nitrogen atom (Figure XVI).

Thesis objectives

The aim of my thesis was to determine the action mechanisms involved in the cytotoxic effect of WN197 on three adenocarcinoma representing cancers with a high prevalence and difficult to treat: MDA-MB-231, HeLa, and HT-29 cell lines. This research takes part in the context of effectiveness' determination of copper-complexes inhibitors of topoisomerases to envision new anticancer chemotherapies. In a first part, the determination of DNA damage and the action on topoisomerases with the type of inhibition (Type I and/or II topoisomerases targeted; and poison or catalytic inhibitor) were ascertained using DNA binding experiments and in vitro topoisomerases tests. In a second part, impacts of WN197 on the cell cycle and the targeted molecular actors were analyzed with flow cytometry, immunoprecipitation, and Western blots. In a third part, the type of cell death activated, the potential effects on resistant cancer cells, including a human cell-line and a vertebrate embryonic model system were investigated. Finally, the effects of WN197 compared to its ligand WN170 allowed us to conclude that WN197 is a promising tool in future cancer chemotherapies, alone or in combination with other compounds and types of therapies. The last part of the manuscript includes the main scientific articles and reviews I have published as main author.

P a g e | 58

Materials & Methods P a g e | 59

MATERIALS & METHODS

CELLULAR MODEL

Cell lines

WN197 effect on cell viability and mechanism of action were determined on three adenocarcinoma cell lines: MDA-MB-231 (breast cancer cells), HeLa (cervix cancer cells) and HT-29 (colon cancer cells). MDA-MB-231 is a triple-negative breast cancer cell line (ER -, PR -, HER2 -) hormono-independent towards growth. These cells express a high level of mutated p53 and are tumorigenic in mice [START_REF] Bartek | Genetic and immunochemical analysis of mutant p53 in human breast cancer cell lines[END_REF]Leroy et al., 2014). The second epithelial cell line, HeLa, originated from Henrietta Lacks, and was the first immortalized human cell line (1951). The cells are HPV positive (HPV-18) and express wild-type p53, but the papillomavirus leads to p53 degradation [START_REF] May | Endogenous HeLa p53 proteins are easily detected in HeLa cells transfected with mouse deletion mutant p53 gene[END_REF]Leroy et al., 2014). The third cancer line is the colon cancer line HT-29. These epithelial cells were isolated from a primary tumor of a patient in 1964. They express a mutated p53 [START_REF] Ikediobi | Mutation analysis of 24 known cancer genes in the NCI-60 cell line set[END_REF]Leroy et al., 2014). The three cancer cell lines were selected for deepen research because a low IC50 was obtained with the WN197 compound on these cells but also based on the high incidence their cancer represents. MCF-10A, a breast non-cancerous cell line, is an epithelium cell line isolated from an anonymous woman mammary gland. BT-549 is a triple-negative breast cancer cell line originating from a woman metastasized ductal tumor. SUM-159 is a triple-negative breast cancer cell line derived from a mouse xenograft of a transplanted primary human invasive infiltrating ductal carcinoma metastatic nodule. H69AR is a small cell lung cancer (SCLC) cell line derivative from the NCI-H69. A treatement with increasing doses of doxorubicin (up to 0.8 µM) for 14 months created this multi-drug resistance (MDR) cell line, notably resistant to anthracyclines [START_REF] Mirski | Multidrug resistance in a human small cell lung cancer cell line selected in adriamycin[END_REF].

HeLa, MDA-MB-231, HT-29, H69AR, and MCF-10A, cell lines originate from ATCC (Manassas, VA, USA). BT-549 were gifted by Dr. Lagadec (CANTHER, Lille), and SUM-159 by Dr. Groux (UGSF, Villeneuve d'Ascq).

Cell Culture

Cell lines were maintained at 37°C in a humidified atmosphere with 5% CO2 in DMEM medium (Lonza, Basel) for adenocarcinomas, or RPMI-1640 medium (Lonza, Basel) for H69AR, supplemented with 10% fetal bovine serum (Dutscher, Dernolsheim, France) for adenocarcinomas, or 20% for H69AR, 1% Zell Shield (Dutscher, Bernolsheim, France) and 1% non-essentials amino-acids (Lonza, Basel, Switzerland). MCF-10A were maintained in MEBM medium (Lonza, Basel, Switzerland) supplemented with MEGM (Lonza, Basel, Switzerland). P a g e | 60

CHEMICAL REAGENTS AND MATERIALS

All commercial reagents and solvents were used without further purification. Cisplatin was purchased from Alfa Aesar; rapamycin from Abcam; doxorubicin, nocodazole and DMSO from Sigma-Aldrich. Stock solutions were prepared in DMSO. Doxorubicin is a Top2 inhibitor that can intercalate in DNA, induce ROS production and apoptosis (Cappetta et al., 2017;Sritharan and Sivalingam, 2021). Cisplatin is an alkylating agent that induces DNA damage and S phase arrest in the cell cycle as long as apoptosis (Dasari et Tchounwou, 2014). Nocodazole is a mitotic spindle poison, that induces a M phase arrest in the cell cycle [START_REF] Blajeski | G(1) and G(2) cell-cycle arrest following microtubule depolymerization in human breast cancer cells[END_REF].

MTS ASSAY

Principle

The MTS assay is a colorimetric test used to determine cell viability. MTS is a tetrazolium salt, reduced by viable cells via mitochondrial dehydrogenases into a colored formazan (Aqueous soluble tetrazolium/formazan MTS as an indicator of NADH-and NADPH-dependent dehydrogenase activity) [START_REF] Kamiloglu | Guidelines for cell viability assays[END_REF]. The brown color is directly proportional to the percentage of viable cells.

Protocol

Cell viability was determined using CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS, Promega). 2.10 3 cells per well were seeded in 96-well plate for 24 h before treatment for 72 h with 0 to 100 µM of WN197, WN170 or cisplatin. After an incubation of 2 h with 20 µL of CellTiter solution at 37°C in 5% CO2, the production of reduced MTS (3-(4,5dimethylthiazol

-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)
in formazan was measured at 490 nm (SPECTROstar Nano, BMG LABTECH). IC50 were calculated P a g e | 61

using GraphPad Prism V6.0 software. Statistical differences between WN197 and WN170 were ascertained by a Student t-test (***p<0.005).

TOPOISOMERASE ACTIVITY IN VITRO TEST

Principle

Topoisomerase activity in vitro assay is based on the capacity of topoisomerase to relax supercoiled DNA (SCDNA). Relaxed (RDNA) and SCDNA subjected to an electric current have different migration profiles in a 1% agarose gel. Topoisomerase inhibitors (poisons or catalytic inhibitors) impede DNA relaxation, altering the migration profiles of DNA. Differentiation between catalytic and poison inhibitors is determined by the ability of a poison to stop topoisomerase reaction after the DNA cleavage, leading to an accumulation of stabilized nicked or linear DNA (NDNA or LDNA) due to the formation of a ternary complex between the DNA, the topoisomerase, and the poison.

Protocol

Topoisomerase I (Top1) activity assay was performed using the drug screening kits protocol (TopoGEN, Inc.). The reaction mixture was composed of supercoiled plasmid DNA pHOT1 (250 ng), 10X TGS buffer (10 mM Tris-HCl pH 7.9, 1 mM EDTA), 5 units of Top1, the compound, and a final volume adjusted to 20 µL with H2O. WN197 was tested at concentrations ranging from 0.2 to 2 µM. Camptothecin (CPT, 10 µM) was used as a positive control (poison inhibitor of Top1 activity), etoposide (VP-16, 100 µM) as negative control (inhibitor of Top2 activity), and 1% DMSO alone as vehicle control. Relaxed pHOT1 DNA (100 ng) was used as migration control. The addition of proteinase K (50 µg/mL) for 15 min at 37°C allowed Top1 degradation to visualize the cleavage products (nicked DNA). Reaction products were separated by electrophoresis in a 1% agarose gel containing ethidium bromide (0.5 µg/mL) for 1 h at 100 V in TAE (Tris-Acetate-EDTA; pH 8.3) buffer.

Topoisomerase II Relaxation Assay Kit (Inspiralis, Inc.) was used to measure topoisomerase II (Top2) activity. The reaction mixture was composed of supercoiled plasmid DNA pBR322 (1 µg), 10X assay buffer , 125 mM NaCl, 10 mM MgCl2, 5 mM DTT, 100 µg/mL albumin), 30 mM ATP, 5 units of Top2α or Top2β, the compound, and a final volume adjusted with H2O to 30 µL. Etoposide (VP-16, 100 µM) was used as positive P a g e | 63 control, and camptothecin (10 µM) as negative control. The mixtures were incubated at 37 °C for 30 min and the reactions stopped by the addition of 5 µL 10% SDS. Reaction products were separated by electrophoresis in a 1% agarose gel for 1 h at 100 V in TAE buffer, and stained with ethidium bromide (0.5 µg/mL) for 15 min. After rinsing with water, the DNA migratory profiles were visualized under UV light (ChemiDocTM XRS+, BioRad).

FLUORESCENCE

Principle

DNA damage were analyzed by immunofluorescence localization of a marker protein.

After DNA damage, the DDR pathway is activated, resulting in the activation of the ATM/ATR kinases. These proteins phosphorylate histone H2AX on serine 139 (γH2AX), on the DNA break sites [START_REF] Hakem | DNA-damage repair; the good, the bad, and the ugly[END_REF]Bonner, 2008;[START_REF] Kinner | Gamma-H2AX in recognition and signaling of DNA double-strand breaks in the context of chromatin[END_REF]. γH2AX is detectable by immunofluorescence as fluorescence dots (foci). The amount of γH2AX foci is directly related to the amount of DNA breaks, allowing to compare the number of breaks between different conditions. Autophagy was visualized by fluorescence, using the autophagy assay kit from Abcam (ab139484). The special dye allows a green fluorescent signal emission at 530 nm after excitation at 488 nm, specifically in the lysosomes, and with a higher intensity when incorporated into pre-autophagosomes, autophagosomes, and autolysosomes. The target(s) or physiological mechanism of the probe is not specified by the seller. P a g e | 64

Protocol

For γH2AX detection, 2.10 5 cells seeded on glass coverslips in 6-well plate were treated with 0.5 µM of WN197 or WN170, 5 µM of doxorubicin, 20 µM of cisplatin as positive controls, or 0.1% DMSO as a solvent control for 30 min or 24 h. Cell fixation was performed with 4% paraformaldehyde (Sigma-Aldrich) for 5 min and followed by permeabilization with 0.1% Triton in PBS (Sigma-Aldrich) for 10 min and saturation of unspecific sites with 1% BSA in PBS (Sigma) for 1 h at room temperature. Cells were incubated overnight at 4°C with anti-γH2AX mouse antibody (S139, 1:1000, Cell Signaling, by Ozyme) and washed 3 times with 1% BSA/PBS. Cells were then incubated with secondary anti-mouse IgG (Alexa Fluor® 488, 1:2000, Thermo-Fisher Scientific Biosciences GMBH) for 1 h at room temperature in the dark, washed 3 times before nuclei were stained with DAPI (6-diamidino-2-phenylindole, 1 µg/mL, Molecular Probes, by Thermo Fisher Scientific Biosciences GMBH). Images were captured under a Leica fluorescent microscope (TISBIO platform, UGSF, Lille), and γH2AX foci were counted with ImageJ (Fiji Software, v1.52i) on 30 cells from 3 independent experiments and quantified with GraphPad Prism V6.0 software. Statistical significances (mean ± SD) were performed by a two-way ANOVA followed by Dunnett's multiple comparison test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

For autophagy detection, the autophagy assay kit was used as described by the manufacturer (Abcam, ab139484). 2.10 5 cells were seeded on glass coverslips and treated for 24 h with 0.5 µM of WN197, rapamycin as positive control, or 0.1% of DMSO as a solvent control. Cells were rinsed twice with 1X assay buffer and incubated with the green detection reagent and Hoechst dye (20 mM, nucleus stain). Cells were fixed by 4% paraformaldehyde (Sigma-Aldrich) for 10 min and rinsed three times with 1X assay buffer. Images were captured under a Leica fluorescent microscope, and γH2AX green spots were counted with ImageJ (Fiji Software, v1.52i) on 30 cells from 3 independent experiments and quantified with GraphPad Prism V6.0 software. Statistical significances (mean ± SD) were performed by a two-way ANOVA followed by Dunnett's multiple comparison test (*p<0.05; ***p<0.001; ****p<0.0001). and cell doublets (high density and height) are excluded from the gate of interest (P1). Right: cells from the gate P1 are distributed according to the intensity of their fluorescent signal, and divided into three different pools corresponding to cell cycle phases (representation of the control distribution of a human proliferating cell population with cell cycle phases repartition as follow: ~45% in G0/G1, ~20% in S, ~35% in G2/M).

FLOW CYTOMETRY

Principle

For apoptosis detection, annexin-V levels were measured by flow cytometry. During apoptosis, the phosphatidylserine (PS) is translocated early from the inner to the outer part of the membrane. Annexin V has a high affinity to PS, therefore, during apoptosis, cells exposing PS to the external environment are ligated to annexin V proteins. Annexin V binds to FITC (fluorescein isothiocyanate) detected by flow cytometry. Propidium iodide (PI) is used as a co-marker to differentiate cells in early versus late apoptosis since only dead cells have a permeabilized membrane. Therefore, annexin V -PI -cells are cells that don't proceed through apoptosis, annexin V + PI -are cells in early apoptosis and annexin V + PI + are cells in late apoptosis. Annexin V -PI + cells can also represent necrotic cells or cell debris as annexin V finally enters any damaged cell membrane. 2.6.2 Protocol 7.5.10 5 cells plated for 24 h were treated with 0.5 µM WN197 or WN170, 20 µM of cisplatin (S phase arrest control), 83 nM of nocodazole (M phase arrest control), or 0.1% DMSO (solvent control). For the dose titration experiments, cells were treated for 24 h with increasing concentrations of WN197. For kinetic experiments, cells were treated with 0.5 µM of WN197 or WN170 from 4 to 48 h. Cells were detached using trypsin (Biowest) for 5 min at 37°C, centrifuged at 1,000 G for 10 min, resuspended in PBS, and fixed with 70% ethanol at -20°C for 24 h, before they were centrifugated (1,000 G, 10 min), resuspended in PBS, and treated for 15 min at room temperature with RNase (200 µg/mL, Sigma). Finally, incubation with propidium iodide (10 µL/mL, Molecular Probes, by Thermo Fisher Scientific Biosciences GMBH) at 4°C for 30 min was performed before flow cytometry (BD FACSCalibur, Becton Dickinson) analysis. For each sample, 10,000 events (without cell doublets and cellular debris) were considered. The cell cycle repartition was analyzed with Graphpad Prism V6.0 software.

P a g e | 67

Statistical significances (mean ± SD) were determined by two-way ANOVA followed by Dunnett's multiple comparison test (*p<0.05; **p<0.01; ***p<0.005; ****p<0.001).

For apoptosis detection featured by annexin V-propidium iodide (PI), MDA-MB-231, HeLa, and HT-29 cells were cultivated to 80% of confluence (7.5.10 5 cells), incubated or not for 24 h with WN170 (0.5 µM), WN197 (0.5 µM), camptothecin (20 µM; CPT) or doxorubicin (5 µM; Doxo), trypsinized for 5 min at 37°C, and washed in ice-cold PBS. Cell suspensions were treated with 1:1 PI and annexin V-FITC reagent (Apoptosis Detection Kit, BD) and incubated for 15 min at RT in the dark. The binding buffer (1X) was added to each tube before they were analyzed by flow cytometry (CytoFLEX LX, Beckman Coulter) with Kaluza analysis software (v2.1.1).

WESTERN BLOTS AND IMMUNOPRECIPITATIONS

Principle

Following DNA damage, a kinases cascade initiates the activation of the DNA damage response (DDR) pathway, with ATM and ATR phosphorylating H2AX, Chk1 and Chk2 [START_REF] Hakem | DNA-damage repair; the good, the bad, and the ugly[END_REF]Bonner, 2008) Depending on the cell cycle phases, different Cdc25 are activated, and subsequently different Cyclin/Cdk couples. In our experiments, markers from the G2/M transition and M phase were analyzed by Western blots to refine the results obtained by flow cytometry: Cdc25C, Cdk1/Cyclin B, and histone H3. Histone H3 phosphorylation on S10 by active MPF is necessary for DNA condensation during mitosis, making it a suitable M phase marker (Hans et Dimitrov, 2001).

Cdc25C is phosphorylated on S216 before the M phase by the DNA damage cascades as seen in the introduction page 34-36, and can bind to the chaperon protein 14-3-3 (Figure H). This Cdc25C sequestration prevents it from dephosphorylating Cdk1, and therefore from activating the MPF complex and entry of cells in the M phase. Cdc25C immunoprecipitations were realized and Western blots were proceeded against 14-3-3 to verify the complexation of the two proteins. During the M phase, active Cdc25C is not phosphorylated on S216 (inhibitory phosphorylation), which allows MPF (Cyclin B/Cdk1) activation. When cells are accumulated or stopped in the M phase, the Cdk1/Cyclin B complex is activated and no 14-3-3 protein is found on Western blots after a Cdc25C immunoprecipitation. In case of an arrest in the other phases of the cell cycle, Cdc25C is inactivated by phosphorylation on S216, and recognized by 14-3-3 to which it binds, allowing a detection of both partners after immunoprecipitation and Western blots.

Apoptosis markers analyzed by Western blots are: activated PARP (cleaved), activated caspase 3 (cleaved) and the cytosolic cytochrome c. For autophagy, markers including the degradation of p62, the synthesis of Beclin-1, and the switch from LC3-I to LC3-II (LC3-I coupled to phosphatidylethanolamine) (Kroemer et al., 2009) were analyzed by Western blots. During autophagy, mTOR gets complexed to a phosphorylated form of RAPTOR (S792) in the rapamycin-sensitive mTOR complex 1 (mTORC1). After DNA damage, the mTORC1 complex is P a g e | 69

inhibited by RAPTOR phosphorylation (on multiple sites including S792) in a negative feedback loop to induce autophagy (Dunlop et al., 2011;[START_REF] Ma | mTORC1 pathway in DNA damage response[END_REF]. This activation is similar to the mTORC1 inhibitor rapamycin [START_REF] Kim | mTOR: a pharmacologic target for autophagy regulation[END_REF]. Immunoprecipitations against mTOR from the mTORC1 complex were proceeded and Western blots against mTOR and phosphorylated RAPTOR were done.

Protocol

7.5.10 5 cells were seeded in a 25 cm 3 flask for 24 h and treated with 0.5 µM of WN197 or WN170, 20 µM of cisplatin, 5 µM of doxorubicin, or 0.1% DMSO (solvent control). After 24 h, they were lysed in RIPA buffer (1% Triton X-100; 50 mM TRIS-HCl pH 4; NP40 2%; 0.4% Na-deoxycholate; 0.6% SDS; 150 mM NaCl; 150 mM EDTA; 50 mM NaF) supplemented with 1% of protease inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitors (Roche SAS by Merck). For cytochrome c analysis, 7.5.10 5 cells were seeded for 24 h and treated for 3 h, 16 h, 24 h or 48 h with 0.5 µM of WN197, and for 24 h or 48 h with 5 µM of doxorubicin as a positive control. Cells were lysed in a glass grinder at 4°C in homogenization buffer (25 mM MOPS at pH 7.2, 60 mM b-glycerophosphate, 15 mM para-nitrophenylphosphate, 15 mM EDTA, 15 mM MgCl2, 2 mM DTT, 1 mM sodium orthovanadate, 1 mM NaF, 1 mM phenylphosphate, 10 mg/mL leupeptin, 10 mg/mL aprotinin, 10 mg/mL soybean trypsin inhibitor, 10 mM benzamidine). Samples were centrifuged for 10 min at 12,000 G and the protein concentration of supernatants was determined using the Bradford assay (BioRad) and a spectrophotometer reading at 595 nm (SPECTROstar Nano, BMG LABTECH).

Proteins were denatured in 2X Laemmli buffer (65.8 mM TRIS-HCl pH 6.8; 26.3% glycerol; 2.1% SDS; 0.01% bromophenol blue; 4% β-mercaptoethanol, BioRad) at 75°C for 10 min. 15 µg of proteins were separated on 4-20% SDS PAGE gels (mini protean TGX, BioRad), for 1 h at 200 V in denaturing buffer (0.1% SDS; 0.3% TRIS base; 1.44% glycine). Proteins were transferred onto nitrocellulose membrane (Amersham Hybond, Dutscher) by wet transfer P a g e | 70 (0.32% TRIS; 1.8% glycine; 20% methanol, Sigma-Aldrich), for 1 h at 100 V. Membranes were saturated with 5% low fat dry milk in TBS (15 mM de TRIS, 140 mM de NaCl: Sigma) added with 0.05% Tween (Sigma-Aldrich), and incubated overnight at 4°C with specific primary antibodies: rabbit polyclonal antibodies against ATM (Cell Signaling technology (CST, Ozyme), 1/1000), ATR (CST, 1/750), phosphorylated ATR (S428, CST, 1/1000), Beclin-1 (CST, 1/800), Cdc25C (CST, 1/1500), phosphorylated Cdc25C (S216, CST, 1/1000), phosphorylated Cdk1 (Y15, CST, 1/1500), phosphorylated Chk1 (S317, CST, 1/1000), phosphorylated Chk2 (T68, CST, 1/1000), cleaved caspase 3 (CST, 1/1000), phosphorylated H2AX (S139, CST, 1/750), histone H3 (CST, 1/1000), phosphorylated H3 (S10, CST, 1/1000), phosphorylated p53 (S15, CST, 1/1000), p53 (CST, 1/1000), p21 (CST, 1/1000), LC3 (CST, 1/50), mTOR (CST, 1/1200), RAPTOR (CST, 1:1500), phosphorylated RAPTOR (S792, CST, 1/1000); mouse monoclonal antibodies against phosphorylated ATM (S1981, Santa Cruz Biotechnology (SCB), 1/200), Chk1 (SCB, 1/1000), Chk2 (SCB, 1/ 200), Cdk1 (CST, 1/1000), 14-3-3 (SCB, 1/1000), cyclin B2 (CST, 1/1500), p62 (SCB, 1/100); goat polyclonal antibodies against β-actin (SCB, 1/1200); and cocktail antibodies against cleaved PARP (Abcam, cell cycle and apoptosis cocktail, 1/1500). After three washes of 10 min in TBS-Tween, nitrocellulose membranes were incubated for 1 h with the appropriate horseradish peroxidase-labeled secondary antibodies: anti-rabbit or anti-mouse antibodies (Invitrogen, Thermo Fisher Scientific Biosciences, 1/30,000) or anti-goat antibodies (SCB, 1/30,000). Secondary antibodies were washed in TBS-Tween three times for 10 min and the signals were revealed with a chemiluminescent assay (ECL Select, GE Healthcare, Dutscher) on hyperfilms (Amersham hyperfilm MP, Dutscher). β-actin or histone H3 were used as loading controls. Signals were quantified with Image J (Fiji Software, v1.52i), and normalized to respective loading control.

For immunoprecipitation, protein samples were pre-cleared with protein A sepharose (20 µL of 50% beads/200 µL of cell lysate, Sigma-Aldrich) for 1 h at 4°C under gentle rocking. After brief centrifugation, supernatants were incubated with antibodies against 14.3.3 (Santa Cruz Biotechnology), Cdc25C (Thermo Fisher Scientific Biosciences GMBH, 1/200) or mTOR (CST, 1/200) at 4°C for 1 h under rotation and followed by incubation with protein A sepharose (20 mL of 50% bead slurry, Sigma-Aldrich) for 1 h at 4°C under rotation. Samples were rinsed 3 times with RIPA buffer. Pellets were collected by brief centrifugation, resuspended in 2X Laemmli buffer, and heated at 100°C for 10 min before SDS-PAGE and Western blots were performed.

TOXICITY ON XENOPUS LAEVIS EMBRYOS

Principle

Xenopus laevis amphibian originates from South Africa. Xenopus eggs and embryos are easy to obtain (high number of gametes, and rapid external development), making them P a g e | 71 perfect developmental models to study diverse aspects of embryogenesis and to assess the effects of various drugs and treatments [START_REF] Ishibashi | Xenopus as a Model Organism for Biomedical Research[END_REF][START_REF] Slaby | Cadmium but not lead exposure affects Xenopus laevis fertilization and embryo cleavage[END_REF]. At the midblastula transition (after the 12th cell cycle, NF stage 9 (Nieuwkoop and Faber), and 7 h after fertilization), Xenopus embryos can activate large-scale zygotic transcription, zygotic cell cycle are modified, and multiple pathways are activated to respond to DNA damage [START_REF] Finkielstein | The midblastula transition in Xenopus embryos activates multiple pathways to prevent apoptosis in response to DNA damage[END_REF]. WN197 toxicity was tested on Xenopus embryonic development.

Animals and embryos handling

All animal experiments were performed according to the rules of the European Community Council guidelines (86/609/EEC) for laboratory animal experimentation. The vertebrate animal protocol was approved by the local institutional review board (Comité d'Ethique en Expérimentation Animale, Hauts-de-France, G59-00913). Mature Xenopus laevis females, purchased from the CRB-University of Rennes I (Rennes, France) and housed in PHExMAR, at the University of Lille, were anesthetized by immersion in 1 g/L MS222 solution (tricaine methane sulfonate, Sigma Aldrich). After anesthesia in 3g/L MS222 solution, males were sacrificed, and testis were surgically removed and stored in MMR (Marc's modified Ringer's; 100 mM NaCl, 2 mM KCl, 1 mM MgSO4, 2 mM CaCl2, 0.1 mM EDTA, 5 mM HEPES, pH 7.8 at 4°C). Ovulation was induced by injection of human chorionic gonadotropin (hCG) ranging from 500 U to 700 U, into the dorsal lymph sac of the female frogs. After 20 h, smooth pressure was applied on the female ventral side to help the egg-laying process. For fertilization, eggs were collected, and immediately placed in contact with a piece of sliced testicle in sterile water with or without doxorubicin or WN197 at 0.5 or 5 µM under gentle agitation for 10 min. The eggs thick jelly coat was removed using an incubation under gentle rocking with L-cysteine (2%). Eggs were rinsed 3 times, exposed or not to doxorubicin or WN197 at 0.5 or 5 µM, and development was followed at 22°C. The number of dead embryos appearing as swollen structures with disorganized pigments was counted at different

RESULTS

EFFECT OF WN197 ON ADENOCARCINOMAS' DNA

To determine if WN197 induces DNA damage in the adenocarcinoma cell lines MDA-MB-231, HeLa, and HT-29, immunofluorescences detection of γH2AX (phosphorylated form) were proceeded. DNA breaks were visualized by γH2AX foci (dots) in the cell nucleus. In control with DMSO treatment (solvent control, 0.5%) for 24 h, MDA-MB-231 and HeLa presented no γH2AX foci, and HT-29 a very low quantity of dots in the range of the background noise (Figures 123). After doxorubicin (5 µM) or cisplatin (20 µM) treatments (positive controls) cell lines displayed a large amount of γH2AX foci. WN197 or WN170 addition at a concentration close to their IC50 (0.5 µM) produced γH2AX foci. A lower number of foci were observed for WN170 in MDA-MB-231 and HT-29 cells, and a small portion in the HeLa cells compared to WN197 that generated more γH2AX foci in the three cell lines.

γH2AX foci were quantified (Figure 4A). In the DMSO controls, a mean of 10, 9 and 7 foci per cell was obtained respectively for MDA-MB-231, HeLa, and HT-29. Doxorubicin and cisplatin induced a significant increase in the mean of foci per nucleus compared to the negative controls: 90 and 60 foci per cell in MDA-MB-231, 127 and 76 in HeLa, 70 and 30 in HT-29. In the WN170 condition, the mean of foci per cell was not significant compared to the control with 30 foci in MDA-MB-231, 25 in HeLa, and 15 in HT-29. After WN197 treatments, the mean of foci was significantly higher compared to the control and WN170, and equal to doxorubicin and cisplatin conditions: 100 γH2AX foci were obtained in MDA-MB-231, 98 in HeLa, and 70 in HT-29.

Western blots were performed to confirm the results obtained by immunofluorescence (Figure 4B). No γH2AX signal was visible in the control conditions for the three cell lines. On the contrary, γH2AX signals were detected in the doxorubicin and cisplatin positive control conditions. For WN170, a signal was obtained 1.9 times superior to the control in the MDA-MB-231 (quantification by densitometry), 2.6 times superior in HeLa, and 1.4 times superior in HT-29. With WN197, the signals were higher (2.8, 3.5, 4.8

respectively).

To determine if WN197 could induced early DNA damage in the cancer cells, immunofluorescence experiments against γH2AX were realized after 30 min of treatment and the foci quantified (Figure 4C). In the untreated conditions, a mean of 4 foci was obtained in MDA-MB-231, 31 in HeLa, and 3 in HT-29. A significant number of foci was observed after doxorubicin and WN197 treatments, with an average of 63 and 84 foci respectively in MDA-MB-231, 106 and 87 in HeLa, and 27 and 13 in HT-29.

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EFFECT OF WN197 ON HUMAN TOPOISOMERASE ACTIVITY

To determine whether WN197 was capable to inhibit topoisomerase 1 (Top1), in vitro topoisomerase activity tests were realized (Figure 5A). The test relies on the ability of Top1 to relax a supercoiled plasmid DNA (SCDNA). In presence of Top1, SCDNA had a relaxed profile on the agarose gel that migrated further compared to RDNA (relaxed DNA). When camptothecin (CPT), a well-known Top1 inhibitor, was added to the enzymatic reaction, the relaxation of the DNA was disturbed and a part of the DNA remains supercoiled. Etoposide (VP-16), a Top2 inhibitor (negative control), and DMSO (solvent control) had no inhibitory effect on DNA relaxation. WN197 inhibited DNA relaxation from 0.2 µM to 2 µM, indicating a Top1 inhibition.

Top1 inhibitors can act as poison or catalytic inhibitors. A poison will prevent DNA religation by Top1, inducing the accumulation of a nicked DNA (NDNA) (Pommier et al., 2015). The addition of proteinase K into the reaction allowed the detection of NDNA on an agarose gel. NDNA was visible in presence of CPT, a Top1 poison (Figure 5A). NDNA was also visible in presence of WN197 at 0.2 µM. At higher doses of WN197 (0.5 µM to 2 µM), the DNA migration profile was disturbed with no presence of NDNA, showing that WN197 is not a poison at higher doses but could act as a catalytic inhibitor by intercalation in the DNA. WN197 effect on Top2α and Top2β was also assayed (Figure 5B and5C). In the presence of Top2α (Figure 5B) or Top2β (Figure 5C), a plasmid SCDNA was relaxed (topoisomers). Etoposide (VP-16), a Top2 inhibitor, avoided the relaxation of DNA. DMSO (solvent control) and CPT (Top1 inhibitor) did not affect DNA relaxation by Top2α and Top2β. WN197 inhibited the relaxation of DNA by Top2α at 2 µM and had a Top2β inhibitory effect at 1 µM and 2 µM as no topoisomers (relaxed DNA) were visible, but the migration profile is disturbed. DNA intercalation of WN197 could explain this disturbed migration. (Surova and Zhivotovsky, 2007). Western blots of DDR effectors have been proceeded to verify its activation in MDA-MB-231, HeLa, and HT-29 (Figure 6). Activating phosphorylation of ATR (S428) and ATM (S1981) were found in the three cell lines treated with WN170, WN197, and the positive controls doxorubicin and cisplatin after 24 h of treatments, with higher signals compared to the untreated cells. The ATM and ATR signals were higher after WN197 treatment compared to WN170 and positive controls, except for ATM in doxorubicin treated HeLa. The downstream proteins of the DDR pathway, Chk1 and Chk2, were also activated (S317 and T68 phosphorylation respectively) in the cisplatin, WN170 and WN197 conditions in the three cell lines, to higher levels compared to the control. Subsequent phosphorylation on S15 of the effector p53 was also visualized: a higher signal was found after 24 h of treatment with WN197 in the three cell lines compared to the untreated controls. The same higher results were obtained for the quantities of p21, a target gene of p53. In doxorubicin and cisplatin treated cell lines, quantities of p53 and p21 were not increased except for p53 in MDA-MB-231 and p21 in HT-29 cells. were treated for 24 h with doxorubicin (5 µM, Dox), cisplatin (20 µM, Cis), WN170 (0.5 µM), or WN197 (0.5 µM). Western blots were performed to detect ATM, ATR, Chk1, Chk2, p53, and their active phosphorylated forms, and p21. β-actin was used as a loading control and relative protein levels were quantified by densitometry using Image J software (Fiji Software, v1.52i). Results are representative of three independent experiments. P a g e | 82

EFFECT OF WN197 ON THE ADENOCARCINOMAS' CELL CYCLE

MDA-MB-231, HeLa, and HT-29 cells were exposed for 24 h to different treatments and analyzed by flow cytometry to investigate possible cell cycle phase accumulation (Figure 7A). Untreated cells showed a classical cell cycle repartition in the three cell lines with a mean of 50.52% cells in G0/G1 phases, 29.80% in the S phase, and 19.68% in the G2/M phases. Cisplatin, a well-known S phase blocking control [START_REF] Wagner | Cisplatin-Induced DNA Damage Activates Replication Checkpoint Signaling Components That Differentially Affect Tumor Cell Survival[END_REF], induced significant accumulation of 79.79%, 59.61%, and 85.53% cells in S phase for MDA-MB-231, HeLa, and HT-29 cells, respectively. Nocodazole, a mitotic spindle poison and a control for M phase accumulation, induced 70.17%, 88.61%, and 39.68% cells in G2/M phase for MDA-MB-231, HeLa, and HT-29, respectively. After treatment with WN170, MDA-MB-231 did not present a perturbated cell cycle (21.08% of cells in the G2/M phase), while an accumulation in the G2/M phase was observed for the HeLa and HT-29 (54.19% and 48.06% respectively). An accumulation in the G2/M phase was also obtained after a WN197 treatment with 51.29% for the MDA-MB-231, 70.51% for the HeLa, and 74.40% for the HT-29, which was significantly higher compared to the untreated controls, WN170, and nocodazole in HT-29.

To determine the sufficient dose of WN197 to obtain the G2/M accumulation, MDA-MB-231, HeLa, and HT-29 were treated for 24 h with increasing doses of WN197 (Figure 7B). A significant accumulation in the G2/M phase was obtained from 0.5 µM to 1 µM in MDA-MB-231, 0.25 µM to 0.5 µM for the HeLa, and 0.25 µM to 1 µM for the HT-29.

A kinetic treatment with WN197 at 0.5 µM was realized on the three adenocarcinoma cell lines to determine the earliest-induced G2/M accumulation (Figure 7C). The G2/M accumulation is obtained from 8 h to 48 h in MDA-MB-231, 12 h to 48 h in HeLa, and 12 h to 24 h in HT-29. The cell cycle arrest phase was further determined by Western blot analysis of major cell cycle regulators: Cdk1, cyclin B, Cdc25C phosphatase, and histone H3 (Figure 8A). The inhibitory phosphorylation on Cdk1's Y15 was increased after 24 h of treatment with cisplatin, WN170, and WN197 compared to the untreated controls in the three adenocarcinoma cell lines. No increase in the signal was obtained after doxorubicin treatment, while a decrease was obtained in the three cell lines for nocodazole, and also in the doxorubicin condition for HeLa and HT-29. The cyclin B level was increased after a treatment with WN170 and WN197 in the three cell lines, with cisplatin in MDA-MB-231 and HeLa, with nocodazole in MDA-MB-231 and HT-29, but a decrease was observed in doxorubicin conditions. Cdc25C inhibitory phosphorylation on S216 was also assayed. The phosphorylation was enhanced by cisplatin and WN197 treatments compared to untreated conditions in the three cell lines, and was slighter compared with WN170. On the contrary, a decrease of this phosphorylation was obtained after doxorubicin and nocodazole treatments, except for HT-29. All cell cycle markers were increased to higher level in WN197 condition compared to WN170. Finally, the S10 phosphorylated form of histone H3, an M phase marker, was increased only in the nocodazole condition for the three adenocarcinoma cell lines.

Cdc25C complexation with 14-3-3 was assayed by immunoprecipitation and visualized by Western blots (Figure 8B). After a Cdc25C immunoprecipitation, no 14-3-3 is found bound to the protein in the untreated conditions, while it was after cisplatin and WN197 treatments in the three cell lines. 14-3-3 reverse immunoprecipitations were performed in HeLa to ascertain the results, and Cdc25C was also found in cisplatin and WN197 treatment conditions. P a g e | 85 Cells were treated for 24 h with doxorubicin (5 µM), cisplatin (20 µM), WN170 (0.5 µM), WN197 (0.5 µM), or nocodazole (84 nM). β-actin was used as a loading control. For H3 S10 phosphorylation, respective H3 total levels were used as loading controls. Relative protein levels were expressed by densitometry using Image J software (Fiji Software, v1.52i). (B) 14-3-3 and Cdc25C immunoprecipitations were realized in cell lines treated for 24 h with cisplatin (20 µM) or WN197 (0.5 µM). Results are representative of three independent experiments.

EFFECT OF WN197 ON ADENOCARCINOMAS' CELL DEATH

Apoptosis, a classical induced cell death after DNA damage and topoisomerase inhibition, was assayed by Western blots (Figure 9A-C). The activated cleaved caspase 3 (early apoptosis marker) was found after 24 h of treatment with doxorubicin, cisplatin and nocodazole in MDA-MB-231, HeLa, and HT-29, while no signal was obtained in the untreated control, WN170 and WN197 conditions (Figure 9A). The inactivated cleaved PARP (late apoptosis marker) was only found in doxorubicin treated condition for the three cell lines. A time-course detection of cleaved PARP and cytochrome c release in the cytoplasm at 3, 16, P a g e | 8624, 48, and 72 h was proceeded, and no signal was obtained after WN197 treatments, compared to doxorubicin apoptosis positive control at 24 and 48 h (Figures 9B,C). In addition, annexin V-Propidium iodide (PI) tests were realized (Figure 9D): a low percentage of cells were found in the early and late apoptosis phases when untreated (controls) for the three cell lines, with a mean of 6.89% for MDA-MB-231, 18.44% in HeLa, and 25.72% in HT-29. After WN170 or WN197 treatments, a mean of respectively 8.6% and 13.6% cells were found in apoptosis in MDA-MB-231, 27.2% and 21.9% HeLa, and 20.5% and 23.4% in HT-29. We then investigated whether autophagy was induced by WN197 and WN170. In the three adenocarcinoma cell lines, several autophagy markers were detected: p62 degradation, Beclin-1 synthesis, and LC3-II formation (Figure 10A). A p62 signal was obtained for the untreated cells but none after rapamycin (autophagy inducer), WN170 or WN197 treatments for 24 h in the three cell lines. On the contrary, Beclin-1 signal was obtained only in the treated cells. LC3-I associated to phosphatidyl-ethanolamine (LC3-II form) was not obtained in the untreated control cells or after treatment with WN170 in MDA-MB-231 and HeLa, but found after rapamycin and WN197 addition in the three cell lines, and only in HT-29 for WN170.

The complexation of mTOR to its phosphorylated (S792) RAPTOR partner occurs in the mTORC1 complex during autophagy and was visualized by immunoprecipitations (Figure 10B). In cells treated with doxorubicin (apoptosis inducer), or untreated, mTOR is not complexed with RAPTORS792. Differently, the two proteins are complexed after 24 h of treatment with rapamycin, an autophagy inducer, and after the treatment with WN197. Finally, autophagic vacuoles (lysosomes, phagosomes) were visualized by immunofluorescence and quantified (Figure 11A,B). In MDA-MB-231, a mean of 7 vacuoles per cell was obtained in the untreated control, while a significant number of vacuoles were obtained after 24 h of treatment with rapamycin or WN197, with a mean of 19 and 22 vacuoles per cell respectively. For HeLa, a mean of 8 vacuoles per cell was obtained in the control, 353 with rapamycin, and 111 with WN197, both significant results compared to the control. For HT-29, a mean of 2 vacuoles per cell was obtained, and 18 and 17 vacuoles per cell were scored after rapamycin and WN197 treatments respectively, also significant compared to the control.

EFFECT OF WN197 ON TUMORIGENIC AND NON-TUMORIGENIC BREAST CELLS

MTS viability assay was proceeded on non-cancerous MCF-10A cells (Table 1). The obtained IC50 for WN197 was 1.080 µM, compared to formerly obtained IC50 on adenocarcinomas with 0.144 µM, 0.22 µM, and 0.358 µM respectively for the MDA-MB-231, HeLa, and HT-29 cell lines. The concentration required to inhibit 50% of the MCF-10A viability is significantly higher compared to the mean obtained on the three adenocarcinomas IC50. To complete the results obtained on breast cells (cancerous MDA-MB-231, MCF-7 (see IC50 introduction §1.2.2, page 55); and non-cancerous MCF-10A), MTS viability assays were performed on two other triple-negative breast cancer cell lines: BT-549 and SUM-159 (Table 2). The IC50 obtained on BT-549 were 0.676 µM and 0.691 µM after WN197 and WN170 treatments respectively. The IC50 obtained on SUM-159 were 0.679 µM and 0.689 µM after WN197 and WN170 treatments respectively. No significative differences between the two compounds are obtained.

EFFECT OF WN197 ON MULTI-DRUG RESISTANT H69AR

MTS viability assay was proceeded on the multi-drug resistant cancer cells H69AR (Table 3). IC50 were 0.683 µM for WN197, and 4.966 µM for WN170. The value obtained for WN170 was increased compared to the IC50 mean of non-resistant adenocarcinomas (mean of P a g e | 90 0.550 µM), while this value stays close to the IC50 mean obtained for WN197 on non-resistant adenocarcinomas (mean of 0.306 µM).

EFFECT OF WN197 ON XENOPUS LAEVIS EMBRYOS

WN197 cytotoxicity was determined on Xenopus laevis embryos by comparing the course of early development after treatment or not (Figure 12). After treatments with 0.5 or 5 µM of doxorubicin, the treated embryo/control embryo ratio was significantly decreased at the neurula stage (mean of 0.38 and 0.41 respectively) compared to the control (mean of 0.77), and the tailbud stage (mean of 0.20 and 0.18 respectively) compared to the control (mean of 0.74). No significant decreases were obtained after treatments with 0.5 or 5 µM of WN197, with a mean ratio of 0.95 and 0.92 at the gastrula stage, 0.88 and 0.83 at the neurula stage, and 0.85 and 0.79 at the tailbud stage compared to the control mean of 0.86. I studied the properties of the WN197 organometallic compound derived from indenoisoquinolines during my thesis, and analyzed its effects on cancerous cells and in nontumorigenic models, in regards to DNA damage, cell cycle arrest, cell death, and topoisomerase activity (Figure i).

The results obtained by immunofluorescence after 30 min and 24 h of treatment with WN197 showed a high number of γH2AX foci, in MDA-MB-231, HeLa, and HT-29 cancer cells, that highlights DNA breaks induced by WN197. Phosphorylation of H2AX histone was realised in particular after double-strand breaks (DSB), notably after topoisomerase inhibition by poisons, and served as a topoisomerase inhibitor marker (Pommier and Cushman, 2008). In addition, DNA breaks were higher in number after treatments with WN197 compared to WN170, and the results were confirmed by Western blots.

Parallelly, in vitro topoisomerase activity tests revealed that the WN197 compound inhibited topoisomerase 1 (Top1) at low doses, corresponding to the IC50 (0.2 µM), acting as a poison inhibitor, while at higher doses (> 1 µM) it behaved as a catalytic inhibitor. WN197 inhibited Top2α and Top2β as a catalytic inhibitor at higher doses (2 µM and 1 µM, respectively). In a previous research published in Nathalie Wambang's thesis, WN197 was demonstrated to intercalate in DNA by melting temperature measurement (ΔTm) and ethidium bromide (EtBr) competition test (Kapp) [START_REF] Wambang | Nouveaux Complexes Organométalliques d'Indéno[1,2-c]isoquinoléines Anticancéreux : Synthèse, Caractérisation et Evaluation Biologique en tant que Ligands de l'ADN et Inhibiteurs de Topoisomérase[END_REF][START_REF] Molinaro | A Novel Copper(II) Indenoisoquinoline Complex Inhibits Topoisomerase I, Induces G2 Phase Arrest, and Autophagy in Three Adenocarcinomas[END_REF]. These results comforted that WN197 acted as a topoisomerase catalytic inhibitor, by intercalation in the DNA. The same mechanism was observed for anthracyclines, capable to switch from a poison activity to a catalytic inhibitory activity at high doses (Pommier et al., 2010).

The activation of the DDR (DNA Damage Response) pathway was demonstrated by Western blots after treatment of the three cancer cells with WN197. ATM/ATR, Chk1/Chk2, p53 and p21 were activated by phosphorylation and p53 and p21 were upregulated. The DDR pathway was shown to induce a G2/M accumulation of the adenocarcinoma cells after WN197 treatments by flow cytometry. The G2/M accumulations were visible starting from 0.5 µM of WN197 for MDA-MB-231 and 0.25 µM for HeLa and HT-29. The cell accumulation in G2/M was lost at 1 µM of WN197 for HeLa and HT-29 and can be related to a different mechanism of action at high doses, due to the intercalation property of WN197. This dose correlated with the in vitro topoisomerase activity tests results. The G2/M accumulation was significative after 8 h of treatment with WN197 for MDA-MB-231, and 12 h for HeLa and HT-29 cell lines. WN170 induced a significant lower accumulation of cells in G2/M at the same dose (0.5 µM).

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Figure i. Deciphering of the molecular mechanisms of the novel copper (II) indenoisoquinoline complex WN197. WN197 inhibits topoisomerase 1 at low doses in a poison mode and forms a ternary complex with the topoisomerase and DNA, leading to strand break accumulation. Phosphorylated H2AX (γH2AX) localizes at the DNA damage sites. The DNA damage response pathway is activated: ATM and ATR kinases are phosphorylated, and subsequently activate Chk1 and Chk2, leading to Cdc25C phosphorylation on serine 216 (S216) and its binding to 14-3-3. Consequently, Cdk1 remains phosphorylated on tyrosine 15 (Y15), impeding the activation of the MPF (Cdk1/Cyclin B) and the phosphorylation of H3 on serine 10 (S10). Cancer cells accumulate in the G2 phase of the cell cycle. The DDR also leads to an increase in p53 and p21 followed by an autophagic cell death characterized by the phosphorylation of RAPTOR on serine 792 (S792) in the mTORC1 complex, the synthesis of Beclin-1, the formation of LC3-II (complex LC3-I/PE), and the degradation of p62.

P a g e | 94

To understand the mechanisms involved in the cell cycle arrest, the amount and the phosphorylation state of the phosphatase Cdc25C were determined by Western blots. After 24 h of treatments with WN197, we observed an increase in the S216 inhibitory phosphorylation of Cdc25C. This phosphorylation allowed the recognition and binding by 14-3-3 verified by immunoprecipitations and prevented the MPF activation (Cdk1/Cyclin B). Indeed, as shown by Western blots, the inhibitory phosphorylation of Cdk1 (Y15) was maintained after treatment with WN197. In addition, an accumulation of Cyclin B was found. Cancer cells can no longer progress in the cell cycle. The histone H3 is only phosphorylated by active MPF in mitosis to allow DNA condensation, making it an M phase marker (Hans and Dimitrov, 2001). After 24 h of treatment with WN197, no histone H3 phosphorylation (S10) was observed, showing that cells were arrested in the G2 phase of the cell cycle.

When DNA breaks are too important, apoptosis is classically activated to induce cell death. Moreover, most of the topoisomerase inhibitors induce apoptosis (Larsen et al., 2003;Pommier, 2013;see §1.1.6, pages 49-53). We have determined by Western blots, immunoprecipitations, and annexin V-propidium iodide analyses that apoptosis is not activated after treatment with WN197, nor WN170. Only a few numbers of topoisomerase inhibitors induce autophagy (e.g. SN-38 in HSC-4 oral squamous cell carcinoma, [START_REF] Tamura | Analysis of type of cell death induced by topoisomerase inhibitor SN-38 in human oral squamous cell carcinoma cell lines[END_REF] as WN197 did in the three adenocarcinomas MDA-MB-231, HeLa, and HT-29. Autophagy was shown by the degradation of p62, the synthesis of Beclin-1, the switch from the LC3-I to the LC3-II form, in addition to the complexation of mTOR to RAPTOR phosphorylated on S792 in the mTORC1 complex, and the formation of autophagic vacuoles visualized by fluorescence. Autophagy cell death has been discussed regarding the gain it could bring compared to apoptosis in cancer treatments leading to cytoprotective effects towards surrounding normal cells around a targeted tumor. However, contradictory opinions have been raised considering autophagy is also protecting cancer cells [START_REF] Kondo | Autophagy and cancer therapy[END_REF], allows cells survival in nutrient deprived situation, and contribute to cancer resistance [START_REF] Hippert | Autophagy in cancer: good, bad, or both?[END_REF][START_REF] Smith | Autophagy, cancer stem cells and drug resistance[END_REF].

As patients bearing triple-negative breast cancer cannot undergo hormonotherapy, it was of prime importance to focus on this type of cancer and determine the toxicity of WN197 towards other breast cancerous cells and a non-cancerous cell line. The IC50 obtained on MDA-MB-231 for WN197 is the lowest with 0.144 µM in comparison to the four other cancer cell lines tested (Wambang, 2016, see §1.2.2, page 55). We also found low IC50 for BT-549 and SUM-159, two other triple-negative breast cell lines. Furthermore, the IC50 obtained on MCF-10A, a non-cancerous breast cell line, is significantly higher compared to all the adenocarcinomas' IC50 obtained, with 1.080 µM. These interesting results allow to envision WN197 as a possible treatment for breast cancer therapy, especially against triple-negative breast cancer, bypassing toxicity on surrounding healthy cells.

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Another approach was to determine if WN197 was able to avoid cancer cell resistance, by testing the viability of H69AR by MTS, a pulmonary MDR (multi-drug resistant) cell line. The IC50 obtained, 0.683 µM, showed that WN197 kept its cytotoxicity on MDR cells, leading to the conclusion that WN197 could be used as a secondary treatment for patients to overcome multi-drug resistance. WN170 did not manage to keep its low cytotoxicity on H69AR, with an IC50 of 4.966 µM. This result, in addition to those obtained for WN170 on the DDR, cell cycle and cell death, showed that the symmetric indenoisoquinoline structural core containing copper atoms brings specificity and increases the activity of WN197 compared to WN170. Previous studies mentioning the presence of metal atoms like iron (Kondratskyi et al., 2017), ruthenium (Wambang et al., 2016), or platin (Dasari and Tchounwou, 2014), demonstrate their increased efficiency and raised interest of organometallic compounds in cancerology (see §1. 1.6, page 49).

Finally, we showed that WN197 had no effect on Xenopus laevis embryonic development, that displays DDR and cell cycle proteins [START_REF] Garner | Studying the DNA damage response using in vitro model systems[END_REF], compared to detrimental effects obtained with doxorubicin. This brings a promising future for WN197 as a chemotherapeutic drug.

PERSPECTIVES

Compound specificity

To confirm the effect of WN197 on topoisomerases, docking evaluations of the compound in complex with the DNA and the topoisomerase are underway in collaboration with Dr. De Ruyck (UGSF). Besides, cell lines with siRNA directed against Top1 have been produced by Dr. Calsou's team (IPBS) and could be used as another way to confirm that the organometallic compounds are specific and do not target other cellular effectors. Indeed, if WN197 cytotoxic effect on cancer cells is lost while Top1 has been silenced, it means no other molecular effectors are targeted.

Although WN197 inhibits Top1 and induces DNA breaks shown by the phosphorylation of H2AX, this mechanism is not exclusive. Other mechanisms can be parallelly activated to provoke DNA breaks, like ROS (Reactive Oxygen Species). ROS production is induced by Top2 inhibitors like anthracyclines, in addition to their poison activity (Pommier et al., 2010;Pommier 2013), but also by other Top1 and Top2 inhibitors (see compounds from Table IV in the introduction). Furthermore, organometallic compounds tend to induce ROS production ( §1. 1.6, page 49). Nevertheless, ROS is mainly related to apoptosis [START_REF] Simon | Role of reactive oxygen species (ROS) in apoptosis induction[END_REF]. The fact that no apoptosis is found after treatment with WN197 might be a clue about ROS production, but other deaths can be linked to ROS, including autophagy [START_REF] Gibson | A matter of balance between life and death: targeting reactive oxygen species (ROS)induced autophagy for cancer therapy[END_REF]. Potential ROS production after treatments with WN197 will be measured with oxidizable P a g e | 96 probs, like CellROX TM that gets oxidized in presence of ROS, and emits measurable fluorescence [START_REF] Soares | A tryptophanol-derived oxazolopiperidone lactam is cytotoxic against tumors via inhibition of p53 interaction with murine double minute proteins[END_REF].

Cancer resistances

We have shown with the results on H69AR cells that WN197 is keeping its cytotoxic effect on cancer cells that have developed a resistance to previous chemotherapeutic agents (adriamycin, daunomycin, epirubicin, menogaril, mitoxantrone, acivicin, etoposide, gramicidin D, colchicine, vincristine, and vinblastine). Nevertheless, H69AR cells do not overexpress MDR transporters responsible for a part of cancer resistance [START_REF] Cole | Overexpression of a transporter gene in a multidrug-resistant human lung cancer cell line[END_REF]. A cell line overexpressing P-glycoproteins could further be used to test the capability of WN197 to overpass this resistance [START_REF] Peng | Overexpression of P-glycoprotein induces acquired resistance to imatinib in chronic myelogenous leukemia cells[END_REF].

Combined anticancer therapies

To enhance the cytotoxicity of drugs in chemotherapy and ensure a response to the drugs, the association of WN197 to other compounds, such as PARP inhibitors and the use of synthetic lethality action mode is another perspective. PARP is required for DNA repair, and the combination of PARP inhibitors like olaparib with the Top1 inhibitor SN-38 showed an interesting synergic effect [START_REF] Tahara | The use of Olaparib (AZD2281) potentiates SN-38 cytotoxicity in colon cancer cells by indirect inhibition of Rad51mediated repair of DNA double-strand breaks[END_REF]. Another synergic effect is obtained when veliparib, a PARP inhibitor, is associated with irinotecan [START_REF] Murai | Rationale for poly(ADP-ribose) polymerase (PARP) inhibitors in combination therapy with camptothecins or temozolomide based on PARP trapping versus catalytic inhibition[END_REF]. A phase I trial showed that the combination of the PARP inhibitor veliparib with topotecan was effective but not tolerated by patients due to hematological toxicities [START_REF] Kummar | Phase I study of PARP inhibitor ABT-888 in combination with topotecan in adults with refractory solid tumors and lymphomas[END_REF]. Another phase I clinical trial showed that the combination of veliparib with etoposide was well tolerated in SCLC [START_REF] Owonikoko | A phase 1 safety study of veliparib combined with cisplatin and etoposide in extensive stage small cell lung cancer: A trial of the ECOG-ACRIN Cancer Research Group (E2511)[END_REF]. An additional phase I trial showed that olaparib PARP inhibitor in combination with the pegylated liposomal doxorubicin could generate good response in patients with ovarian cancer with good tolerance, with only 2 patients out of 44 exhibiting high-grade dose-limiting toxicity (Del [START_REF] Conte | Phase I study of olaparib in combination with liposomal doxorubicin in patients with advanced solid tumours[END_REF].

The combination of topoisomerase inhibitors with ATR inhibitors has also been tested to inhibit the DDR that could repair the DNA damage induced by topoisomerase inhibitors. The ATR inhibitor M6620 in combination with topotecan is in a current phase II clinical trial against SCLC [START_REF] Thomas | Phase I Study of ATR Inhibitor M6620 in Combination With Topotecan in Patients With Advanced Solid Tumors[END_REF].

Combination of WN197 to TDP1/2 inhibitors is also an interesting prospect. TDP1 and TDP2 are enzymes responsible for the reparation of DNA breaks induced by topoisomerase inhibitors (Pommier 2016). They act by cleaving the Top1 (TDP1) or Top2 (TDP2) tyrosyl-DNA covalent bond, and leave a 3' or 5'-phosphatase ends repaired by polynucleotide kinase phosphatase, DNA polymerases, and ligases (Pommier 2014;[START_REF] Kawale | Tyrosyl-DNA phosphodiesterases: rescuing the genome from the risks of relaxation[END_REF]. Inhibitors of TDP1 and TDP2 exist, such as neomycin, rolitetracycline, and deazaflavin (Huang et al., 2012;[START_REF] Kawale | Tyrosyl-DNA phosphodiesterases: rescuing the genome from the risks of relaxation[END_REF]. The combination of TDP inhibitors alongside P a g e | 97

topoisomerase inhibitors could have a synergic effect by inducing DNA damage and inhibiting their reparation.

For all these reasons, synergetic effects could be tested for WN197 with additional inhibitors described hereby.

Organoids models of cancer

To pursue the research of WN197 as an anti-cancerous compound, tests on organoids can be proceeded. In an ongoing effort to develop alternatives and reduce animal experimentations, organoids are good compromise because they possess a topology and cell to cell interaction alike in vivo tissues. The WN197 compound can be tested on cancerous organoids but also non-cancerous organoids. Preliminary results were obtained on cancerous colon organoids (TraCo2) and non-cancerous colon organoids (Col). Organoids were treated with 1 µM of WN197 or 0,1% DMSO (solvent control) for 72 h and pictures were taken every 3 h. The results showed that the cancerous colon had a dead morphology after treatment with WN197, while the non-cancerous colon did not present a dead morphology (Appendice 4). The results will be confirmed with further experiments at various doses and times.

Prospects for organoids experiments are the development of colorimetric assays with tetrazolium salt like MTS or CCK-8 to evaluate organoids viability [START_REF] Zeng | Inhibition of autophagy with Chloroquine enhanced apoptosis induced by 5-aminolevulinic acid-photodynamic therapy in secondary hyperparathyroidism primary cells and organoids[END_REF]. After the treatment of organoids with various doses of WN197 for various times, the tetrazolium salt can be used and measured by spectrophotometry at the corresponding wave length to determine IC50. The other prospect on organoids is to determine the presence of apoptosis or autophagy proteins after WN197 treatment. However, adjustments will be necessary as these experiments remain difficult to carry out in a context where the organoids are cultured in small quantities in a surrounding matrix.

Structural enhancement

Structural modifications of WN197 could be undertaken to enhance its efficiency. For example, the addition of sugar bounds that could increase the drug permeability. The addition of a carbohydrate chain has been previously shown to enhance the antiproliferative activity of indenoisoquinoline (Beck et al, 2014).

Another possible approach would be the addition of a light-activating unit to the drug. The topoisomerase 1 inhibitor, SN-38, has been modified with the addition of a phototriggered moiety (nitrovanillin) and a cancer targeting unit (biotin). This drug, PT-1 (phototherapeutic agent 1), is activated upon light excitation and therefore permits the activation of the topoisomerase inhibitor at determined location to limit side effects [START_REF] Shin | Programmed activation of cancer cell apoptosis: A tumor-targeted phototherapeutic topoisomerase I inhibitor[END_REF]. Finally, my thesis results demonstrate that WN197 is a novel anti-cancerous agent by inhibiting topoisomerase 1 at low doses, less toxic for healthy cells, that could be an interesting candidate as a new chemotherapeutic drug for pre-clinical studies. Topoisomerases, targets of inhibitors used in chemotherapy, induce DNA breaks accumulation leading to cancer cell death. A newly synthesized copper(II) indenoisoquinoline complex WN197 exhibits a cytotoxic effect below 0.5 µM, on MDA-MB-231, HeLa, and HT-29 cells. At low doses, WN197 inhibits topoisomerase I. At higher doses, it inhibits topoisomerase IIa and IIb, and displays DNA intercalation properties. DNA damage is detected by the presence of gH2AX. The activation of the DNA Damage Response (DDR) occurs through the phosphorylation of ATM/ATR, Chk1/2 kinases, and the increase of p21, a p53 target. WN197 induces a G2 phase arrest characterized by the unphosphorylated form of histone H3, the accumulation of phosphorylated Cdk1, and an association of Cdc25C with 14.3.3. Cancer cells die by autophagy with Beclin-1 accumulation, LC3-II formation, p62 degradation, and RAPTOR phosphorylation in the mTOR complex. Finally, WN197 by inhibiting topoisomerase I at low concentration with high efficiency is a promising agent for the development of future DNA damaging chemotherapies.

Keywords: indenoisoquinoline, copper(II) complex, adenocarcinoma, topoisomerase, cell cycle, autophagy Abbreviations: ATM, ataxia telangiectasia mutated; ATR, ataxia telangiectasia related; BSA, bovine serum albumin; Cdc25, cell division cycle 25; Cdk1, cyclin dependent kinase 1; Chk1/2, checkpoint kinases 1/2; c-IAP1, cellular inhibitor of apoptosis protein 1; DDR, DNA damage response; DNA-PK, DNA-dependent protein kinase; DSB, double strand break; H2AX, H2A histone family member X; IC 50 , half maximal inhibitory concentration; Kapp, apparent dissociation constant; mTOR, mammalian target of rapamycin; MTS, 3-( 4

INTRODUCTION

Adenocarcinomas are the most diagnosed cancers. Among them, breast and cervix, respectively the first and fourth most represented cancers in women, and colorectal cancers the second and third most represented cancers respectively in women and men (1). Current treatments include chemotherapy with agents that generate DNA damage to trigger cancer cell division arrest and associated programmed cell death of tumours (2,3).

Topoisomerases (Top) regulate DNA topology during replication, transcription, and chromosomal segregation (4)(5)(6). To relieve torsional strain, these DNA-interacting enzymes cleave one or two DNA strands before the religation step (7,8). Human Top are subdivided into three subgroups including IA (Top3a and Top3b), IB (Top1 nuclear and Top1 mitochondrial), and IIA (Top2a and Top2b), type I Top cause single-strand breaks (SSB) while type II Top generate double-strand breaks (DSB) (9). In anticancer therapy, inhibition of Top allows DNA cleavage, prevents the religation reaction, and leaves cancer cells with DNA breaks. Top1 and Top2 are mainly targeted due to their overexpression in many cancers including breast, cervix, and colorectal cancers (10)(11)(12)(13). The increased quantity and activity of Top in highly dividing cells directly correlate with positive responses to Top inhibitory treatments (12,14,15). The primary cytotoxic lesions in cancer cells result from collisions between the trapped Top and the replication forks (16)(17)(18). DNA breaks further trigger the activation of DNA Damage Response (DDR) pathways, leading to cell cycle arrest and to death if DNA damage is too severe (19,20). The DDR pathways start with the recruitment and the phosphorylation of histone H2AX on serine 139 (gH2AX) by phosphoinositide 3-kinase related kinase family members ATM, ATR, and DNA-PK (21,22). Consecutively, Chk1 and Chk2 kinases are activated, inhibit phosphatase Cdc25 (23), and induce a cell cycle arrest followed in most cases by apoptosis (20).

Top inhibitors display different action mechanisms. Poisons target the DNA/topoisomerase cleavage complex, form a ternary complex (interfacial inhibition) inhibiting DNA religation, and result in persistent DNA breaks (24). Catalytic inhibitors either intercalate into DNA in the Top fixation site or are ATP competitors or hydrolysis inhibitors to provoke an antineoplastic effect (25). A small number of Top inhibitors are approved for clinical use. The Top2 poison doxorubicin and its isomer epirubicin from the anthracycline family are first-line antineoplastic agents used against many different types of solid tumors, leukemias, and lymphomas (26,27), with main side effects including cardiotoxicity and t-AML (treatment-related acute myelogenous leukemia) (28)(29)(30). At high doses (up to 10 µM), doxorubicin becomes a DNA intercalator and contributes to increase DNA breaks (31,32). Top2 poison etoposide (VP-16) also induces t-AML (9). The Top1 poison camptothecin derivatives, topotecan and irinotecan, are used to treat solid tumors including ovary, cervix, pancreatic, lung, and colorectal cancers (33). However, their use in chemotherapy is limited by their instability, the need for long-term chemotherapies, and by severe side effects including hematotoxicity, vomiting and diarrhea (34). Unlike camptothecins, the Top1 inhibitors indenoisoquinolines are chemically stable, are not substrates for drug efflux transporters and as such are promising Top inhibitors (35,36). Indenoisoquinoline derivatives (LMP400, LMP776, and LMP744) are in phase I/II clinical trials (35,36).

Since the discovery of platinum anticancer properties and the use of cisplatin, a platinum-based alkylating agent, and its derivatives in chemotherapy (37)(38)(39), other metal-based drugs have been designed and developed for their cytotoxic effects on tumour cells (40)(41)(42). Transition metals from the d-block of the periodic table (groups 3 to 12) (43)(44)(45)(46) are particularly suitable for this purpose as they adopt a wide variety of coordination geometries (47). Among them, copper modifies the backbone of the complexed ligand and grants better DNA affinity (48)(49)(50).

Copper derivatives interact with DNA using noncovalent interactions with the major or the minor DNA grooves, intercalation, or electrostatic binding to enhance DNA damage, and display antitumor activity (51). Some copper complexes inhibit either or both Top1 and Top2 and results in severe DNA damage, cell cycle arrest, and death in cancer cells (52,53).

As a part of an ongoing effort to develop new efficient anticancer organometallic drugs and to palliate limitations in drug resistances and/or side effects, the synthesis of a novel copper(II) complex of indenoisoquinoline ligand, named WN197, is established based on previous studies (54,55). This organo-copper complex effects were investigated on breast triple-negative MDA-MB-231, cervix HeLa, and colon HT29 cell lines representative of three most prevalent adenocarcinomas, and associated with poor prognostics. WN197 exerts a specific cytotoxic effect at low concentration (IC 50 below 0.5 µM) on the three cell lines and significantly below the value of human non-tumorigenic epithelial cell line MCF-10A (IC 50 1.08 µM). WN197 acts as a Top1 poison and displays DNA intercalation properties. The action mechanism of WN197 is further deciphered to bring insights into its efficiency. DNA damage is detected by the presence of a rapid increase in nuclear phosphorylated H2AX (after 30 min of treatment with 0.5 µM) and the main DDR kinases are activated by phosphorylations. Cell cycle arrest in the G2 phase is confirmed by the inhibitory phosphorylation of Cdk1 on tyrosine 15, an accumulation of cyclin B, and the unphosphorylated form of histone H3. Furthermore, the cell cycle is halted in G2 by inhibitory phosphorylation of Cdc25C on serine 216 associated with a binding to the 14.3.3 chaperon. Cancer cells halt in G2, die by autophagy detected through an increase in Beclin-1, and a decrease in the LC3-I/ LC3-II ratio and the p62 marker. Moreover, the RAPTOR component in the mTORC1 complex is phosphorylated on serine 792, a feature of autophagic-induced cell death.

MATERIALS AND METHODS

Chemical Reagents and Materials

All commercial reagents and solvents were used without further purification. Cisplatin is purchased from Alfa Aesar (Heysham, UK); rapamycin from Abcam (Cambridge, UK); doxorubicin, nocodazole and DMSO from Sigma-Aldrich (Saint-Quentin-Fallavier, France). Stock solutions were prepared in DMSO. Melting points were determined with a Barnstead Electrothermal (BI 9300) capillary melting point apparatus and are uncorrected. Elemental analyses were performed with a varioMICRO analyser. Thin layer chromatography (TLC) was carried out on aluminium-baked (Macherey-Nagel GmbH, Düren, Germany) silica gel 60. Column chromatography was performed on silica gel (230-400 mesh). The electronic absorption spectra were acquired on a UV-Vis double beam spectrophotometer SPECORD ® PLUS (Analytik Jena GmbH, Germany). The molar conductance measurement was carried out using a CDRV 62 Tacussel electronic bridge, employing a calibrated 10 -2 M KCl solution and 10 -3 M solutions of compounds in DMSO. Purities of all tested compounds were ≥95%, as estimated by HPLC analysis. High Resolution Mass Spectrum (HR-MS) was measured at REALCAT (Universitéde Lille) on a Synapt G2Si (Waters) equipped with an ion mobility cell.

WN197 Copper(II) Indenoisoquinoline Complex Synthesis

WN170 was synthesized according to the literature procedure (56). To a solution of WN170 (160 mg, 0.443 mmol) in dry methanol (8 mL) was added dropwise a solution of CuCl 2 (59 mg, 0.443 mmol) in MeOH (7 mL). After stirring at room temperature for 10 h, the reaction mixture was filtered off to yield an orange precipitate which was washed with MeOH and dried under vacuum (8 h

Cell Culture

HeLa, MDA-MB-231, HT-29 and MCF-10A cell lines originate from ATCC (Manassas, VA, USA), and were maintained at 37°C in a humidified atmosphere with 5% CO 2 in DMEM medium (Lonza, Basel, Switzerland) supplemented with 10% fetal bovine serum (Dutscher, Dernolsheim, France), 1% Zell Shield (Dutscher, Bernolsheim, France) and 1% non-essentials aminoacids (Lonza, Basel, Switzerland). MCF-10A were maintained in MEBM medium (Lonza, Basel, Switzerland) supplemented with MEGM (Lonza, Basel, Switzerland). All cell lines culture media were added with 1% Zell Shield (Dutscher, Bernolsheim, France).

Cell Viability Assay

Cell viability was determined using CellTiter 96 ® AQ ueous One Solution Cell Proliferation Assay (MTS, Promega, Charbonnièresles-Bains, France). 2.10 3 cells well were seeded in 96-well plate for 24 h before treatment with 0 to 100 µM of WN197, WN170 or cisplatin for 72 h. After a 2 h incubation with 20 µL of CellTiter solution at 37°C in 5% CO 2 , the production of reduced MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium) in formazan was measured at 490 nm (SPECTROstar Nano, BMG LABTECH, Ortenberg, Germany). IC 50 were calculated using GraphPad Prism V6.0 software.

Statistical differences between WN197 and WN170 were ascertained by a Student t-test (**p<0.01 and ****p<0.0001).

Immunofluorescence for Nuclei Foci 2.10 5 cells seeded on glass coverslips were treated with 0.5 µM of WN197 or WN170, 5 µM of doxorubicin, 20 µM of cisplatin as positive controls, or 0.1% DMSO as a solvent control for 30 min or 24 h. Fixation was performed with 4% paraformaldehyde (Sigma-Aldrich, Saint-Quentin-Fallavier, France) for 5 min and followed by permeabilization with 0.1% Triton in PBS (Sigma-Aldrich, Saint-Quentin-Fallavier, France) for 10 min and saturation of unspecific sites with 1% BSA in PBS (Sigma) for 1 h at room temperature. Anti-gH2AX mouse antibody (S139, 1:1000, Cell Signalling, by Ozyme, Saint-Cyr-L'E ́cole, France) was incubated overnight at 4°C, washed 3 times with 1% BSA/PBS. Cells were incubated with secondary anti-mouse IgG (Alexa Fluor ® 488, 1:2000, Thermo-Fisher Scientific Biosciences GMBH, Villebon-sur-Yvette, France) for 1 h at room temperature in the dark, washed 3 times before nuclei were stained with DAPI (6-diamidino-2-phenylindole, 1 µg/ mL, Molecular Probes, by Thermo Fisher Scientific Biosciences GMBH, Villebon-sur-Yvette, France). Images were captured under a Leica fluorescent microscope, and gH2AX foci were counted with ImageJ (Fiji Software, v1.52i) on 30 cells from 3 independent experiments and quantified with GraphPad Prism V6.0 software. Statistical significances (mean ± SD) were performed by a two-way ANOVA followed by Dunnett's multiple comparison test (**p<0,01; ***p<0,001; ****p<0,0001).

Electrophoresis and Western Blot 7.5.10 5 cells were seeded for 24 h and treated with 0.5 µM of WN197 or WN170, 20 µM of cisplatin, 5 µM of doxorubicin, or 0.1% DMSO (solvent control). After 24 h, they were lysed in RIPA buffer (1% Triton X-100; 50 mM TRIS-HCl pH 4; NP40 2%; 0.4% Na-deoxycholate; 0.6% SDS; 150 mM NaCl; 150 mM EDTA; 50 mM NaF) supplemented with 1% of protease inhibitor cocktail (Sigma-Aldrich, Saint-Quentin-Fallavier, France) and phosphatase inhibitors (Roche SAS by Merck, Kenilworth, NJ, USA).

For cytochrome C analysis, 7.5.10 5 cells were seeded for 24 h and treated for 3 h, 16 h, 24 h or 48 h with 0.5 µM of WN197, and for 24 h or 48 h with 5 µM of doxorubicin as positive control. Cells were lysed in a glass grinder at 4°C in homogenization buffer (25 mM MOPS at pH 7.2, 60 mM b-glycerophosphate, 15 mM paranitrophenylphosphate, 15 mM EDTA, 15 mM MgCl2, 2 mM DTT, 1 mM sodium orthovanadate, 1 mM NaF, 1 mM phenylphosphate, 10 mg/mL leupeptin, 10 mg/mL aprotinin, 10 mg/mL soybean trypsin inhibitor, 10 mM benzamidine).

Samples were centrifuged for 10 min at 12,000 G and protein concentration of supernatants were determined using the Bradford assay (BioRad, Marnes-la-Coquette, France) at 595 nm (SPECTROstar Nano, BMG LABTECH, Ortenberg, Germany). Proteins were denatured in 2X Laemmli buffer (65.8 mM TRIS-HCl pH 6.8; 26.3% glycerol; 2.1% SDS; 0.01% bromophenol blue; 4% b-mercaptoethanol, BioRad, Marnes-la-Coquette, France) at 75°C for 10 min. 15 µg of proteins were separated on 4-20% SDS PAGE gels (mini protean TGX, BioRad, Marnes-la-Coquette, France), for 1 h at 200 V in denaturing buffer (0.1% SDS; 0.3% TRIS base; 1.44% glycine). Proteins were transferred onto nitrocellulose membrane (Amersham Hybond, Dutscher, Bernolsheim, France) by wet transfer (0.32% TRIS; 1.8% glycine; 20% methanol, Sigma-Aldrich, Saint-Quentin-Fallavier, France), for 1 h at 100 V. Membranes were saturated with 5% low fat dry milk in TBS added with 0.05% Tween (Sigma-Aldrich, Saint-Quentin-Fallavier, France), and incubated overnight at 4°C with specific primary antibodies: rabbit polyclonal antibodies were against ATM (Cell Signaling technology (CST, by Ozyme, Saint-Cyr-L'E ́cole, France), 1/1000), ATR (CST, 1/750), phosphorylated ATR (S428, CST, 1/1000), Beclin-1 (CST, 1/800), Cdc25C (CST, 1/ 1500), phosphorylated Cdc25C (S216, CST, 1/1000), phosphorylated Cdk1 (Y15, CST, 1/1500), phosphorylated Chk1 (S317, CST, 1/1000), phosphorylated Chk2 (T68, CST, 1/1000), cleaved caspase 3 (CST, 1/1000), phosphorylated H2AX (S139, CST, 1/750), histone H3 (CST, 1/1000), phosphorylated H3 (S10, CST, 1/1000), phosphorylated p53 (S15, CST, 1/1000), p53 (CST, 1/1000), p21 (CST, 1/1000), LC3 (CST, 1/50), mTOR (CST, 1/ 1200), RAPTOR (CST, 1:1500), phosphorylated RAPTOR (S792, CST, 1/1000); mouse monoclonal antibodies against phosphorylated ATM (S1981, Santa Cruz Biotechnology (SCB), Santa Cruz, CA, USA, 1/200), Chk1 (SCB, 1/1000), Chk2 (SCB, 1/ 200), Cdk1 (CST, 1/1000), 14-3-3 (SCB, 1/1000), cyclin B2 (CST, 1/1500), p62 (SCB, 1/100); goat polyclonal antibodies against bactin (SCB, 1/1200); and cocktail antibodies against cleaved PARP (Abcam, Cambridge, UK, cell cycle and apoptosis cocktail, 1/1500). After three washes of 10 min in TBS-Tween, nitrocellulose membranes were incubated 1 h with the appropriate horseradish peroxidase-labeled secondary antibodies: anti-rabbit or anti-mouse antibodies (Invitrogen, by Thermo Fisher Scientific Biosciences GMBH, Villebon-sur-Yvette, France, 1/30,000) or anti-goat antibodies (SCB, 1/30,000). Secondary antibodies were washed in TBS-Tween three times for 10 min and the signals were revealed with a chemiluminescent assay (ECL Select, GE Healthcare, Dutscher, Bernolsheim, France) on hyperfilms (Amersham hyperfilm MP, Dutscher, Bernolsheim, France). b-actin or histone H3 were used as loading controls. Signals were quantified with Image J (Fiji Software, v1.52i), and normalized to respective loading control. The means of 3 independent experiments were calculated.

In Vitro Activities of Human Topoisomerases I and II

Topoisomerase activities were examined in assays based on the relaxation of a supercoiled DNA into its relaxed form. Topoisomerase I (Top1) activity was performed using the drug screening kits protocol (TopoGEN, Inc., Buena Vista, CO, USA). The reaction mixture was composed of supercoiled pHOT1 DNA (250 ng), 10X TGS buffer (10 mM Tris-HCl pH 7.9, 1 mM EDTA), 5 units of Top1, a variable amount of compound to be tested, and a final volume adjusted to 20 µL with H2O. WN197 was tested at concentrations ranging from 0.2 to 2 µM. Camptothecin (10 µM) was used as a positive control (poison inhibitor of Top1 activity), etoposide (100 µM) as negative control (inhibitor of Top2 activity), and 1% DMSO alone as vehicle control. Relaxed pHOT1 DNA (100 ng) was used as migration control. The addition of proteinase K (50 µg/mL) for 15 min at 37°C allowed Top1 degradation to visualize the cleavage products (nicked DNA). Reaction products were separated by electrophoresis in a 1% agarose gel containing ethidium bromide (0.5 µg/mL) for 1 h at 100 V in TAE (Tris-Acetate-EDTA; pH 8.3) buffer.

Topoisomerase II Relaxation Assay Kit (Inspiralis, Inc., Norwich, UK) was used to measure topoisomerase II (Top2) activity. The reaction mixture was composed of supercoiled pBR322 DNA (1 µg), 10X assay buffer (50 mM Tris-HCl (pH 7.5), 125 mM NaCl, 10 mM MgCl2, 5 mM DTT, 100 µg/mL albumin), 30 mM ATP, 5 units of Top2a or Top2b, variable amount of compound to be tested, and a final volume adjusted with H2O to 30 µL. Etoposide (VP-16, 100 µM) was used as positive control, and camptothecin (10 µM) as negative control. The mixtures were incubated at 37 °C for 30 min and the reactions stopped by the addition of 5 µL 10% SDS. Reaction products were separated by electrophoresis in a 1% agarose gel for 1 h at 100 V in TAE buffer, and stained with ethidium bromide (0.5 µg/mL) for 15 min. After destaining in water, the DNA migratory profiles were visualized under UV light (ChemiDocTM XRS+, BioRad, Marnesla-Coquette, France).

Melting Temperature Measurement

Melting temperatures were obtained as described (54,55). 20 µM solutions of WN170 or WN197 were prepared in 1 mL of BPE buffer (2 mM NaH 2 PO 4 , 6 mM Na 2 PO 4 , 1 mM EDTA, pH 7.1) in the presence or not of 20 µM DNA from calf thymus (42% GC bp, Merck, Kenilworth, NJ, USA). Absorbances were measured at 260 nm (Uvikon 943 coupled to Neslab RTE111) every minute over the range of 20 to 100°C with an increment of 1°C per minute. All spectra were recorded from 230 to 500 nm. Tested compound results are referenced against the same DNA concentration in the same buffer. The Tm values were obtained from the first derived plots.

Ethidium Bromide Competition Test

Fluorescence titrations were determined as described (54,55). Ethidium bromide/WN170 or WN197 molar ratio of 12.6/10 at concentrations ranging from 0.05 to 10 µM were used in a BPE buffer (pH 7.1). The excitation wavelength was set at 546 nm and the emission was monitored over the range of 560 to 700 nm (SPEX Fluorolog). IC 50 values for ethidium bromide (EB) displacement were calculated using a fitting function incorporated into GraphPad Prism 6.0 software. The apparent binding constants were calculated using the equation K app = (1.26 (K app (EB)/IC 50 ) with K app (EB) =10 7 M -1 and IC 50 in mM.

Flow Cytometry

7.5.10 5 cells plated for 24 h were treated with 0.5 µM WN197 or WN170, 20 µM of cisplatin (S phase arrest control), 83 nM of nocodazole (M phase arrest control), or 0.1% DMSO (solvent control). For the dose titration experiments, cells were treated for 24 h with increasing concentrations of WN197. For kinetic experiments, cells were treated with 0.5 µM of WN197 or WN170 from 4 to 48 h. Cells were detached using trypsin (Biowest, Nuaille, France), centrifuged at 1,000 G for 10 min, resuspended in PBS, and fixed with 70% ethanol at -20°C for 24 h, before they were centrifugated (1,000 G, 10 min), resuspended in PBS, and treated for 15 min at room temperature with RNase (200 µg/mL, Sigma). Finally, incubation with propidium iodide (10 µL/mL, Molecular Probes, by Thermo Fisher Scientific Biosciences GMBH, Villebon-sur-Yvette, France) at 4°C for 30 min was performed before flow cytometry (BD FACSCalibur, Becton Dickinson, Le Pont-de-Claix, France) analysis. For each sample, 10,000 events (without cell doublets and cellular debris) were considered. The cell cycle repartition was analyzed with Graphpad Prism V6.0 software. Statistical significances (mean ± SD) were determined by two-way ANOVA followed by Dunnett's multiple comparison test (****p<0,0001).

Immunoprecipitation

Cell lysates were obtained as described in the Western blot section. Samples were pre-cleared with protein A sepharose (20 mL of 50% beads/200 mL of cell lysate, Sigma-Aldrich, Saint-Quentin-Fallavier, France) for 1 h at 4°C under gentle rocking. After brief centrifugation, supernatants were incubated with antibodies against 14.3.3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1/200), Cdc25C (Thermo Fisher Scientific Biosciences GMBH, Villebon-sur-Yvette, France, 1/200) or mTOR (CST, 1/200) at 4°C for 1 h under rotation and followed by incubation with protein A sepharose (20 mL of 50% bead slurry, Sigma-Aldrich, Saint-Quentin-Fallavier, France) for 1 h at 4°C under rotation. Samples were rinsed 3 times with RIPA buffer. Pellets were collected by brief centrifugation, resuspended in 2X Laemmli buffer, and heated at 100°C for 10 min before SDS-PAGE and Western blots were performed.

RESULTS

Organocopper Synthesis

The synthesis of WN197 is described in Figure 1. Indenoisoquinoline WN170 was first obtained in a four-step reaction. Condensation of the benzo[d]indeno [1,2-b]pyran-5,11dione with a primary aminoalcohol was followed by tosylation of the alcohol function. The substitution of the tosyl group by the protected ethylenediamine and the consecutive deprotection of the Boc group led to WN170 in 68% global yield. Complex WN197 was then synthesized by reacting methanolic solutions of indenoisoquinoline derivative WN170 and CuCl 2 . After purification, WN197 was obtained in 70% yield.

WN197 Displays a Cytotoxic Activity on Three Adenocarcinoma Cell Lines at Low Doses

Cells viability was assayed on the triple-negative breast cancer cells (MDA-MB-231), the cervix cancer cells (HeLa), and the colorectal cancer cells (HT-29) (Table 1). IC 50 obtained are respectively 0.144 µM, 0.22 µM, and 0.358 µM for WN197 below the cisplatin IC 50 values ranging from 10 to 40 µM. The copper-free indenoisoquinoline ligand, WN170, affected cell viability at higher doses (0.875 µM for MDA-MB-231, 0.630 µM for HeLa, and 0.479 µM for HT-29 cells), showing that the presence of the copper metal significantly enhances the anticancer effect of the indenoisoquinoline core for MDA-MB-231 and HeLa cell lines. A significantly higher IC 50 (1.080 µM) is obtained on MCF-10A compared to the adenocarcinoma cell lines (Table 2).

WN197 Induces DNA Damage

To determine whether WN197 affects DNA integrity, detection of g H 2 A X D N A b r e a k m a r k e r w a s p e r f o r m e d b y immunofluorescence. gH2AX foci were visualized in the nucleus at 0.5 µM of WN197, a concentration close to the IC 50 determined previously, in MDA-MB-231, HeLa, and HT-29. After 24 h of treatment, the average number of gH2AX foci per cell were respectively 99, 98, and 70 for MDA-MB-231, HeLa, and HT-29 cells (Figure 2A). The number of gH2AX foci was close to the result obtained for the Top2 inhibitor, doxorubicin, (average of 95 foci per cell), and higher than the number of gH2AX foci triggered by an alkylating agent, cisplatin (average of 55 foci per cell). WN197 induced more DNA damage than the indenoisoquinoline WN170 (average of 23 foci per cell). Controls with DMSO solvent showed a low number of foci (average of 9 foci per cell for the 3 adenocarcinomas) compared to treated conditions (Figure 2B).

These results were further confirmed by Western blot analysis (Figure 2C). Untreated cells showed a low gH2AX signal while a

FIGURE 1 | WN197 synthesis.
strong signal was observed after doxorubicin, cisplatin, and WN197 treatments. As observed by immunofluorescence, the gH2AX signal is weaker in the WN170 condition compared to the WN197 condition, indicating that the WN197 compound induces more DNA damage than WN170 at the same concentration (0.5 µM).

Foci were detected as soon as 30 min after treatment (Figure 2D). The number of gH2AX foci was close to the result obtained at 24 h with an average of foci per cell of 84, and 87 for MDA-MB-231, HeLa, and lower to 13 for HT-29 cells after WN197 treatment.

WN197 Is a Concentration-Dependent Topoisomerase Inhibitor

To determine whether the Cu(II)-complex WN197 is a topoisomerase inhibitor, in vitro human topoisomerase activity tests were realized. The topoisomerase I (Top1) test relies on the ability of Top1 to relax supercoiled DNA, and the absence of relaxed DNA implies inhibition of Top1 activity. In the presence of Top1, supercoiled DNA showed a relaxed profile (Figure 3A). Camptothecin, a well-known Top1 inhibitor, disturbed DNA relaxation in the reaction, and part of the DNA remained supercoiled. Increasing doses of WN197 from 0.2 to 2 µM showed a decrease quantity of relaxed DNA, indicating disruption of Top1 activity. The solvent control, DMSO, and VP-16 (etoposide, a Top2 inhibitor) displayed no effect on Top1-induced DNA relaxation showing no inhibitory effect on Top1 activity.

Top1 inhibitors can act either as catalytic inhibitors by DNA intercalation at the Top1 fixation site or as poisons, forming a ternary complex (DNA + Top1 + compound) (24,25), preventing DNA religation and inducing accumulation of nicked DNA. The addition of proteinase K to the Top1-DNA relaxation test allows the release of nicked DNA that can be resolved and detected on agarose gel. The short half-life of the nicked DNA is stabilized and detectable after addition of a Top1 poison, camptothecin (Figure 3A). Nicked DNA was also observed in presence of 0.2 µM of WN197, indicating a Top1 poison activity (Figure 3A). At higher concentrations (0.5 µM, 1 µM, 2 µM), the inhibition of Top1 activity without nicked DNA accumulation indicates that WN197 does not act as a Top1 poison.

The effect of WN197 on Top2a and Top2b activities were also assayed. The same principle based on the inhibition of topoisomerase-induced DNA relaxation was used (Figure 3B). In the presence of Top2a or Top2b, the supercoiled DNA is relaxed (topoisomers). VP-16 (etoposide, Top2 inhibitor) disturbed DNA relaxation in the reaction, as seen by the presence of supercoiled DNA in the gel, while camptothecin had no inhibitory effect, as expected. WN197 disrupted the Top2a-induced DNA relaxation only at 2 µM, and the Top2b at 1 and 2 µM, higher doses than the concentration necessary to inhibit Top1 activity, indicating a concentration-dependent mechanism of action.

WN197 Intercalates in DNA

Melting curves and fluorescence measurements were performed to comfort results obtained in Figure 3, and ascertain WN197 intercalation in DNA.

Drugs ability to protect calf thymus DNA (CT DNA, 42% GC bp) against thermal denaturation was used as an indicator of the capacity of indenoisoquinoline derivatives to bind and stabilize the DNA double helix. The Cu(II) indenoisoquinoline complex WN197 displayed a slightly higher DTm value compared to the metal-free indenoisoquinoline WN170 (respectively 16.6°C and 16.1°C, drug/ DNA ratio 0.5), showing a better binding affinity with DNA (Table 3).

The binding affinities, determined using a fluorescence quenching assay based on DNA binding competition between the intercalating drug ethidium bromide and the tested molecules, were used to gain insight into the DNA binding affinity. The apparent DNA binding constant Kapp value of the Cu(II) complex (15.005 ± 0.290 10 7 M -1 ) is higher compared to the original ligand value (2.436 ± 0.883 10 7 M -1 ). These results are in agreement with the DTm values showing that the complexation of indenoisoquinoline ligand by copper allows a stronger interaction with DNA (Table 3).

WN197 Activates the DNA Damage Response Pathway

The activation of molecular effectors of the DDR pathways involved in SSB and DSB was analysed by Western blot (Figure 4). Activating phosphorylation of ATR (S428) and ATM (S1981) occurred in the three cell lines MDA-MB-231, HeLa, and HT-29 treated with WN197 compared to the untreated cells. The subsequent activating phosphorylation of Chk1 (S317) and Chk2 (T68) were observed, confirming the DDR pathway activation. In the doxorubicin, cisplatin, and WN170 these phosphorylations also occurred while in untreated controls they were always lower or absent.

p53 facilitates cell cycle arrest by targeting p21 WAF1/CIP1 . After WN197 treatment, p53 and phosphorylated p53 were increased in MDA-MB-231, HeLa and HT-29 cells (respectively by factors 34.8, 3.2, and 1.6 for p53 and by 58.3, 1.6 and 5.5 for phosphorylated p53), while p21 was highly increased in HT-29 cells (by a factor 8.3) compared to MDA-MB-231 and HeLa (respectively 1.3 and 2.2). The WN170 values are slightly identical except for p53 and p21 in MDA-MB-231 (respectively factors 0.4 and 1.0). In doxorubicin and cisplatin treated cell lines, p53 and p21 were not increased except for p53 in MDA-MB-231 and p21 in HT-29 cells.

WN197 Induces a Cell Cycle Arrest in G2 Phase

The cell cycle repartition following the DDR pathway activation was monitored by flow cytometry in cells exposed for 24 h to present after doxorubicin treatment (positive apoptotic control) in Supplementary Figure S1.

To determine the lower dose necessary to induce a G2/M phase accumulation, flow cytometry experiments were performed with increasing concentrations of WN197 and results are shown in Figure 5C. A G2/M phase accumulation was significantly induced by WN197 from 0.5 to 1 µM for MDA-MB-231, 0.25 to 0.5 µM for HeLa and 0.25 to 1 µM for HT-29.

A kinetic of treatment with WN197 (0.5 µM) was realized on the three adenocarcinoma cell lines by flow cytometry to determine the earliest-induced G2/M accumulation (Figure 5D). After 8 h of treatments, the cell cycle was modified for MDA-MB-231 with a significant accumulation in G2/M. A later effect after 12 h and 16 h of treatment was observed respectively for HT-29 and HeLa.

Cell cycle arrest phase was further determined by Western blot analysis of major cell cycle regulators: Cdk1, cyclin B, Cdc25C phosphatase, and histone H3 (Figure 6A). The Cdk1/ cyclin B complex that forms the also called MPF (M-phase Promoting Factor) is required for the transition from G2 to M phase of the cell cycle. During the G2/M transition, Cdk1 is activated by dephosphorylation of its threonine 14 and tyrosine 15 residues (inhibitory phosphorylations) by the active Cdc25C phosphatase that requires prerequisite dephosphorylation on threonine 161 (59,60). In comparison to the untreated control, the phosphorylation of Cdk1 on tyrosine 15 was increased after cisplatin, WN170 or WN197 treatments in the three adenocarcinoma cell lines, while it decreased after treatments with doxorubicin or nocodazole in HeLa and HT-29 and was slightly identical in MDA-MB-231 treated with doxorubicin. The cyclin B amount was increased after WN197 treatment in the three cell lines. Cdc25C was decreased in MDA-MB-231 and HT-29, and increased in HeLa after treatments with WN197 compared to untreated conditions. The inhibitory phosphorylation of Cdc25C on serine 216 was enhanced by WN197 treatments compared to untreated conditions in the three cell lines. On the contrary, a decrease of this phosphorylation was obtained after nocodazole treatments, consistent with the former detection of an activated form of MPF except for HT-29. Finally, histone H3 phosphorylation on serine 10 is involved in mitotic chromatin condensation and is a marker for entry in the M phase after activation of the Cdk1/ Cyclin B complex (61). In WN197 treated cells, histone H3 was A B FIGURE 6 | WN197 arrested the cell cycle in G2. (A) Western Blot analysis of cells treated for 24 h with doxorubicin (5 µM), cisplatin (20 µM), WN170, WN197 (0.5 µM), or nocodazole (84 nM). b-actin was used as a loading control. For H3 phosphorylation, respective H3 total levels were used as loading controls. (B) 14-3-3 and Cdc25C immunoprecipitations were realized in cell lines treated for 24 h with cisplatin (20 µM) or WN197 (0.5 µM). Relative protein levels were expressed by densitometry using Image J software (Fiji Software, v1.52i). Results were representative of three independent experiments. not phosphorylated on serine 10, showing that cancer cells were stopped in the G2 phase before they could reach the M phase. On the contrary in nocodazole treated adenocarcinoma lines in which an arrest in the M phase occurs, histone H3 was phosphorylated on serine 10.

Furthermore, as seen in Figure 6B, Cdc25C phosphorylated on serine 216 was trapped by 14-3-3 as shown by Cdc25C or 14-3-3 immunoprecipitations realized in HeLa, and Cdc25C immunoprecipitations in MDA-MB-231 and HT-29 cells after 24 h of treatment with 0.5 µM of WN197. The binding was observed after cisplatin treatment but not in untreated controls.

WN197 Induces Autophagy

Apoptosis is often activated after DNA damage (25,62). However, the early apoptosis marker cleaved caspase 3 and the late apoptosis marker cleaved PARP were not detected after treatments with WN197 and WN170 in contrast to doxorubicin and cisplatin treatments (Figure 7A). A time-course detection of cleaved PARP and cytochrome C release in the cytoplasm at 3,16,24,48, and 72 h compared to doxorubicin apoptosis positive control at 24 and 48 h (Figures 7B,C) and annexin V tests (Figure S2) confirm apoptosis is not triggered by WN197. These data indicate that apoptosis is not the programmed cell death activated.

We then determined whether WN197 and WN170 could induce autophagy. In the three adenocarcinoma cell lines, several autophagy markers (63) were detected. p62/sequestosome-1 was degraded, Beclin-1 was synthesized and LC3-I association with phosphatidyl-ethanolamine that forms LC3-II was increased as shown by accumulation of LC3-II after 24h of treatment with 0.5 µM of WN197 and WN170 (Figure 7D). The same changes were observed with the inhibitor of mTOR pathway, rapamycin which is known to activate the autophagy process. Moreover, immunoprecipitation carried on the mTOR complex showed that the RAPTOR component was phosphorylated on serine 792 after treatment with 0.5 µM of WN197, as seen in positive controls treated with 500 nM of rapamycin, and compared to negative controls treated with doxorubicin (Figure 7E).

DISCUSSION

This study aims to develop and understand the molecular properties of a new organometallic compound WN197, derived from the topoisomerase 1 inhibitor indenoisoquinoline. Previous studies highlighted action specifically correlated to the presence of a metallic atom like copper (53), iron [e.g. ferrocen/ferroquine (43,64)], ruthenium [e.g. indenoisoquinoline (55) and various complexes (65,66)], or platin [e.g. cisplatin (67)], and demonstrate the interest of these organometallic compounds in cancerology. More recently, a class of topoisomerase inhibitor, the indenoisoquinoline derivatives, were developed and selected for their high stability and non-drug substrate for efflux transporters involved in cell resistance (35,68). These promising compounds are in phase I/II clinical trials (36,68). However, constant efforts are made to increase their efficiency.

The addition of a carbohydrate moiety to indenoisoquinoline derivatives significantly improves the binding affinity to DNA due to a stronger interaction through hydrogen bonds (69). Hereby, we synthesised a new copper indenoisoquinoline derivative. The copper(II) addition to the indenoisoquinoline backbone significantly enhance the toxicity on triple-negative breast MDA-MB-231 and cervix HeLa cancer cell lines. Those two cell lines are related to breast and cervix cancers with high mortality rates in women. In addition, the toxicity is obtained at lower doses compared to human non-tumorigenic epithelial cell line MCF-10A. The use of low doses in chemotherapy could be of particular interest and represent an advantage with less risk of adverse side effects. Further experiments will help to determine if WN197 has specificity at the cellular level.

The viability assays showed that low doses are necessary to induce cell death in breast, cervix, and colon cancer cell lines, from three of the most prevalent adenocarcinomas. The IC 50 are under the values obtained for most other Top1 inhibitors that usually range from concentration of 1 to 10 mM except for thiosemicarbazone or pyrimidine-derived compounds (53). The medium value of 0.5 mM, close to the IC 50 for the three adenocarcinoma cell lines, was further chosen to decipher the molecular pathways involved in the anti-proliferative effect of WN197. Topoisomerases are overexpressed in M phase in cancer cells and generate a high number of DNA breaks under the action of Top inhibitors (12,14,15). Cells overexpressing topoisomerases have shown better responses to Top inhibitors (70,71). Using low doses of the compound could be useful to avoid unwanted normal cell death. Such strategies of low minimal but necessary anti-tumorigenic doses are often employed for anthracycline to limit cardiotoxicity (72,73).

We determined the extent of DNA damage induced by the new compound, with immunofluorescence and Western blot analysis of a front-line activated marker of DNA breaks, the gH2AX histone. The recruitment of gH2AX normally occurs at the site of DNA breaks after exposition to Top1 or Top2 poisons (74,75). Higher level of DNA breaks is observed with WN197 compared to the control copper-free compound WN170, proving that the presence of a metal atom increases the efficiency to induce DNA damage. DNA breaks appear early around 30 min after addition of the product. In parallel, in vitro tests reveal that WN197 inhibits Top1 at low doses, corresponding to the IC 50 , and Top2 at higher doses up to 1 mM showing a dose-dependent action. The copper complex WN197 is a Top1 poison that forms a ternary complex with the DNA (interfacial inhibition) as indenoisoquinoline derivatives (24).

After DNA damage is induced, DDR effectors are activated, as shown in Western blot experiments. The upstream kinases ATM, ATR, Chk1, and Chk2 are phosphorylated after 24 h of treatment with 0.5 µM of WN197, a prerequisite for their activation (76,77). Both SSB (ATR, Chk1) and DSB (ATM, Chk2) markers are detected at a concentration capable to inhibit Top1. Top1 are known to generate SSB and Top2 DSB. However, Top1 poisons produce SSB that can be converted into DSB, the most dangerous type of DNA break, at the replication fork stalling (78,79) intercalation of WN197 into DNA. At doses above 1 µM, the compound exerts a dual Top1/Top2 inhibitory activity and intercalation properties as demonstrated by the melting curves a n d t h e fl u o r e s c e n c e m e a s u r e m e n t s . T h e p l a n a r indenoisoquinoline skeleton of WN197 displays an increased intercalation into DNA compared to WN170. The high affinity of the Cu(II) complex with DNA can be attributed to the pcation interaction between the base pairs and the atom of Cu(II) coordinated with ligands, but also to the capability to increase the p-p interaction between the base pairs of DNA and a second ligand molecule (80,81). At high doses, DNA intercalation could avoid topoisomerase access to its fixation site similarly to a catalytic inhibitor. Such mechanism is found with anthracyclines such as doxorubicin whose poison activity at low doses is lost for an intercalating catalytic inhibitory activity at high doses. Due to a strong affinity for DNA duplexes, those anthracycline compounds prevent Top2 binding to DNA (75,82).

To determine the exact arrest phase in the cell cycle, analyses were further conducted. To allow the G2 to M phase transition, Cdc25C dephosphorylates on residues tyrosine 15 and threonine 14, leading to its activation (83,84). Cdk1 activation in the MPF complex phosphorylates histone H3 on serine 10 to allow DNA condensation during mitosis (61). After 24 h of treatments, an increase in the inhibitory serine 216 phosphorylation of Cdc25C is detected. This phosphorylation is recognized by 14-3-3 (85) to form a complex with Cdc25C, as shown in the three adenocarcinomas, by immunoprecipitation. Sequestration of Cdc25C by 14.3.3 impedes Cdk1 dephosphorylation on tyrosine 15 and histone H3 phosphorylation does not occur on serine 10 in the three cell lines after treatment with WN197 for 24 h. The cancer cell lines lack the required MPF activation and H3 phosphorylation to allow an M phase entry and remain arrested in G2. In addition, cyclin B accumulates in our experiments concomitantly and is not destroyed by the proteasome as expected at the end of the M phase (86,87). p53 and its target the cell cycle inhibitor p21 are increased after WN197 treatments. p53 is involved in cell-cycle arrest by a transcriptional activation of p21 capable to inhibit Cdk1/ cyclin B and cell-cycle progression through mitosis (88)(89)(90). p53 also targets 14-3-3 and blocks G2/M transition (91). Altogether, the results demonstrate that WN197 at low doses with a Top1 poison activity arrest adenocarcinoma cells in G2. After DNA damage have been induced, activation of the DDR pathways normally ensures repairs but when damage is too extended, cells undergo a programmed death (92,93). While most of the actual topoisomerase inhibitors induce apoptosis (25,62), WN197 triggers autophagy. Among topoisomerase I inhibitors, a camptothecin derivative irinotecan and an indenoisoquinoline compound NSC706744 were reported to activate autophagy with the absence of apoptosis (94,95). After 24 h of treatment with low doses of WN197 (0.5 mM), autophagy markers are detected by Western blots: synthesis of Beclin-1 (96), increase in LC3-II/LC3-I ratio (97), and degradation of p62 (98). It was previously shown, after DNA damage, that the mTORC1 complex was inhibited by RAPTOR phosphorylation (on multiple sites including serine 792) in a negative feedback loop to induce autophagy (99,100). We further show autophagy is triggered through the phosphorylation of RAPTOR in the mTOR complex. This mechanism of activation is similar to the mTORC1 inhibitor rapamycin (101). Our results show that under WN197 treatment from 3 to 72 h, cells die by a caspase-independent mechanism as classical markers annexin V staining, caspase 3 and PARP cleavage, cytoplasmic cytochrome C released were not detected. It also has to be noted, no sub-G1 cells were detected after WN197 treatment while they were after doxorubicin known to induce apoptosis. Previous data on breast cancer cells have showed autophagy could mask and delay apoptosis but was associated with an early release of cytochrome C from mitochondria which is not the case in our experiments (102). Cytochrome C is not released when autophagy is triggered and mitochondria degraded in autophagosomes ( 103). Several studies have described autophagy as dependent on wild-type p53 depletion or inhibition (104). WN197 action is associated with an increase in p53 and p53 phosphorylation. However, the inducedautophagy does not dependent on the cell lines p53 status. HeLa cells express wild-type p53 that end up as functionally null when targeted to degradation by E6 endogenous papillomavirus protein, while MDA-MB-231 and HT-29 display p53 mutations resulting in positive gain of function ( 105). Nevertheless, WN197 inducedautophagy is in agreement with an increase of p21 level and the G2 arrest detected our experiments in cancer cells. Several antiapoptotic effects of p21 can explain the choice of an autophagic cell death instead of apoptosis. High levels of p21 are known to block Cdk1/cyclin B and to inhibit apoptosis through down-regulation of caspase-2 (106), stabilization of anti-apoptotic cellular inhibitor of apoptosis protein-1, c-IAP1 (107), and inhibition of procaspase 3 activity (108). Another additional mechanism through Beclin-1 could play an important role in apoptosis inhibition and autophagy. Beclin-1 protein expression was shown necessary to block the apoptotic cascade after induced-DNA damage (102, 109) and to activate autophagy under low doses of chemotherapeutics (rapamycin, tamoxifen) in breast and ovarian cancers (110,111).

CONCLUSION

Copper(II) indenoisoquinoline complex WN197 displays an anticancerous activity at low doses inhibiting Top1. MDA-MB-231 (triple negative breast cancer cells), HeLa (cervix cancer cells), and HT-29 (colon cancer cells), cancer cells accumulate DNA breaks and arrest in the G2 phase of the cell cycle. This arrest is characterized by the inactivation of the Cdc25C phosphatase through phosphorylation on serine 216 and binding to 14.3.3 that consequently leaves in its inactive form the MPF (a phosphorylated form of Cdk1 associated to accumulated cyclin B). Autophagy is further processed by the RAPTOR effector phosphorylation in the mTOR complex, and associated to p21 overexpression. WN197 appears as a new efficient drug to counteract cancer cells when used at low doses. The action mechanism of the copper complex is summarized in Figure 8. Its use in chemotherapy could particularly benefit patients with cancer cells overexpressing topoisomerases or sensitize cancer cells to other DNA modifying agents including DNA adducts inducer, methylating agents, or PARP inhibitors (112,113).

FIGURE 8 | Deciphering of the molecular mechanisms of the novel copper(II) indenoisoquinoline complex WN197. WN197 inhibits topoisomerases I at low doses in a poison mode and forms a ternary complex with the topoisomerase and DNA, leading to strand breaks accumulation. Phosphorylated H2AX (gH2AX) localizes at the sites of DNA damage. The DNA damage response pathway is activated: ATM and ATR kinases are phosphorylated, and subsequently activate Chk1 and Chk2, leading to Cdc25C phosphorylation on serine 216 (S216), and to its binding to 14-3-3. Consequently, Cdk1 remains phosphorylated on tyrosine 15 (Y15), impeding the activation of the MPF (Cdk1/Cyclin B) and the phosphorylation of H3 on serine 10 (S10). Cancer cells arrest in the G2 phase of the cell cycle. The DDR also leads to an increase in p53 and p21 followed by an autophagic cell death characterized by the phosphorylation of RAPTOR on serine 792 (S792) in the mTORC1 complex, the synthesis of Beclin-1, the formation of LC3-II (complex LC3-I/PE (phosphatidylethanolamine)), and the degradation of p62.

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Publication (review 1)

Proteins from the DNA Damage Response: Regulation, Dysfunction, and Anticancer Strategies

Introduction

The genome is constantly harmed by spontaneous damage caused by endogenous factors produced by normal cellular physiological conditions such as bases alteration, aberrant DNA enzyme function or oxidation, and by a large variety of exogenous genotoxic factors [1]. Cells have evolved a complex network of hundreds of proteins, named the DNA damage response (DDR), to ensure genome integrity and the expression of dedicated proteins to each cell type. The quality control mechanisms of DDR senses DNA damage, coordinates DNA repair with cell cycle arrest, and ensures cell death when repairs are not possible [2]. The detection of DNA damage involves the recruitment of various repair protein complexes depending on the type of break. Classically, phosphatidylinositol-3kinase-related kinases ATM (ataxia-telangiectasia mutated), ATR (ATM-and Rad3-related), and DNA-PKcs (DNA-dependent protein kinase, catalytic subunit) are activated [3]. These kinases phosphorylate numerous targets at the DNA damage sites including CHK1/2 (checkpoint kinases 1/2) and histone H2AX. Substrates of CHK kinases are effectors for DNA repair, transcription, and cell-cycle control, such as BRCA1, NBS1, P53, CDC25, and CDKs (cyclin-dependent kinase). The cellular outcome depends on the types but also on the severity of DNA damage, the cell cycle state, chromatin modifications, posttranslational events, and non-coding RNA. Cells with excessive or unrepairable DNA Cancers 2021, 13, 3819. https://doi.org/10.3390/cancers13153819 https://www.mdpi.com/journal/cancers undergo an apoptotic P53-dependent death or another type of programmed cell death that does not rely on caspase activation. Many natural cellular events rely on DNA breaks/repairs and use part of the DDR network to fulfill specific physiological functions. Despite physical and chemical agents, several biological agents induce severe DNA damage. When DDR processes are damaged or bypassed, DNA damage elicits mutations and heritable changes resulting in pathologies (e.g., immunodeficiency, inflammation, neurodegeneration, aging, cardiovascular diseases, and cancer). When and how the DDR complex signaling network of proteins is controlled to ensure the right cell outcome; and how the vulnerabilities are exploited in precision medicine to target and treat cancers (from single inhibitor to combined treatments) are hereby highlighted and updated in the present review.

DDR Proteins Activation Is Function of the Type of DNA Damage

Cells encounter tens of thousands of DNA lesions every day arising from endogenous cellular functions and exogenous environmental factors. Intracellular and external DNA damaging events create more than a single type of lesion. DNA damage inflicted to the DNA's double helix includes base damage that does not involve breakage of the phosphodiester backbone or single-strand DNA breaks (SSBs) and double-strand breaks (DSBs). Single base alterations are generated by depurination, deamination, alkylation (usually by guanine methylation), oxidation (production of 8-oxo-7, 8-dihydroguanine), hydrolysis or chemical bonds cleavage in DNA, base analog incorporation, and stable covalent DNA adducts formation. Two-base alterations are formed by a thymine-thymine dimer or by cross-linking under the effect of a bifunctional alkylating agent. DNA strand breaks arise from oxidative or DNA replication stress, transcriptional stalling, failure to repair processes, and abnormal high effectors activation [4,5]. SSBs are converted into DSB lesions during DNA replication [6] (Figure 1).

Cancers 2021, 13 2 of 40 unrepairable DNA undergo an apoptotic P53-dependent death or another type of programmed cell death that does not rely on caspase activation. Many natural cellular events rely on DNA breaks/repairs and use part of the DDR network to fulfill specific physiological functions. Despite physical and chemical agents, several biological agents induce severe DNA damage. When DDR processes are damaged or bypassed, DNA damage elicits mutations and heritable changes resulting in pathologies (e.g., immunodeficiency, inflammation, neurodegeneration, aging, cardiovascular diseases, and cancer). When and how the DDR complex signaling network of proteins is controlled to ensure the right cell outcome; and how the vulnerabilities are exploited in precision medicine to target and treat cancers (from single inhibitor to combined treatments) are hereby highlighted and updated in the present review.

DDR Proteins Activation Is Function of the Type of DNA Damage

Cells encounter tens of thousands of DNA lesions every day arising from endogenous cellular functions and exogenous environmental factors. Intracellular and external DNA damaging events create more than a single type of lesion. DNA damage inflicted to the DNA's double helix includes base damage that does not involve breakage of the phosphodiester backbone or single-strand DNA breaks (SSBs) and double-strand breaks (DSBs). Single base alterations are generated by depurination, deamination, alkylation (usually by guanine methylation), oxidation (production of 8-oxo-7, 8-dihydroguanine), hydrolysis or chemical bonds cleavage in DNA, base analog incorporation, and stable covalent DNA adducts formation. Two-base alterations are formed by a thymine-thymine dimer or by cross-linking under the effect of a bifunctional alkylating agent. DNA strand breaks arise from oxidative or DNA replication stress, transcriptional stalling, failure to repair processes, and abnormal high effectors activation [4,5]. SSBs are converted into DSB lesions during DNA replication [6] (Figure 1).

Replication Stress and DNA Damage

DNA replication disturbances or the slowing/stalling of the replication fork progression during DNA synthesis generate replication stress. Replication stress is a source of massive DNA damage. It arises as a consequence of normal cellular reactions involving DNA, or upon exposure to external agents. Exogenous factors affecting DNA are numerous, ranging from physical agents such as ionizing radiation to genotoxic chemicals (organic compounds such as aromatic substances, or inorganic compounds such as heavy metals [7]) and biological agents including pathogens (bacteria, DNA, and RNA viruses), fungi, and plants [8][9][10]. Those agents also increase the ROS formation that enhances DNA damage [11].

The endogenous factors responsible for replication stress are numerous. DNA sequences can form special structures such as secondary hairpin loops, G-quadruplexes, display damage, or replication/transcription conflict, R-loops, topological stress constraints, or dysregulation of replicative proteins such as RPA, a natural barrier for replication forks [12]. Imbalance in the cellular pool of dNTPs also affect DNA polymerases inactivation and require two different RAD51-mediated pathways for restart and repair [13].

Damaged DNA bases, as a consequence of natural reactions such as depurination, base oxidation, and bulky adducts, are not recognized as templates by DNA polymerases, creating a source of replication stress [14]. On the contrary, interstrand crosslinks do not generate extensive areas of ssDNA (single stranded DNA) and are repaired by the FA (Fanconi Anemia) pathway [15].

Replication forks operate on genomic regions with special chromatin structures such as telomeres and centromeres that represent a challenge for the replisome. R-loops, formed by a DNA/RNA duplex and a ssDNA, arise during transcription and replication-transcription collision. They promote transcription-associated recombination and genome instability in mitotic and meiotic cell cycles [16] and the exposed ssDNA is processed into a DSB by transcription-coupled NER (nucleotide excision repair) [17].

Replication/transcription collisions can lead to transcription-associated recombination and chromosomal rearrangements. To minimize these encounters, both processes are temporally and spatially separated in different cell cycle phases S and G1 [18]. The THO/TREX (TRanscription-Export) and THSC/TREX-2 complexes that mediate the processing of mR-NAs and nuclear export are required to avoid replication-transcription conflicts and may prevent R-loop formation [19].

Physiological DNA Breaks Involved in Normal Cellular Processes

Several natural events, necessary for cellular integrity, require programmed DNA lesions and a partial DDR recruitment [20] (Figure 1).

The replication stress checkpoint enables the DDR to act in coordination with the replisome to protect the stalled forks from degradation and to restart broken forks. Replication forks stalling triggers the activation of the ATR-CHK1 pathway for stabilization and is followed by the release of CHK1 from the chromatin, allowing for the phosphorylation and degradation of CDC25A and cell cycle S phase arrest [21][22][23].

During the course of the cell cycle, in the replicative and the mitosis phases, DNA is naturally cut and religated by topoisomerases (Top) to control DNA topology. To allow DNA or RNA polymerase action, topoisomerases (Top1 and 2) bind to DNA, cut the phosphate backbone of, respectively, one or two strands (SSB or DSB), and untangle DNA before the DNA backbone is resealed. Mitotic DDR functions to coordinate microtubule/kinetochore attachment and spindle assembly [24]. Top2 is necessary for chromosomal segregation before anaphase, and for chromosomal structure maintenance [25]. In mitosis, damage of condensed chromosomes activate initial upstream effectors such as H2AX-MDC1 (mediator of DNA damage checkpoint 1) platform, and the downstream signaling cascade is restrained (inhibition of CHK2), waiting for further progression into the cell cycle [26,27].

DNA lesions also occur through enzymatic reactions and chemical modifications from intracellular metabolism, the release of free radicals, reactive oxygen species (ROS), reactive nitrogen species (RNS), and reactive carbonyl species (RCS) that trigger a chain of reactions and result in a production of exocyclic adducts to DNA bases [28].

The production of male and female gametes for sexual reproduction involves a natural DSB formed during the meiotic recombination process by the SPO11 topoisomerase [29,30]. Meiotic DSBs occur in prophase from the leptotene to the pachytene stage of the first meiotic division. They are essential to allow chromosomal DNA exchange between nonhomologous sister chromatids when they are paired to create genetic diversity [31]. A failure in this process directs germ cells to aneuploidy and leads to congenital disorder or pregnancy loss. During spermatogenesis, when histones are substituted by protamines, Top2 generates temporal DNA breaks to relax DNA that needs to be repaired properly before the end of spermiogenesis to avoid mature spermatozoa with fragmented DNA [32,33]. During meiotic sex chromosome inactivation (MSCI) in males, the sequestration of DDR factors (ATR and H2AX histone) to the XY chromatin away from the autosome is critical for meiosis progression [34].

The variable exon regions encoding the antigen recognition sites of B and T lymphocyte's receptors, respectively involved in humoral and cellular-mediated immunity, are generated by a V (variable), D (diversity), or J (joining) genes recombination during early development. In response to an antigen, class switch recombination modifies the immunoglobulin genes in B lymphocytes [35]. V(D)J recombination starts by the formation of a DSB in DNA by RAG1 and RAG2 (recombination activating gene 1/2) proteins and are repaired through the recruitment of the non-homologous end-joining (NHEJ) pathway composed by Ku70/80, DNA-PK, XRCC4 (X-ray repair cross-complementing protein 4)/DNA-ligase IV, and Artemis [36][37][38]. The class switch recombination (CSR) breaks are initiated in B-cells by a specific activation-induced cytidine deaminase (AID) that mediates DNA deamination. Unlike the V(D)J recombination, the CSR depends on non-canonical BER and MMR to generate DSBs that are finally joined by NHEJ for blunt ends and alt-NHEJ for staggered breaks. DDR factors such as H2AX, ATM, 53BP1, and NBS1 are also involved in CSR [39]. CSR and V(D)J recombination represent a paradigm to study regulations among the various DNA repair pathways. A SAGA deubiquitination module was recently shown to promote DNA repair and class switch recombination through DNA-PK and ATM-induced γH2AX formation [40]. These proteins were found to function upstream of various double-stranded DNA repair pathways. Interestingly, IgG were found to be produced in non-immune cells (spermatozoids) through CSR with RAG1/2 and AID, suggesting a role in fertilization, but also that gametes are far more complex in their DDR regulation [41].

Telomeres located at chromosomal ends protect chromosomes from end-to-end fusions and abnormal mitosis segregation. Telomeric DNA presents a single-stranded overhang protected by a specialized capping complex, called shelterin, composed by TRF1 and TRF2 (telomere repeat-binding factor 1 and 2), TIN2 (TRF1-interaction factor 2), RAP1 (repressor activator protein 1), TPP1, and POT1 (protection of telomere 1), that blocks the recognition by the DDR repair machinery [42]. After each replication, telomeres are partly deprotected, recruit a non-canonical DDR in the G2 phase of the cell cycle, and reconstruct the end protection complex. Only the MRN complex (MRE11/RAD50/NBS1) and the ATM kinase are activated, while the downstream diffusible DDR signaling proteins, CHK2 and P53, are not, avoiding downstream cell-cycle inhibition [43].

Structural Organization of the DDR

The type of ends and the structures of the DNA breaks dictate the selection and the recruitment of repair factors. The choice of the cell response and its outcome depends on factors from the DNA repair pathway, the cell cycle checkpoints, the expression level of effectors from the P53 family, the chromatin state, the genomic location of the break, the post-translational modifications undergone by cytoplasmic or nuclear proteins, and RNA metabolism [44,45].

Selection of the Repair Mechanisms

After the detection of DNA damage, repair mechanisms are activated depending on the break types (Figure 1). The critical steps of the DNA repair process are the recognition and the signaling of the DNA lesions. DNA breaks can be repaired by direct reversal of the chemical reaction of the DNA damage using O 6 -methylguanine methyltransferase (MGMT), or by removal and replacement of the damaged bases [46]. Repair processes involve an important number of effectors, multiple interactions, and post-translational modifications [47,48].

For SSBs, the following three main excision repair pathways exist: NER (nucleotide excision repair), BER (base excision repair), and MMR (mismatch repair). NER includes XPC/A key components for the recognition of the DNA lesion, ERCC1, ERCC4 (XPF), and XPG for excision (excision repair cross-complementation group 1/4/6), recruitment of PCNA (proliferating cell nuclear antigen) by RFC (replication factor C) to allow the copy of the undamaged strand by DNA polymerases (δ, ε, and/or κ), and finally ligated by the ligase-I or ligase-III-XRCC1 complex [49]. The repairs of the lesions throughout the genome are processed by GG-NER (global genome NER) and those blocking transcription by TC-NER (transcription coupled NER). BER is activated for bulky adducts. BER starts with OGG1 (8-Oxoguanine glycosylase), forming abasic (AP) sites cleaved by AP endonucleases, resulting in single-strand breaks further processed by single nucleotide replacement or synthesis and by the recruitment of PARP1/2, XRCC1, ends with polymerase β and ligase-I and III. The MMR, or strand-specific mechanism, recognizes and repairs erroneous insertion, deletion, or miss-paired bases, includes MSH (MutS Homolog) and MLH/PMS proteins. A translesion synthesis process (TLS) allows the DNA replication machinery to bypass DNA lesions such as AP sites or thymine dimers. In TLS, PCNA is ubiquitinated by RAD6 and RAD18 and allows the binding of polymerases (η, ι, κ, ζ, REV1) [50].

DSB repairs are processed by a non-homologous end joining (NHEJ), an alt-NHEJ also named micro-homology mediated end joining (MMEJ), single-strand annealing (SSA), or by a homologous directed repair (HDR) including the homologous recombination (HR). NHEJ mobilized in the G1 phase of the cell cycle requires the recognition of DNA damage by Ku70/80, the recruitment of APLF (Aprataxin and PNK-like factor), 53BP1 (P53-binding protein 1), DNA-PK, end processing by Artemis, the recruitment of PAXX (paralogue of XRCC4 and XLF) and polymerase δ, µ, and ligation by ligase-IV with partner XRCC4 and XLF (XRCC4-like factor). Alt-NHEJ/MMEJ is initiated by PARP1 and MRE11 nuclease to resect DNA-ends that generate single-stranded overhangs annealed at microhomologies and recruits polymerase θ. SSA requires RAD52 for annealing in addition to the ERCC1/4 endonucleases complex and ligase I. HR is activated in the S and G2 phases of the cell cycle and requires the ATM-MRN complex, CtIP (CtBP-interacting protein), RPA (replication protein A), BRCA1 (breast cancer 1), and PALB2 (partner and localizer of BRCA2) recruitment of BRCA2 that acts with RAD52 to mobilize RAD51 and PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3). In case of DSBs, CHK2 phosphorylates BRCA1, serving not only as a scaffold protein but also as a clock for HR repair [51]. The selection between the different DSB repair processes is, in part, driven by the length of the homologies [52,53].

Interstrand crosslinks (ICLs) are DNA strands linked with covalent bonds preventing the separation of DNA strands that are repaired by the FA (Fanconi anemia) pathway using FANCs proteins and effectors from the NER, TLS, HR, factors responsible for Fanconi anemia, and structure-specific endonucleases [54].

During gametogenesis, the DDR has a specific organization. In spermatogenesis, HR acts during the S phase to resolve the DNA damage and NHEJ in G1. Haploid spermatids use an Alt-NHEJ pathway due to the absence of the main components of the canonical NHEJ. Spermatozoa possess a truncated BER containing only the first enzyme, the 8-oxoguanine DNA glycosylase, OGG1, to remove oxidized base adducts after oxidative damage (8-Oxoguanine lesions) [55]. Meiotic recombination in oocyte arrested in meiosis I uses HR, while oocytes in meiosis II accumulate transcripts from all the DNA repair pathways to be used after fertilization in the early embryo before transcription starts at the four-cell stage, and the canonical DDR is activated [56,57].

It was recently shown that during sleep, chromatin motion and dynamic in neuronal nuclei match DNA repairs but require a threshold of DNA damage. The general mechanism for this relationship remains to be deciphered. On the contrary, sleep deprivation affects NER, BER, HR repair processes and ERCC1, OGG1, and XRCC1 genes are decreased [58,59].

Cell Cycle Checkpoints

Checkpoint pathways arrest the cell cycle progression until DNA integrity is restored by the repair mechanisms [60]. Phosphatidylinositol 3' kinase-related kinases (ATM, ATR, DNA-PK) phosphorylate multiple substrates. For example, a DSB allows the recruitment and the phosphorylation of the MRN (MRE11-RAD50-NBS1) complex and the ATM activation that further phosphorylates several targets: SMC1 (structural maintenance of chromosomes protein 1) activates the S-phase checkpoints and halts DNA replication, the BRCA proteins involved in HR repair, and NBS1 necessary for the ATM-dependent phosphorylation of CHK1 and CHK2. CHK1 and CHK2 phosphorylate phosphatases CDC25B/C and CDC25A promoting, respectively, a cell cycle arrest in G2/M and G1/S. These arrests involve the binding of CDC25 to 14-3-3 and a self-accelerated proteasomal degradation of CDC25. Therefore, the activation of the cyclin/CDK complexes by dephosphorylation on Thr14 and Tyr15 does not occur. Moreover, the phosphorylation of MDC1 (mediator of DNA damage checkpoint 1) by ATM, activates the S and G2/M cell cycle checkpoints and is facilitated by the phosphorylated histone H2AX (γH2AX). The phosphorylation of P53, by ATM and CHK2, activates P21 WAF1/CIP1 and Bax genes (BCL2 associated X protein), and, respectively, arrests cells in G1 or triggers apoptosis. Phosphoprotein phosphatases PP1, PP2A, PP2Cδ (WIP1), PP4, and PP6 restrain the response intensity with an action on several targets involved in recognition, signaling or recombination processes. PP5 has a contrasting activity by stimulating ATM signaling and restraining DNA-PK activity [61,62]. WIP1 is necessary for BRCA1 recruitment to the DNA lesion, 53BP1 dephosphorylation, and to promote HR repair [63] (Figure 2).

Cells are sensitive to DNA damage during mitosis and hypersensitive in antephase when they prepare to enter the M phase [64]. Even though the DDR is partially inactivated in mitosis, H2AX is phosphorylated, and the DNA-PK and MRN complexes, MDC1, and the DNA topoisomerase II binding protein 1 (TopBP1) are recruited [45]. After a DSB, mitosis progression is not halted unless a DNA break affects the centromeric or telomeric regions, while a reversible arrest is initiated in the G1 and G2 phases [65]. In the yellow panel: after DNA damage, induced for example by topoisomerases misregulation, repair pathways are selected, and major sensors/effectors are recruited to the damaged lesions. These recruitments are accompanied by many post-translational modifications of proteins with major impacts on the cell response to DNA damage, e.g., O-GlcNAcylation of H2AX and NDC1 at the DNA damage repair site, ubiquitination and NEDDylation of Ku70/80 from the NHEJ and/or PARylation of NBS1 in the MRN complex from HR, and sumoylation of TIP60 in HR. The chromosomal state and the regions damaged mobilize different repair processes that are facilitated by chromatin decompaction through the action of HMGN1 on histone H3, histones methylation/acetylation, and ING proteins (e.g., ING2) action on phosphorylated P53. Topoisomerases misregulation induces persistent DNA breaks. The cell cycle phase controls the selected DSB repair process: HR acts in S phase. CDH1, a cofactor of APC/C (anaphase-promoting complex/cyclosome, a multi-subunit E3 ubiquitin ligase) involved in cyclin degradation, is activated by CDC14 and by DNA damage in G2, while normally inactive. CDK12 represses transcription by inhibition of RNA POLII (polymerase II). The replication factor CDT1 is proteolyzed in G1 providing a checkpoint control to avoid replication. In the blue panel: early signaling by PI3K (ATM, ATR, and DNA-PK) activates secondary kinases. CHK1 and CHK2 lead to cell cycle checkpoint regulation and arrest the cell cycle, respectively, in G2/M after CDC25C inactivation and G1/S after CDC25A inhibition. Another PI3K, PTEN, triggers cell death. Phosphatases tightly regulate phosphorylation at several levels of the signaling cascades, and counter-balance kinases. Recruited repair effectors, such as ATM, regulate miRNA biogenesis (orange) at the transcriptional and post-transcriptional levels. In return, miRNAs regulate other components of the signaling pathways. RNA/DNA hybrids facilitate the recruitment of DDR effectors to DSB. In the red panel: different cell death processes are triggered by different pathways. P53 oscillations initiate a caspase cleavage cascade and apoptosis, while the inhibition of the mTORC1 complex directs the cell outcome to autophagy. Levels of ATP and NAD dictate necrosis and senescence, and activation of the RIP3 kinase conducts to necroptosis. Senescence is only triggered when P21, P16, and CDK4/6 are activated with IFN-associated inflammatory background and results in the secretion of SASP (senescence-associated secretory phenotype) factors.

The cell cycle controls the competition between DSB resection-dependent repair pathways HR, SSA, Alt-NHEJ, and NHEJ. A DSB repair proceeds with a copy of the missing information from the sister chromatid during the S and G2 phases with the phosphorylation of the members of the MRN complex, BRCA1 and CtIP by the ATM kinase, favoring the three resection-dependent DSB repair pathways (HR, alt-NHEJ, or SSA). HR depends on CDK/cyclin and the phosphorylation of multiple substrates, including the MRN complex and CtIP. In the G1 phase, RIF1 and 53BP1 localize to the DSB, inhibit the recruitment of BRCA1, block the DNA end resection pathways, and promote NHEJ. The HR process competes with the NHEJ in interphase. The regulation of DNA-PK during the cell cycle determines the choice of the DNA repair pathway. After a DSB, in the G1 phase of the cell cycle, DNA-PK autophosphorylation on Ser2056 and activation is facilitated by its interaction with TIP60 histone acetyltransferase (HAT), and the repair process executes a NHEJ. In the S phase, the SUMO2 (small ubiquitin-related modifier 2) modification of TIP60 mediated by the PISA4-E3-ligase blocks the TIP60-induced DNA-PK activation, and HR repair is favored [58].

Cell cycle chromatin modifications regulate the coupling between DNA replication and mitosis. The initiation of DNA replication is controlled by CDT1 licensing factors. CDT1 accumulates from the M to the G1 phase and is degraded in the S phase (after poly-ubiquitination by Cullin-4-ring E3 ubiquitin ligase) to ensure a correct onset of DNA replication and prevent re-replication. In response to DNA damage, CDT1 is proteolyzed in G1 providing a checkpoint control [66].

Cell Death and P53 Signaling

The cellular outcome after the kinases cascades depends on the types and the severity of the DNA damage. Cells with excessive DNA damage or unrepairable DNA undergo a programmed cell death involving a P53-dependent mechanism such as apoptosis, or other types of cell death that do not rely on caspase-dependent activation (Figure 2).

P53 accumulates after a negative feedback loop involving an MDM2 (Murine double minute 2) inhibitor with a ubiquitin ligase activity and WIP1 phosphatase to induce cellular senescence or apoptosis. The P53 level oscillates and the oscillation numbers directly relate to the DSB quantities and to the cell death outcome that depends on the genetic context of the damaged cells [67]. A failure in these oscillations allows the cell to escape the G1 arrest. When P53 is mutated in a high-grade serous carcinoma, their reliance on the G2/M checkpoint is increased [68]. P53 maintains a cell arrest, initiates senescence through the activation of P21 WAF1/CIP1 , inhibits cyclin-dependent kinases, and the binding of retinoblastoma protein to E2F transcription factor to regulate the G1/S transition. P53 phosphorylation on Ser46 is specifically linked to the pro-apoptotic expression of PUMA and NOXA proteins, the release of mitochondrial cytochrome C, the cleavage of caspases, and apoptosis [69,70]. An exception is the mature spermatozoa that do not complete apoptosis as their nucleus and mitochondria are localized in different compartments [71]. Autophagy is mediated by ATM and nuclear P53, which activates the transcription of PTEN (phosphatase and tensin homolog), AMPK, and Sestrin that engages TSC2 (Tuberous Sclerosis Complex 2) and AKT to inhibit mTORC1. The inhibition of the DNA-PK or ATR/CHK1/RhoB-mediated lysosomal recruitment of a TSC complex also results in autophagy [72]. mTORC1 connects to the cell cycle checkpoint through the GATOR1 complex component NPRL2, which arrests the cell cycle in G1/S in P53 positive cells and in S or G2/M in P53 negative cells. mTORC1 also negatively controls ATM via S6K1/2 signaling by up-regulating two miRNAs (miR-18a and miR-421) targeting ATM mRNA [73,74]. The cellular depletion of ATP and NAD causes oxidative stress, activation of the RIPK3 (receptor-interacting protein kinase 3), and necroptosis [75]. In dividing cells, DNA lesions interfere with DNA synthesis (replication forks stalling) and the incompletely replicated DNA causes mitotic catastrophe when cells try to break through G2-arrest into the M phase [76].

Another member of the P53 family is involved in oocytes facing cumulative DNA damage when arrested in the prophase of the first meiosis division for many years in ovaries [77]. Prophase 1 oocytes in primordial follicles are unable to respond immediately to DNA damage, while all components necessary for the DNA repair including direct lesion reversal, NER, BER, MMR, HR, and NHEJ processes are present [56,78]. They depend on a G2/M DDR and P63, a member of the P53 family, activated by ATM and CHK1. During their meiotic arrest, DNA-damaged oocytes undergo a specific P63-dependent apoptotic death [79] only for severe DNA damage [80]. Later, in metaphase I and II oocytes of growing follicles, the DNA damage-induced arrest is overridden by the spindle assembly checkpoint (SAC) [81,82].

Chromatin State and Chromosomal Damage Location

The native chromatin state (heterochromatin, just transcribed DNA) and the type of damage (blunt end, overhang, adducts) influence the choice and the kinetic of the repair processes.

Chromatin compaction, such as heterochromatin, limits the accessibility of DNA damage response proteins to the DNA damage sites, making these regions difficult to repair. Chromatin local relaxation by histone H1 and high mobility group proteins (HMGs) is an early event of the DDR. HMGN1 binds to nucleosomes and reduces histone H3 phosphorylation on Ser10, a necessary site to maintain the chromatin condensed state in mitosis. TIP60 acetylates histone H2AX, a prerequisite to both its phosphorylation and ubiquitination. The phosphorylation of H2AX is mediated by ATM, ATR, or DNA-PK on the nucleosomes flanking accidental DSBs but also in natural processes of meiosis and the immune system during DNA recombination. This is an indirect effect of ING (inhibitor of growth) proteins, as ING2 on P53 is essential for the chromatin-induced decompaction allowing accessibility of the repair proteins to DNA damage sites and providing the necessary signal to translocate ATM and the MRN complex to the damaged DNA site [83]. A DSB in actively transcribed chromatin is preferentially halted and repaired by HR and end resection in G2, as they are absent in G1 [84,85]. Inactive lamina heterochromatin prefers alt-EJ [86]. Nuclear position dictates the DNA repair pathway choice. Transcriptionally active chromatin recruits HR at DNA DSBs. Open chromatin (in promoters and enhancers) is more permissive for end resection [87]. Specific chromatin locations have repair pathway preferences. Damage in the telomeres tends to inhibit NHEJ and to select alt-NHEJ or HR repair processes; centrosomes, the microtubules-organizing centers, rely on HR or MMEJ (resection mechanisms) [45,88].

Post-Translational Modifications

Proteins from the DDR are controlled by post-translational modifications, revealing a complex organizational response. Post-translational modifications modulate the chromatin structure and provide docking sites to recruit and accumulate DDR repair proteins at the DNA damage site. Phosphorylation is one of the central proteins post-translational modification and also the most studied in the initiation and the execution of the DDR response. Many different DDR proteins involved in checkpoint control and DNA repair are regulated by a cycle of phosphorylation/dephosphorylation and by the presence of DDRkinase/phosphatase molecular switches. The kinases ATM/ATR/DNA-PK and CHK1/2 cascades are central networks in DDR activation.

Following DNA damage, several phosphatases participate in the DNA damage checkpoint activation and regulate several phosphorylations at multiple levels by dephosphorylating sensor kinases, ATM, ATR, and DNA-PK, transducer kinases CHK1/2, histones H2AX and H3, and effectors such as P53 and MDM2 [89]. PP1, PP2A, WIP1, and PP5 dephosphorylate ATM, allowing its recruitment to damaged sites [89,90]. PP5 is required for the ATR-dependent phosphorylation of CHK1 [91]. PP2A, PP5, and PP6 dephosphorylate DNA-PK on multiple sites to enhance its activity [92][93][94]. WIP1 dephosphorylates CHK1, promoting its release from chromatin and an increase in its kinase activity [95], and antagonizes CHK2 [96]. PP2A, PP4, PP6, and WIP1 dephosphorylate γH2AX, and PP1 dephosphorylates H3, leading to the transcriptional repression of cell cycle-regulated genes [62]. PP1 associates to PNUTS (protein phosphatase 1 nuclear-targeting subunit) [97] and WIP1 dephosphorylates and inactivates P53 [98]. PP1 dephosphorylates and positively regulates BRCA1 [99]. PP5 dephosphorylates 53BP1, leading to its release from DNA damage sites, while normally recruited to DNA damage to coordinate DDR factors localization and activation [100]. CDC14 triggers nuclear accumulation and the activation of YEN1 at anaphase to resolve recombination repair intermediates [101].

Distinct ways of regulation are used by phosphatases to regulate upstream DDR signals. WIP1 dampens the DDR with a feedback circuit, while PP2A provides finetuning and acts with P53 in a negative feedback loop [102] that is necessary for efficient DNA repair and post-repair restart of cell division [103]. PP2A keeps CHK2 inactive in normal conditions but, following DNA damage, CHK2 is released and followed by a late reconstitution of the PP2A/CHK2 interaction, increasing the threshold necessary for CHK2 activation [104]. Phosphatases are submitted to the DDR kinetic regulation working on different repair substrates during different time periods after DNA damage. Indeed, PP4 dephosphorylates and maintains RPA2 in a hypo-phosphorylated form immediately after DNA damage to avoid competition for ssDNA binding with other factors such as RPA1 [61,105,106].

Several other covalent modifications including ubiquitylation, sumoylation, poly-ADP-ribosylation (PARylation), neddylation, acetylation, and methylation facilitate protein recruitments to the damaged DNA [47]. SUMO and ubiquitin conjugates compete for lysine residues in target proteins [107]. The poly ADP-ribose polymerases (PARPs) modify proteins by linear or branched chains of ADP-ribose units originating from NAD rapidly after the induction of DNA breaks. More than 90% of PAR addition is mediated by PARP1. PARylated proteins are recruited through their PAR binding site. PARylation is capable of prime ubiquitination at the site of DNA lesions for mediators such as CHFR ubiquitin E3 ligases [108]. Neddylation is the link of the ubiquitin-like protein NEDD8 through its glycine carboxy-terminal to a lysine of the targeted protein, such as Ku70/80 and DNA-PK [109,110]. HATs (histone acetyltransferases) and HDACs (deacetylases) catalyze the addition or removal of acetyl groups on lysine residues, altering the chromatin structure and providing binding signals for proteins with recognition domains, to mediate key DDR activities [111]. Histone and non-histone lysine methylation can alter the protein's interaction with chromatin and recognition by reader proteins [112]. Similar to phosphorylation, the O-linked N-acetylglucosamine linkage (O-GlcNAcylation) is realized on serine or threonine residues [113]. OGT (O-GlcNAc transferase) relocates to a DNA-damaged site, allowing for the O-GlcNAcylation of the H2AX histone and MDC1 and restrains the DDR to a specifically damaged site. The inhibition of OGT also delays DSB repair, reduces cell proliferation, and increases cell senescence [114].

ncRNAs as Emerging Regulators

Local RNA synthesis or processing, and non-coding RNA have emerged as novel pathways in the regulation of DDR. Transcription is globally impaired in response to DNA damage, with ATM/DNA-PK triggering the ubiquitination of RNAPII by NEDD4 ligase and proteasomal degradation but opening chromatin at the site of the DSB locally to allow RNA synthesis [115,116]. Moreover, DNA damage affects mRNA splicing and stability, and mRNA splicing affects DDR. Serine/arginine-rich splicing factors (SRSFs) and heterogeneous nuclear ribonucleoproteins (hnRNP) are phosphorylated by ATM/ATR. HnRNP is co-recruited with the BCRCA1/PALB2/BRCA2 complex that further recruits Rad51 and regulates proper splicing at the DNA damaged site [117].

R-loops are three-stranded structures formed by an RNA/DNA hybrid and a displaced single-stranded DNA that plays multiple roles in gene expression, DNA replication, immunoglobulin CSR, and DNA repair [118]. In normal conditions, several mechanisms prevent the deleterious effects of an accumulation of deregulated R-loops. The THO/TREX (TRanscription-EXport) and TREX-2 mRNA export complexes function in proper packaging of mRNA into export-competent mRNPs, minimizing the possibility for a nascent mRNA to re-hybridize with the strand of transcribed DNA [19]. DNA topoisomerases relax DNAnegative supercoils and SRSF1 packs nascent mRNA into ribonucleoprotein complexes in a topoisomerase 1-dependent manner [119,120]. An SR protein, the alternative splicing factor/splicing factor 2 (ASF/SF2), regulates the formation of R-loops [121]. However, the accumulation of diverse RNA-containing structures, including RNA/DNA hybrids, generates DNA damage. It has been accepted that R-loops present a block for the replication fork progression, and constitute a major cause of replication stress that gives rise to genetic instability and chromosome fragility [122,123]. R-loop-mediated genomic instability is caused by the impairment of replication fork progression [124] and a deficiency in the molecular factors responsible for R-loops prevention. On the contrary, other studies have shown that RNA/DNA hybrids are involved in DSB repair processes in HR or NHEJ [125][126][127]. Hybrids protect DNA 3'overhangs, preventing an excess of resection [125,128], and contribute to the recruitment of DNA repair factors such as BRCA1 [129], 53BP1 [130], RPA [131], and FA pathway proteins, including FANCD2, FANCA, and FANCM. Senataxin, in complex with BRCA1, is recruited to R-loop-rich termination regions [129]. The R-loop-driven ATR pathway acts at centromeres to promote faithful chromosome segregation [132].

DNA damage induces the production and the control of a variety of non-coding RNA (ncRNA), as follows: microRNA (miRNA), and small or long ncRNA (s/lncRNA). P53 induces lncRNA expression that controls gene expression, cell cycle, and apoptosis; DSB-induced small RNA contributes to HDR; ATM, BRCA1, and P53 regulate miRNA expression; DROSHA/DICER-dependent RNA are involved in the formation of DNA repair foci [133]. A class of sncRNA, the DNA damage response RNAs, generated in a DROSHA/DICER-dependent manner are required to recruit secondary DDR factors at the chromosomal and the telomeres DSBs [134]. Several DNA damage-induced effectors control the transcription processes of non-coding RNA by the regulation of RNA interference factors DROSHA/DICER, and RNA polymerase II [115]. On the reverse, ncRNA plays various roles in damage signaling and repair by a regulation of the quantity of DNA repair proteins, by guiding or by providing a template for the DNA reparation (Figure 2). Nearly all primary DDR-induced effectors are regulated by miRNA and interact through their RNA-binding motifs in response to DSBs [126]. MiRNAs regulate, for example, H2AX (miRNA 24 and 138 [135,136]), PARP1 ), ATM (miRNA 18a and 421 [138]), ATM and DNA-PK (miRNA 101 [139]), BRCA1 (miRNA 182 [140]), RAD51 (miRNA 193b,103 and 107 [141]), BRCA2 (miRNA 1245 [142]), RAD52 (miRNA 302a [143]), and P53 (miRNA 125b and 504 [144,145]). Actual models propose that ncRNAs facilitate DNA repair, either by the hybridization of sncRNA to broken DNA via DDX1 and DHX9 (dead-box helicases) to form specific sequences propagating repair pathways or by the hybridization of lncRNA via RAD52-strand invasion, generating a template for high-fidelity repair [146,147].

LncRNA has proven regulatory modulation of DDR. They attenuate ATM activation and HR repair, sensitizing genotoxic treatment in cancers [53]. MiRNAs and lncRNAs cooperate with P21 WAF1/CIP1 (CDK inhibitor) to affect protein expression [148]. This generates cross-signatures in glioma and could serve malignancy diagnosis and prognosis [149]. In actively transcribed regions, lncRNA serve as template for NHEJ-mediated DSB repair to allow for the faithful transfer of missing information [150].

DDR Alterations and Associated Diseases

The alterations of many DDR members are associated with a range of diverse diseases, including immunodeficiency, inflammation, neurodegeneration, premature aging, cardiovascular or metabolic diseases, and cancers. However, there are reciprocal interactions between DNA damaged/repairing responses. For example, under viral infection, immunity directly relates to inflammation that underlies premature aging, and a high risk of cancer (Figure 3).

Immune Diseases and Inflammation

Immunodeficiencies are associated with DNA repair defects in the process of V(D)J genes and immunoglobulin class-switch recombinations. In immunoglobulin class switch recombination, a DSB is induced by the activation of a cytidine deaminase before broken ends are repaired by NHEJ or MMR. Any deficiency in one of the effectors of these repair complexes impairs the generation of IgG diversity to fight foreign antigens (acquired immunity), and leads to immunodeficiency at various degrees or hyper-IgM syndrome [151].

Different components of the DDR activate the innate immune system through the expression of ligands for the immune cell receptors and promote the production of proinflammatory mediators [152,153]. ATM, ATR, and CHK1 upregulate the formation of a ligand for the NKG2D immune receptor triggered by the stalled DNA replication fork [154].

The association of pre-existing DNA-repair defects (e.g., PARP1 or XRCC1/4) with DNA-damaging antibodies is highly genotoxic. In autoimmune diseases, such as systemic lupus erythematosus, anti-DNA autoantibodies circulate in the blood, cross cell membranes through mechanisms independent of their Fc region, and translocate in the nucleus to inhibit DNA repair and even damage DNA [155][156][157].

DNA damage effectors detect aberrant DNA structures in the nucleus and the cytoplasm, leading to immune signaling activation. For instance, Ku70 and DNA-PK, which are involved in the NHEJ, act as pathogenic DNA-recognition receptors in the cytoplasm and activate proinflammatory interferon, respectively, directly by IRF3 (IFN regulatory factor 3) or indirectly via STING (stimulator of interferon genes) signaling [158]. PARP1 upon genotoxic stress or the ATM activation of IκB kinases in DSBs both induce the translocation of NF-κB into the nucleus and promote the proinflammatory interferon and cytokine mediator's production. A loss of ATM impairs the innate inflammasome-dependent antibacterial immunity due to oxidative stress [159].

Moreover, an infection causes inflammation and enhances the production of ROS and oxidative DNA damage, creating a positive feedback loop that promotes genomic instability, cellular transformation, and carcinogenesis [160]. In the innate immune response, the cGAS-cGAMP-STING pathway senses cytoplasmic DNA, including microbial DNA or DNA released by DNA damage, and allows the production of more interferon and cytokines, the hallmarks of inflammation [161]. This pathway is also involved in the detection of DNA damage induced by autoantibodies in the systemic lupus erythematosus and directly connects this disease to inflammation by chronic interferon release [162].

detection of DNA damage induced by autoantibodies in the systemic lupus erythematosus and directly connects this disease to inflammation by chronic interferon release [162].

Figure 3. DNA damage induces inflammation and immunity that connect to metabolism and aging, with the genesis of mutations leading to cancer formation. The DNA damage response is activated upon DNA damage, arresting the cell cycle by CDK inhibition, and triggering an inflammatory response. cGAS, a cytosolic DNA sensor, triggers an innate immune response that activates STING. In mitosis, the CDK1-cyclin B complex phosphorylates and inhibits cGAS, but upon mitotic exit, dephosphorylation of cGAS by PP1 enables DNA cytoplasmic sensing [163]. cGAS connects DNA damage to the immune system, and subsequent inflammation by the production of interferon (IFN) and cytokines, but also to senescence and cancer [164]. Chronic inflammation can lead to an impairment of the immune system and generate ROS that counteracts DNA repair mechanisms [165]. Immunity triggers an inflammatory process that increases blood pressure, stimulating organ and metabolism damage [166,167]. Metabolism shifts with increased oxygen consumption and the generation of reactive nitrogen and oxygen intermediates are associated with inflammatory and immune responses [168]. Dysbiosis produces inflammation and metabolism syndromes that contribute to senescence. Pathogens can induce DNA damage and trigger immune and inflammatory responses. Aging is associated with adaptative immune and inflammatory responses and cumulative DNA damage and genomic instability that increase mutations [169,170]. Cancer cells evade detection by the immune system using immune checkpoints PD-1/PD-L1 (programmed cell death protein 1/programmed cell death ligand 1) and CTLA-4 (anti-cytotoxic T lymphocyte-associated antigen-4), LAG-3 (lymphocyte activation gene-3), TIM-3 (T cell immunoglobulin and mucin domain containing-3), and TIGIT (T cell immunoglobulin and ITIM domain) [171].

Neurodegeneration and Premature Aging

Neuronal dysfunction and neurodegenerative diseases occur with higher frequency when a persistent DDR is set up in neurons in association with an enhanced cholesterol biosynthesis and impaired Wnt or insulin (GSK3β increase) pathways [172]. In Huntington's, Parkinson's, and Alzheimer's diseases, PARP1 is increased. In Huntington's disease, NHEJ is defective and elevated PARP1 leads to increased AMPK activation, decreasing the CREB (C-AMP response element-binding protein) transcription factor necessary for the survival factor BNFD (brain-derived neurotrophic factor) and, finally, promotes necrotic cell death. In Parkinson's disease, increased levels of PARP1 accelerates the fibrillization and cytotoxicity of α-synuclein, which further activates ATM, H2AX, 53BP1, and NHEJ. In Alzheimer's disease, P53 stabilized by PARylation causes the upregulation of

Neurodegeneration and Premature Aging

Neuronal dysfunction and neurodegenerative diseases occur with higher frequency when a persistent DDR is set up in neurons in association with an enhanced cholesterol biosynthesis and impaired Wnt or insulin (GSK3β increase) pathways [172]. In Huntington's, Parkinson's, and Alzheimer's diseases, PARP1 is increased. In Huntington's disease, NHEJ is defective and elevated PARP1 leads to increased AMPK activation, decreasing the CREB (C-AMP response element-binding protein) transcription factor necessary for the survival factor BNFD (brain-derived neurotrophic factor) and, finally, promotes necrotic cell death. In Parkinson's disease, increased levels of PARP1 accelerates the fibrillization and cytotoxicity of α-synuclein, which further activates ATM, H2AX, 53BP1, and NHEJ. In Alzheimer's disease, P53 stabilized by PARylation causes the upregulation of pro-apoptotic protein [173]. Moreover, Tau hyperphosphorylation, and abundant neurofibrillaries down-regulate DNA damage/repair pathways with abnormal BRCA1 location and cell cycle re-entry inhibition [174].

Due to germline mutations in relevant genes, specific syndromes linked to DNA repair defects are hereditary. Distinct chromosomal instability disorders ataxia telangiectasia, Bloom syndrome, Cockayne syndrome, Fanconi anemia, Nijmegen breakage syndrome, trichothiodystrophy, Werner syndrome, and xeroderma pigmentosum display hypersensitive cells to particular genotoxic compounds. They also share clinical features of growth retardation, neurological disorders, premature aging, and skin alterations [175]. In ataxia telangiectasia, ataxia with oculomotor apraxia 1 and 2, and spinocerebellar ataxia with axonal neuropathy 1, the cerebellum is particularly affected. Neurodegeneration in xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy is associated with NER deficiency [176]. These progeroid syndromes feature some aspects associated with physiological aging at an early age.

Aging

Several lines of evidence indicate that the aging process is an association of DNA damage accumulation due to decreased efficiency in repair processes, mitochondrial dysfunction, metabolism alteration, free radical accumulation, and telomeres shortening [177][178][179]. The persistence of DNA modifications affects DNA polymerization, resulting in the collapse of the replication fork, an arrest in transcription, and the genesis of mutagenic templates during the synthesis of nucleic acid leading to cellular aging [180]. Aged cells accumulate extranuclear DNA, which triggers inflammation initiated by cGAS-STING and senescence [181]. Senescence is associated with the inhibition of mitophagy [182]. The loss of sirtuin NAD-deacetylase activity due to lower NAD+ levels inhibits mitophagy and results in persistent dysfunctional mitochondria and aging [183]. Moreover, proteins from the DDR lack deacetylation, such as ATM, XA, WRN helicase, and Ku70, impairing the repair processes [184].

DNA repair proteins become less efficient with age, especially in germline cells where DNA damage accumulates within the genome [185]. After remaining in the prophase of the first meiotic division for years, the quality of oocytes declines with age. An increasing number of primordial follicles accumulate severe DSBs and are submitted to apoptosis that diminishes the ovarian reserve [186]. In the mid-30s, in the remaining oocytes, aneuploidy increases due to a decreased efficiency in BRCA1 and ATM that is necessary for the regulation of sister chromatid cohesion by a control of the ATM-regulated SMC1 cohesin complex [185] and ROS vulnerability [187]. During the menopause, DNA damage accumulates in oocyte due to the reduction in DNA repair capacities, such as a decrease in BRCA1 [185]. However, human oocytes submitted to potent DNA damaging agents applied chronically at high concentrations extrude their polar body and complete meiosis I, and have a higher acceptance of DNA-damage compared to other species [80].

Cardiovascular and Metabolic Diseases

DNA damage is a hallmark in cardiovascular and metabolism disorders. In failing hearts and cardiovascular diseases, oxidative stress (redox imbalance) and DNA damage are enhanced [123,188]. The BRCA1-deficient cardiomyocytes that have an impaired DSB repair activate p53-mediated proapoptotic signaling, whereas P53 deletion rescues BRCA1deficient mice from cardiac failure. In addition, ischemia induces DSBs and upregulates BRCA1 expression in adult and fetal cardiac tissues [189]. An SSB was shown to cause heart failure by activating the DDR and increasing the expression of inflammatory cytokines via NF-κB signaling. Heart failure was more severe in the mice lacking XRCC1, but was inhibited by the genetic deletion of ATM [190].

In atherosclerosis, where the atherosclerotic plaque instability is associated with inflammatory cells, multiple DNA damage markers are present, including early induced DNA repair enzymes and the phosphorylated forms of ATM and H2AX [191]. The vascular smooth muscle cells show a defective repair of oxidative DNA damage, due to the increased degradation of the BER enzyme, OGG1 [192]. In atherosclerotic cardiovascular diseases such as the Werner or Hutchinson-Gilford progeria syndromes, DSB repairs are defective [193]. Both syndromes are also, respectively, associated with diabetes and lipodystrophy symptoms. The metabolic changes in type 1 and type 2 diabetes increase the levels of genotoxic ROS and dicarbonyls that further form glycation adducts and reduce DNA repair [194].

Chronic diabetic type 2 β-cells have a greater extended DSB [195]. A compromised DNA repair is responsible for diabetes-associated fibrosis in various organs [194].

In obesity, oxidative stress and inflammation induce DNA damage, inhibit DNA repair, alter gene expression to set up metabolism disturbances, and further promote resistance to apoptosis and predispose to cancer [196]. Adipocytes' metabolic and secretory functions are strongly altered by DNA damage [197]. On the contrary, weight loss may prevent obesity and other adverse health effects due to a reduction in DNA damage with a decrease in pro-inflammatory cytokines and insulin levels [198].

Cancers

Various mechanisms related to DNA damage including dysregulation of the DDR pathways and a high level of replication stress are responsible for genomic instability, and linked to pretumor and tumor formation [44]. Aberrant copy numbers of genes correlate with the expression levels of mRNA [199]. Various primary cancers also display mitochondrial DNA mutations involved in ROS production, apoptosis inhibition, DNA repair anomalies, and cell cycle misregulation in the mitotic spindle and the G2/M checkpoint [200,201].

More than 450 proteins from the DDR [2] are related to familial cancer predispositions through diverse somatic mutations or inactivating germline mutations (Table 1). BRCA1 and BRCA2 are associated with a family history of breast and ovarian cancers. ATM is associated with a family history of breast and ovarian cancers with 1.7 times higher risk [202,203]. MSH2/6 is associated with a family history of colon/endometrial cancers. The "ICGC/TCGA Pan-Cancer Analysis of Whole Genomes Project Consortium 2020" has shown that somatic mutations of DDR-related genes were found in a third of cancers [204]. Multiple processes generate somatic mutations in the genome and determine a mutational signature for each type of cancer. These studies have identified defective components from the DNA damage repair, replication, the DNA-maintenance processes [204], and associated mutations, for instance, CDK12 (transcriptional modulator of DDR genes and mRNA processing) with DNA duplicated tandem stretches, or truncated variants of the MMR enzyme MBD4 (methyl-CpG-binding domain protein 4) with a mutational signature of CpG sites [205].

Moreover, interconnections are present between chronic inflammation or infection and generate genotoxic stress and tumor initiation/progression [206,207]. The risk of colorectal cancer is underlined by faster DNA damage in inflammatory bowel disease [208]. Tumor cell proliferation is also linked to the remodeling of the immune microenvironment through the deregulation of rhythmic expression of cyclin genes and the P21 WAF/CIP1 cell cycle inhibitor when the circadian rhythm is disrupted [209]. A virus such as the human papillomavirus reinitiates DNA replication by abrogating the cell cycle checkpoint proteins P53 and Rb (retinoblastoma protein), leading to an unscheduled S phase entry, followed by replication stress, DDR, and carcinogenesis [210].

DDR Targets in Anticancer Therapies

Targeting DDR Proteins to Induce Deficiencies

Several categories of DDR proteins are targeted in anticancer monotherapies to elicit cell death [222,223] (Figure 4). DNA damage signaling inhibitors target kinases: ATM and DNA-PK are among the first activated kinases upon a DSB and, respectively, execute checkpoint signaling or DNA repair, while ATR is recruited by replication stress to facilitate fork stabilization and restart. ATR and ATM, respectively, activate the kinases CHK1 and CHK2, and WEE1. ATM and CHK2 are G1/S checkpoint DDR protein targets, ATR, CHK1, DNA-PK, and WEE1 are S phase regulators, and CHK1 and WEE1 are G2/M checkpoint regulators. Clinical trials are still currently running for this category of inhibitors, such as the trial in phase I for an oral ATR inhibitor BAY-1895344 against an advanced solid tumor [222,224,225].

Another category of inhibitors targets proteins involved in DNA repair such as RAD51 and POLQ, respectively from the HR and MMEJ processes, or . The discovery of PARP inhibitors was a milestone for anticancer chemotherapies directed against DDR proteins. Initially evaluated in patients with BRCA-mutated breast tumors, the inhibition of PARP eliminates its trapping on DNA and elicits death [230].

A third class of inhibitors is raised against proteins involved in DNA metabolism such as inhibitors for RNR (ribonucleotide reductase) that catalyzes a rate-limiting step in the production of deoxyribonucleotides and PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6biphosphatase 3) that converts fruc-tose-6-phosphate to indirectly stimulate glycolysis [231,232].

A fourth category of inhibitors is directed against chromatin regulation. DNA methylation or histone methylation/acetylation are epigenetic modifications that affect DNA repair ability. Histone deacetylase (HDAC) inhibitors lower DNA damage repair proteins from the NHEJ and the HR through the inhibition of ATM and MRN [233,234]. DNA methyltransferase 1 (DNMT1) inhibition induces the depletion of multiple repair protein in the MMR [235].

Targeting the DDR Protein Network with Synthetic Lethality

By exploiting the defects in DNA repair, several categories of DDR proteins are targeted to take advantage of the concept of synthetic lethality and induce cell death. Cells usually repair DNA damage by switching from one alternative repair mechanism to another if a repair pathway is defective. Moreover, a loss of function targeting several DDR pathways renders these cells less susceptible to survival. The concept of synthetic lethality used to target DDR pathways occurs when deficiencies in independent genes are tolerated but when the combinations are lethal [230,236,237].

Several DDR inhibitors are investigated in ongoing clinical trials and the choice of the combination of inhibitors depends on the characteristic of the tumor [238]. Synthetic lethal vulnerabilities are exploited therapeutically by combining inhibitors against a tumor deficiency in the DDR protein: CHK1 inhibitor (MK-8776) is used with gemcitabine

Targeting the DDR Protein Network with Synthetic Lethality

By exploiting the defects in DNA repair, several categories of DDR proteins are targeted to take advantage of the concept of synthetic lethality and induce cell death. Cells usually repair DNA damage by switching from one alternative repair mechanism to another if a repair pathway is defective. Moreover, a loss of function targeting several DDR pathways renders these cells less susceptible to survival. The concept of synthetic lethality used to target DDR pathways occurs when deficiencies in independent genes are tolerated but when the combinations are lethal [230,236,237].

Several DDR inhibitors are investigated in ongoing clinical trials and the choice of the combination of inhibitors depends on the characteristic of the tumor [238]. Synthetic lethal vulnerabilities are exploited therapeutically by combining inhibitors against a tu-mor deficiency in the DDR protein: CHK1 inhibitor (MK-8776) is used with gemcitabine (antimetabolite) in solid tumors [239], WEE1 Inhibitor (AZD-1775) with gemcitabine, cisplatin, or carboplatin (DNA damaging agents) in advanced solid tumors [240] or in combination with a checkpoint kinase CHK1 inhibitor (PF-0047736) in acute lymphoblastic leukemia [241], and a DNA-PK and mTOR dual inhibitor (CC-115) in hematopoietic and solid cancer cells [242].

Synthetic lethality is explored with PARP inhibitors in several cancers such as ATMdeficient tumors from breast and ovaries, aggressive prostate cancers in combination with ATR inhibitors [243,244], and high-risk neuroblastomas under ATR inhibition [245]. POLQ deficiency is synthetically lethal in combination with PARP inhibitors and associated with mutations of ATM, BRCA, Ku, 53BP1, and FA (Fanconi anaemia) [226,229,246]. Other synthetic lethalities include the ATM inhibitor, KU60019, or Top1 inhibitor, irinotecan, in ATRX-deleted neuroblastoma [247]. The application of synthetic lethality also depends on the RAD52 inhibition in cancer cells bearing BRCA1/2 mutations or the suppression of the BRCA1-RAD51 pathway [248]. Moreover, PARP controls the epigenetic modifications of chromatin at the level of DNA and histones, and changes in PARylation modify DNA methylation patterns and directly affect DNA repair [249]. PARP inhibitors show synergistic activity to allow a selective vulnerability with DNMT1 inhibition in acute myeloid leukemia and breast cancer [250], with HDAC inhibitors in triple-negative breast cancers [251], or with the inhibition of chromatin remodeling components of the SWI/SNF (switch/sucrose non-fermentable) complex in cancer cell lines [252].

Multiple components of the DDR are also targeted in cancer stem cells that are particularly resistant to DNA damage to increase their genetic lesions and boost their chemoand radio-sensitivities [253]. Combining PARP and ATR inhibitors, or CHK1 and WEE1 inhibitors are efficient strategies for cancer stem cells sensitization [254][255][256].

Targeting Replication Stress

Replication stress arises when genomic stress affects the progression of DNA polymerase and uncouples DNA polymerization from DNA unwinding. New anti-cancer therapies targeting DNA damage repair mechanisms of the DDR are developed to exploit excessive unsustainable replication stress in cancer cells having a high proliferative index compared to normal cells [226,257]. The ATR-CHK1-WEE1 signaling pathway provides the possibility to enhance fork destabilization and replicative stress, and concomitantly lowers cell cycle checkpoint thresholds and pushes tumor cells into mitotic catastrophe and cell death. A high number of approved inhibitors against CDK, or DNA repair protein such as PARP are in clinical trials one and two against advanced ovarian cancer [68]. A novel target, WRNIP1 (Werner interacting protein 1), involved in an (ATM)-dependent phosphorylation of CHK1, is implicated in counteracting an aberrant R-loop accumulation capable of blocking the replication fork [258].

The antimetabolites that incorporate into DNA in place of thymidine during the S phase, preventing nucleobase addition and terminating chain elongation, also cause DNA replication failure. Antimetabolites are pyrimidine analogs (5-fluorouracil, gemcitabine, floxuridine, or capecitabine) and purine analogs (6-mercaptopurine, 8-azaguanine, fludarabine, and cladribine) that affect DNA and RNA metabolism and arrest cells in the S phase of the cell cycle. A transcriptomic analysis of colon cancer cells' response to 5-fluorouracil showed different cell fate phenotypes in the DNA-damage-induced genes' expression of apoptosis and cell-cycle checkpoints [259]. Similarly, the topoisomerase poisons (forming stable cleavage complex) described in the next paragraph hamper replication and are efficient agents to increase DNA damage.

Replication Stress and Telomere Deprotection

Several telomere-associated factors in the shelterin complex and the CST complex (CTC1-STN1-TEN1) ensure the prevention of replication stress by capping and by regulating telomerase and DNA polymerase alpha-primase [260,261].

Telomere deprotection contributes to replication stress, induces a SAC-dependent mitotic arrest through the activation of aurora B kinase and TRF2 that dissociates from chromosomal ends, triggers structural changes from telomere-loops to linear telomeres, exposes chromosome ends to ATM, and activates the mitotic telomere DDR [65,[262][263][264][265]. Deprotected telomeres induce the transcription of tDDRNAs (telomeric small non-coding DNA damage response RNAs) and subsequent DDR. The depletion of tDDRNA by complementary antisense oligonucleotides results in the inhibition of the DDR at telomeres [266]. The strategy targeting tDDRNAs with oligonucleotides could be exploited as potential biomarkers to detect the DDR activation of telomeric damage or to modulate telomeres' function.

T-oligos (telomere homolog oligonucleotides) are homologous to the 3' single-stranded TTAGGG overhang involved in chromosome end stabilization. They trigger telomere uncapping, or mimic DNA damage and are also promising tools. Telomere overhang oligonucleotides decrease the proliferation of prostate cancer cells [267] and non-small cell lung cancer cells [268], and sensitize mammary tumor cells to radiation [269].

Telomeric DNA G-rich sequences allow for the formation of a G-quadruplex (G4) capable of impeding the progression of the replication fork and promoting telomere fragility and replication stress [260,261,270]. Several classes of G4 interacting agents affect telomere maintenance and are promising targets for cancer therapies [271,272]. G4 ligands alter telomeric chromatin, leading to POT1 dissociation and the consequent activation of the ATR-/ATM-dependent damage pathway at the telomeres [271]. G4 ligands have been successfully developed as favorable agents (tetracarboxymethyl porphyrins) for accurate probing [273] or as specific G4 telomeric porphyrin photosensitizers (TMPipEOPP) to discriminate G-quadruplex from duplex and ssDNA [274]. Employed in combination with PARP1 inhibitor, the RHPS4 G4 ligand reduces cell proliferation, avoids uncontrolled DNA replication, and induces cell death [275]. A basal level of DNA damage and telomere deprotection increase their sensitivity in cancer cells [276]. The CX-5461 G4 ligand, a quarfloxin derivative initially identified as a RNA-Pol I inhibitor [277], was recently shown to act at transcribed regions bearing G4 structures as a DNA structure-driven TOP2 poisons [278,279]. CX-5461 induces replication-dependent DNA damage through the stabilization of G4 DNA structures, which has entered phase I/II of clinical trials for patients with BRCA1/2 deficient tumors [280] and hematologic cancers [281].

Other Oligonucleotides, or Small Interfering RNA

In addition to telomere homolog oligonucleotides, other oligonucleotide therapeutics based on antisense RNAs and small interfering RNA (siRNA) modify the mRNA metabolism [282]. Using siRNA against repair protein XPA (NER) and co-targeting the synthetic lethal relationship with the cell cycle checkpoints, kinase MK2 enhances the antitumor response in P53-deficient cancers [283]. In myeloma, ILF2 antisense oligonucleotide (Interleukin Enhancer Binding Factor, a key modulator of the DNA repair pathway) is a synthetic lethal target with DNA2 (DNA replication helicase/nuclease 2) [284]. A radiolabeled oligonucleotide that targets the RNA-associated telomerase promotes radiation-induced genomic DNA damage in telomerase-positive cancer cells [285]. A short, double-stranded oligonucleotide linked to a cholesterol molecule, AsiDNA™, acts as a decoy, mimics DSBs, and triggers a false DNA break signal to activate and attract DNA repair proteins, preventing their recruitment to the site of genomic DNA damage. The oligonucleotide acts by the overactivation of false signaling of DNA damage through DNA-PK and PARP enzymes and depletion of the DDR. The tumor DNA breaks are not repaired, accumulate damage, and drive cancer cells to death at the onset of replication [286]. The inhibition of ncRNAs overexpressing or the replacement of tumor-suppressive ncRNAs is a strategy if the inflammatory response they produce is bypassed [287].

Topoisomerase Inhibitors

Interference with protein-DNA complexes alters DNA structure and triggers DDRassociated cell death. Topoisomerases (Top) are involved in replication and transcription. Top1 and Top2 relax DNA superhelical tension with, respectively, SSB or DSB cleavages to allow for strand rotation before resealing the DNA. A collision between the replication fork and the cleavage complex of Top1 produces DSBs. Top covalently attaches DNA, forming reversible DNA-Top cleavage complexes during the cleavage step. From the three subgroups of human Top (type IA, IB, and IIA), types IB and IIA are particularly overexpressed in certain cancers [288]. Top inhibitors are either poisons that bind to the DNA-topoisomerase cleavage complex (interfacial inhibition) and form a ternary DNAtopoisomerase-inhibitor complex preventing the religation of the two strands of DNA; or catalytic inhibitors including DNA intercalators, ATP competitors, and inhibitors of ATP hydrolysis [289,290]. When targeted by Top poisons, the stabilization of the DNA-Top cleavage complex blocks DNA replication and generates deleterious DSBs, inducing cell death. The initial inhibitors were camptothecin (Top1 inhibitor, poison), etoposide (Top2 inhibitor, poison), and doxorubicin (Top2 inhibitor, poison) an anthracycline that intercalates, alkylates and crosslinks DNA, producing free radicals, or interferes with the helicase function. Top1 inhibitors, indenoisoquinoline derivatives LMP400 (Indotecan) and LMP776 (Indimitecan) have reached the clinical trial phase [290,291]. The only dual Top1/2 inhibitor used in therapy is aclarubicin, but other dual inhibitors exist against Top2 and other targets such as kinases or proteasome [292]. Tyrosyl-DNA phosphodiesterase 1/2 (TDP1/2) resolve Top-DNA complexes by liberating Top1 or Top2 (respectively TDP1 and TDP2) from the DNA after the degradation of the cleavage complexes. A combination of inhibitors such as the Top1 inhibitor irinotecan with a TDP1 inhibitor also improves the treatment of glioblastoma [293]. A higher response to irinotecan (Top1 inhibitor) is obtained in BRCA2-mutated cancer with acquired resistance to olaparib (PARP inhibitor) showing the downregulation of TDP1 [294]. To render the Top2 inhibitor etoposide specific for cancer cells, a covalent link was realized with a single-stranded oligonucleotide displaying a complementary sequence to a translocated cleavable DNA region only present in promyelocytic leukemia [295].

The structural optimization of Top inhibitors by glycosylation is an option to enhance the DNA damaging efficiency [296]. The addition of a carbohydrate moiety to indenoisoquinoline enhances the binding affinity of the Top1 drug to DNA due to a stronger hydrogen bonding interaction [297]. The covalent conjugation of galactose on doxorubicin increases its effect on liver cancer cells with less myocardial damage [298]. Doxorubicin uptake by the GLUT1 glucose transporters is increased by the conjugation of 2-amino-2-deoxy-D-glucose or succinic acid within cancer cells that overexpress GLUT1 and are dependent on glucose metabolism (the Warburg effect) [299].

DNA Damaging Drugs

The concept of increasing DNA breaks to activate the DDR protein pathway and promote cancer cell death inspired the development of various anticancer drugs. Anticancer compounds that chemically modify DNA bases, intercalate, or crosslink DNA include alkylating agents such as nitrogen mustard or platinum-derived compounds and agents that target the minor groove of DNA instead of the major groove compared to most of the alkylants [300]. Alkylating agents such as estramustine for the treatment of prostate cancer [301] or bifunctional cyclophosphamide [302] result in the covalent transfer of alkyl-groups to DNA, induce bulky DNA damage, and block transcription and replication. The anticancer activity of alkylating agents such as cyclophosphamide is in close interplay with miRNA to determine the outcome of an applied therapy [303]. Platinums such as cisplatin and its derivatives such as carboplatin, oxaliplatin, and picoplatin bind to guanine and adenine residues to form DNA adducts after the aquation of the platinum chloride group. Recently, a map of the sites of cisplatin damage and repairs for the entire human genome were obtained at a single-nucleotide resolution to better understand cancer sensitivity and resis-tance [304]. Other non-traditional alkylators are methylating agents, such as temozolomide used to treat glioblastoma [305]. Numerous metal-derived compounds, organometallics, are developed to generate severe DNA alterations [306,307]. Some ruthenium-containing compounds such as polynuclear ruthenium induce DNA damage and recruit DNA repair effectors such as XPC (Xeroderma Pigmentosum complementation group C) [308]. Certain organometallics target enzymes involved in DNA topology (Top1) [309]. Most metallo-glycopeptides are interesting DNA damaging drugs such as bleomycin that require a metal ion (Fe (II)) to be activated and cleave a DNA-specific sequence in 5'GT and 5'GC dinucleotides [310].

Most metal-containing compounds also belong to the class of ROS-inducing drugs that disrupt redox homeostasis, causing various types of death. Besides doxorubicin and cisplatin, a number of prooxidative drugs are used such as 2-methoxyestradiol, buthionine, or sulfoximine [311]. Cancer cells have an accelerated metabolism compared to normal cells, which generates a high level of ROS and makes them depend on MTH1 (MutT homolog 1), a repair enzyme that hydrolyzes oxidized nucleotides to corresponding monophosphates. The selective inhibition of MTH1 suppresses cancer growth through an accumulation of oxidative damage [312].

DNA Damaging Tools

Molecular tools are capable of introducing programmable DNA breaks. The CRISPR/ Cas9 (clustered regularly interspaced palindromic repeats/associated protein 9) system provides the ability to add or remove specific DNA sequences in the genome to perform site-specific gene editing. In this plasmid-based technology, Cas9 endonuclease works guided by a single-stranded RNA at a specific site on a 20 base pair sequence among the human genome to create DSBs followed by endogenous DDR repair mechanisms NHEJ (error-prone) and HDR (high fidelity) [313]. To control DNA repair, outcomes for genome editing strategies have been developed to locally or globally inhibit or activate DNA repair effectors and favor a high-fidelity repair [314]. The fusion of DNA repair factors such as the HDR enhancer element of CtIP or Geminin to Cas9 ensures a rapid, efficient HDR [315]. Geminin inhibits the replication factor CDT1 during the S and G2 phases to avoid rereplication. The fusion of Cas9 to a dominant-negative 53BP1 inhibits NHEJ and improves HDR frequency [316]. DSB repair factors fused with Cas9 favored MMEJ and HDR in hematopoietic cells [317].

Ionizing radiation creates high levels of clustered DNA damage (including DSB, covalent crosslinking, base damage) that are difficult to repair and exploitable in radiation therapy. Over the years, radiotherapy has been improved by intensity modulation, image guided, and internal access [318,319]. The radiation efficiency is enhanced using drugs that target DNA repair machinery [320]. A new class of drugs, named radiopharmaceuticals, delivers radiation therapy specifically and directly to cancer cells. Despite the exploitation of the natural affinity of radioactive iodine to treat thyroid cancer or radium 223 to treat cancers that have spread to bones, new radiopharmaceuticals target surface molecules specifically present on cancer cells. A radiolabeled somatostatin analog compound, lutetium 177-dotatate, that targets surface receptors, is approved for the treatment of certain neuroendocrine tumors affecting the digestive tract [321], and small molecules such as PSMA (prostate-specific membrane antigen) inhibitors deliver radiotherapeutic nuclides in prostate cancer [322]. A combined strategy with an oncolytic adenovirus that inhibits the DDR, renders external beam radiation therapy more efficient [START_REF] Liu | The Role of CDC25C in Cell Cycle Regulation and Clinical Cancer Therapy: A Systematic Review[END_REF].

Drugging DDR Proteins in Association with Other Therapies

Inhibition of Cell Cycle Progression and Transcription

Cancer cells are rapidly dividing cells and highly susceptible to DNA damage. Despite the fact CDKs activate the DNA damage checkpoint and initiate the DNA repair, they also interfere with the DDR. CDKs inhibition compromises CHK1 function in the DNA damage and the stalled replication [324]. The inhibition of CDK4/6 that commits the G1 and S phases' transition inactivates the retinoblastoma tumor suppressor protein and arrests the cell cycle in G1. The CDK4/6 inhibitor, palbociclib, in combination with PARP inhibitors, added to endocrine therapy, suppresses the DNA damage repair to treat BRCAmutated ER+ (estrogen receptor-positive) breast cancers [325]. In addition to their cell cycle regulatory functions, CDKs, especially CDK7, 8, 9, 12, and 13 phosphorylate and regulate RNA polymerase II-mediated transcription. Their inhibition can deeply affect the expression of DDR genes. CDK12 inhibitor THZ531 induces the RNA Pol II elongation defect and the premature cleavage and polyadenylation of long DDR genes followed by apoptosis in neuroblastoma cells [326]. SR-4835, a CDK12/13 inhibitor, acts in synergy with PARP inhibitors in triple-negative breast cancers [327].

Restoration of the P53 Pathway

P53 is the most frequently mutated gene in more than 50% of all cancers, leading to P53 protein loss or disfunction. Several strategies have been developed to reactivate P53. Murine double minute 2 (MDM2) is an E3 ubiquitin ligase responsible for P53 ubiquitination and inactivation by proteasomal degradation. MDM2 heterodimerizes with MDMX to enhance P53 ubiquitination and degradation.

Some compounds inhibit MDM2 interaction with P53, such as small molecules [328] that act as MDM2 antagonists, inhibit E3 ubiquitination of P53, or stabilize P53 and restore its conformation and DNA-binding ability [329,330]. PRIMA-1 and its analog (APR-246) restore mutated P53 proteins to a normal conformation and re-establish P53 transcriptional activity. The expression of PUMA, NOXA, and BAX are increased. PRIMA-1 is chemically converted intracellularly and binds the cysteine residues of mutated P53 protein, allowing a correct refolding [331,332]. Most P53 mutations are located in the DNA-binding core domain and create a destabilizing cavity that can be corrected by small molecules named PhiKan083 and PhiKan7088, restoring transactivation potential inducing P21 and NOXA expression with the consequent cell cycle arrest and apoptosis [333,334]. Many of those inhibitors have entered clinical trials against hematological malignancies or advanced solid tumors [335].

WIP1 phosphatase is a negative regulator of P53 after the completion of DNA repair [336]. The inhibition of WIP1 suppresses the proliferation of cancer cells by the activation of the P53 pathway. WIP1 depletion by RNA interference sensitizes cancer cells to DNA damage-inducing chemotherapy [337], and WIP1 inhibitor GSK2830371 potentiates the cytotoxic effect of doxorubicin in neuroblastoma [338].

The cell-cycle checkpoint abrogation is also a possible strategy to bypass a mutation in P53. Compared to normal cells, P53 mutated cancer cells lack a normal G1 checkpoint and mostly rely on the G2 checkpoint. The loss of WEE1 activity sensitizes P53 deficient cells to DNA damaging agents and radio sensitization [339]. The inhibition of WEE1 induces a mitotic catastrophe and cell death [340]. WEE1 inhibitor MK1775 was recently reported to suppress tumor proliferation and to potentiate Top1 inhibitor irinotecan action in P53 mutant colonic cancer cell lines [341].

DDR Inhibition Strategies Associated to Cancer Immunotherapies

The expression of immunosuppressive surface proteins allows cancer cells to escape immune detection and destruction. Activated T cells recognize tumor antigens, but their PD-1 receptor engaged to tumor PD-L1 (programmed death-ligand 1) or their CTLA-4 (cytotoxic T-lymphocyte associated antigen 4) interaction with B7 antigen-presenting cells inhibit T cells cytotoxicity. Consequently, antibodies against PD-1, PD-L1, or CTLA-4 are immune checkpoint blockers that potentiate anti-tumorigenic immune responses [342]. Combining a PD-L1 inhibitor with a PARP inhibitor or PARP/PD-L1 dual inhibitors [224] in breast cancers [343] allows a stronger antitumor activity compared to separate agents. In non-small cell lung cancer, pembrolizumab (anti-PD1 antibody) co-administrated with chemo-or radiotherapy showed a marked clinical benefit [344,345]. Regardless of the cancers' tissue of origin, a large proportion of mutant neoantigens in MMR-deficient cancers are sensitive to an immune checkpoint blockade [346]. Around 200 clinical trials are testing a combination of immune checkpoint inhibitors with DNA damaging chemotherapies [226].

DNA fragments are recognized by cGAS sensor (innate immunity), followed by cGAMP synthesis, STING activation, and translocation to the Golgi, where it recruits IKKA (inhibitor of nuclear factor kappa-B kinase subunit alpha) and TBK1 (TANK-binding kinase 1) to induce the transcriptional activation of interferon and chemokines CXCL10 (chemokine (C-X-C motif) ligand 10). In addition to cytosolic DNA detection, cGAS is involved in the control of the replication dynamic by decelerating the replication fork. cGAS-deficient cells are exposed to an acceleration of the replication fork, and to replication stress that renders them hypersensitive to radiation and chemotherapy [347,348].

Pharmacogenomic and Predictive Markers in DDR Strategies

Cancer pharmacogenomic relates the interaction between genetic predisposition and therapeutic drugs responses [349,350]. Genome mapping of chemotherapeutic effects is performed to determine compounds selectivity for genomic regions: topotecan (Top1 inhibitor) and etoposide (Top2 inhibitor) induce similar DNA damage in transcriptional active genomic regions, daunorubicin (anthracycline) evicts histones, while aclarubicin evicts histones from heterochromatin. This different genomic specificity of DNA damageinducing drugs has consequences for compound activity in different tumor types [351]. A prognostic signature related to the DDR pathway and a gene signature of RNA binding proteins was determined to promote an individualized treatment strategy in prostate cancer [352]. When mutated, 73 genes from the HR, FA components, and ATM/ATR kinases determine a hypersensitivity to PARP inhibitors [353]. Single nucleotide polymorphisms in ERCC1 predict the response to DNA damaging platinum chemotherapy for patients with non-small cell lung cancer [354].

The identification of markers is also important to select patients for proper therapy. A polymorphism study on CDK12, which modulates the transcription elongation of RNA polymerase II and affects the expression of DDR genes, reveals it is a possible prognostic biomarker in late-stage ovarian cancer treated with platinum [355]. Markers that predict sensitivity or resistance to induced DNA damage therapy such as the Schlaffen biomarker are useful to predict the accurate response to Top1 and Top2 inhibitors, in colon and ovarian cancers [356]. However, using a single marker to perform personalized therapy is limited and it appears necessary to integrate genomic data of several markers regarding the disease progression. The use of circulating tumor DNA (ctDNA), an emerging field in multiple cancer types, allows the evolutionary dynamics of tumors such as early stage lung cancer [357] or in bladder cancer to be tracked to follow the development of resistance in real-time using a liquid biopsy [358]. DNA-repair gene mutations are highly prevalent in ctDNA from myeloma [359]. The pharmacodynamic activation of the DDR pathway can confirm target engagement in tumors following anticancer treatment. DNA damage recognition and repair proteins (γH2AX, pS343-NBS1, and RAD51) are biomarkers detected in preclinical and clinical treatment with a quantitative multiplex immunofluorescence assay [360]. Mutation and neoantigen burden are developed as potential clinical biomarkers. The microsatelliteinstability-high (MSI-H) that results from a defect in MMR in germline or somatic genes was recently approved as an indicator for immune checkpoint inhibitors [346]. Tumors with microsatellite instability respond better to PD-1 blockade with pembrolizumab in a phase two study [361]. The expression of major DDR kinases including ATM, ATR, CHK1/2, and WEE1 tailor anticancer treatment, such as systemic treatment with PD-L1-targeting monoclonal antibodies (checkpoint inhibitors). PD-L1 expression is upregulated in cancer cells in response to DSBs under genotoxic stress, such as radiotherapy or PARP inhibition. The inhibition of ATR downregulates PD-L1 in a proteasome-dependent manner, attenuates the interaction of PD-L1/PD-1, and sensitizes cancer cells to T-cell-mediated killing [362]. Using siRNA library screen targeting DNA repair genes, PD-L1 induction was shown to be dependent on the ATM/ATR/CHK1 pathway [363].

Viral Therapies to Boost the DDR

Viruses are actively involved in manipulating the DDR pathway targeting the MRN sensor complex of proteins for degradation and providing a rationale for their use in therapies [364]. Viral vectors have high efficiencies for gene transfer and expression. Adenoviral and retroviral vectors are primary used with tissue-specific promoters, facilitating tumor killing [365]. An adenovirus was used to express P53 (gendicine) to treat patients affected by head and neck carcinoma and is now applied to other cancers as gene therapy with adenovirus serotype-5 being the prevalent adenovirus used in clinical trials [366]. The combination of the potent Ad5/35 chimeric virus with either DNA damaging therapies (radiation or chemotherapy) leads to significant increase in the survival of aggressive tumors [367].

Oncolytic viruses such as Enadenotucirev formed as a chimera of two group B adenoviruses, Ad3 and Ad11p, are promising due to their dual action by a direct intratumor spread and oncolysis, in addition to an increase in the antitumor T-cell response [START_REF] Liu | The Role of CDC25C in Cell Cycle Regulation and Clinical Cancer Therapy: A Systematic Review[END_REF]368]. DNA-PK inhibition sensitizes cancer cells to oncolytic alphavirus M1 and improves therapeutic effects in refractory patient tumor samples. Infection triggers the transcription of IFN and the activation of an antiviral response that is abolished by pretreatment with DNA-PK inhibitors and results in an enhanced replication of the virus within malignancies. DNA-PK inhibition further promotes viral induced DDR and increases cell apoptosis [369].

The oncolytic herpes simplex lentivirus has a unique property to target multiple components of the DDR, irrespective of their mutation. This virus generates cancer-selective sensitivity to PARP inhibitors in glioblastoma. Infection induces a loss of RAD51 due to proteasome-dependent degradation that requires viral DNA replication. This synthetic lethal-like interaction is applicable to resistant tumors in which PARP inhibitor treatments block BER and activate DDR and S phase arrest through CHK1 activation, while viral infection induces DSBs, inhibits HR repair through the degradation of RAD51, and alters the cell cycle through CHK1 degradation [370].

Conclusions

The DDR network, guardian of the genome, contains numerous proteins connected by multiple regulatory interactions. Cancers and several other diseases are caused by inherited mutations in DNA or those generated by high levels of DNA damage and repair defects in DDR proteins. To kill cancer cells, the creation of DNA-damaging agents in chemotherapy alone or combined with other agents controls the DDR to optimize anticancer activity. Choosing the right inhibitor to administer with indications from the genomic profile, and/or associated with a predictive effect of the drug would fine-tune these therapies and could adapt to the evolving profiles of tumors.

Introduction

Chemotherapy is a systemic treatment proposed to patients suffering from cancer. It is often a complementary approach to surgery or radiotherapy. The discovery of platinum's inhibitory effect on tumor cell growth in the 1960s [1] was a milestone for anticancer drug application in medicine [2]. Platinum (II) sets at the center of the squared planar structure of cisplatin and is coordinated with two chlorides and two ammonia molecules in a cis configuration. Cisplatin and its derivative drugs (carboplatin of second generation and oxaliplatin of third generation) are used worldwide in clinical applications and several other platinum analogs (lobaplatin, nedaplatin, and heptaplatin) are approved in several countries (Figure 1) [3,4]. However, serious side effects including toxicities on the kidney, heart, ear, and liver, decrease in immunity, hemorrhage, and gastrointestinal disorders limit the use of platinum derivatives [5][6][7]. The appearance of drug resistances, issuing from acquired or intrinsic multiple genetic and epigenetic changes, has also limited the clinical use of platinum-derived drugs [8].

Platinum-based treatment efficiency is challenged by cross-resistance and multiple changes including a decreased accumulation of the drug, a reduction in DNA-drug adducts, a modification in cell survival gene expression, an alteration of DNA damage repair mechanisms, modifications of transporters, protein trafficking, and altered cell metabolism [9][10][11][12][13][14].

Cancers 2020, 12, x 2 of 26 derived drugs [8]. Platinum-based treatment efficiency is challenged by cross-resistance and multiple changes including a decreased accumulation of the drug, a reduction in DNA-drug adducts, a modification in cell survival gene expression, an alteration of DNA damage repair mechanisms, modifications of transporters, protein trafficking, and altered cell metabolism [9][10][11][12][13][14]. To circumvent drug resistance, a possible approach consists of designing and developing new therapeutic metal-based anticancer drugs [15][16][17][18][19][20][21]. Several transition metals from the d-block of the periodic table (groups 3 to 12) and particularly essential trace metals [15,22,23], such as copper [24][25][26][27][28][29], are useful for the implementation of metal-based complexes in anticancer therapies. Copper plays central roles in various cellular processes being an essential micronutrient and an important cofactor for several metalloenzymes involved in mitochondrial metabolism (cytochrome c oxidase), or cellular radical detoxification against reactive oxygen species (ROS) (superoxide dismutase) [30]. Copper is essential for angiogenesis, proliferation, and migration of endothelial cells [31][32][33]. Elevated copper favors tumor growth and metastasis. It is detected in several brain [34], breast [35], colon, prostate [36], and lung [37] tumors and serves as an indicator of the course of the disease [38]. The differences in tumor cells' responses to copper compared to normal cells laid the foundation of copper complexes' (CuC) evolution as anticancer agents. Numerous developed CuC contain different sets of N, S, or O ligands and demonstrate high cytotoxicity and efficient antitumor activity [25]. Different mechanisms are involved in copper drugs' anticancer effect. They act as chelators, and interact with and sequester endogenous copper, reducing its availability for tumor growth and angiogenesis [39]. On the contrary, ionophores trigger intracellular copper accumulation, cytotoxicity, and activate apoptosis inhibitor factor (XIAP) [24,[40][41][42][43][44][45][46]. Other CuC are proteasome inhibitors [47,48]. Several CuC are actually on clinical trials: a number of copper/disulfiram-based drug combinations for therapy and as diagnostic tools (metastatic breast cancer and germ cell tumor), several casiopeínas compounds and elesclomol (leukemia), and thiosemicarbazone-based copper complexes labeled with a radioactive isotope for positron emission tomography imaging of hypoxia (in head and neck cancers) [49].

The cisplatin DNA-targeting principle of action also conditioned the development of anticancer copper-based drugs [4,23,50]. Antitumor activities of copper-based drugs are based on the interactive properties of both copper and the ligand. Copper toxicity results from its redox capacities (Cu(I) and Cu(II) redox states' interconversion in oxidation-reduction cycles), the property to displace other ions from the enzyme binding sites, a high DNA binding affinity, and the ability to promote DNA breaks [28,51]. In most cases, copper modifies the backbone of the complexed ligand and grants better DNA affinity, specificity, and stability [52]. Copper derivatives can interact with DNA without the formation of covalent adducts. The noncovalent interactions with DNA include binding along with the major or the minor DNA grooves, intercalation, or electrostatic binding. Some copper-based drugs generate reactive oxygen species (ROS) that overwhelm cellular antioxidant defenses to produce oxidative damages in the cytoplasm, mitochondria, and DNA [53]. An important class of CuC, actually on focus for chemotherapy, inhibits topoisomerases (Top) 1 and 2, resulting in severe DNA damages, cell cycle arrest, and death [40,[54][55][56][57]. Chemotherapeutics that target Top as poisons convert a transient DNA- To circumvent drug resistance, a possible approach consists of designing and developing new therapeutic metal-based anticancer drugs [15][16][17][18][19][20][21]. Several transition metals from the d-block of the periodic table (groups 3 to 12) and particularly essential trace metals [15,22,23], such as copper [24][25][26][27][28][29], are useful for the implementation of metal-based complexes in anticancer therapies. Copper plays central roles in various cellular processes being an essential micronutrient and an important cofactor for several metalloenzymes involved in mitochondrial metabolism (cytochrome c oxidase), or cellular radical detoxification against reactive oxygen species (ROS) (superoxide dismutase) [30]. Copper is essential for angiogenesis, proliferation, and migration of endothelial cells [31][32][33]. Elevated copper favors tumor growth and metastasis. It is detected in several brain [34], breast [35], colon, prostate [36], and lung [37] tumors and serves as an indicator of the course of the disease [38]. The differences in tumor cells' responses to copper compared to normal cells laid the foundation of copper complexes' (CuC) evolution as anticancer agents. Numerous developed CuC contain different sets of N, S, or O ligands and demonstrate high cytotoxicity and efficient antitumor activity [25]. Different mechanisms are involved in copper drugs' anticancer effect. They act as chelators, and interact with and sequester endogenous copper, reducing its availability for tumor growth and angiogenesis [39]. On the contrary, ionophores trigger intracellular copper accumulation, cytotoxicity, and activate apoptosis inhibitor factor (XIAP) [24,[40][41][42][43][44][45][46]. Other CuC are proteasome inhibitors [47,48]. Several CuC are actually on clinical trials: a number of copper/disulfiram-based drug combinations for therapy and as diagnostic tools (metastatic breast cancer and germ cell tumor), several casiopeínas compounds and elesclomol (leukemia), and thiosemicarbazone-based copper complexes labeled with a radioactive isotope for positron emission tomography imaging of hypoxia (in head and neck cancers) [49].

The cisplatin DNA-targeting principle of action also conditioned the development of anticancer copper-based drugs [4,23,50]. Antitumor activities of copper-based drugs are based on the interactive properties of both copper and the ligand. Copper toxicity results from its redox capacities (Cu(I) and Cu(II) redox states' interconversion in oxidation-reduction cycles), the property to displace other ions from the enzyme binding sites, a high DNA binding affinity, and the ability to promote DNA breaks [28,51]. In most cases, copper modifies the backbone of the complexed ligand and grants better DNA affinity, specificity, and stability [52]. Copper derivatives can interact with DNA without the formation of covalent adducts. The noncovalent interactions with DNA include binding along with the major or the minor DNA grooves, intercalation, or electrostatic binding. Some copper-based drugs generate reactive oxygen species (ROS) that overwhelm cellular antioxidant defenses to produce Cancers 2020, 12, 2863 3 of 27 oxidative damages in the cytoplasm, mitochondria, and DNA [53]. An important class of CuC, actually on focus for chemotherapy, inhibits topoisomerases (Top) 1 and 2, resulting in severe DNA damages, cell cycle arrest, and death [40,[54][55][56][57]. Chemotherapeutics that target Top as poisons convert a transient DNA-enzyme complex into lethal DNA breaks [58][59][60][61][62]. However, topoisomerase inhibitors' activity and their multifaceted binding modes to DNA, the effects, and the modulations they produce on the control of cancer cell division necessitate better understanding to optimize their efficiency.

This review focuses on CuC targeting human Top1 and Top2, the molecular mechanism of induced DNA damages, cell cycle arrest, programmed cell death responses, and emerging research strategies.

Copper Complexes as Topoisomerases Inhibitors

DNA topoisomerases have been molecular targets for anticancer agents since their discovery in 1971 [63]. Topoisomerases regulate DNA winding and play essential functions in DNA replication and transcription [59,64]. Topoisomerase 1 (Top1) creates transient single-DNA nicks, while topoisomerases 2 (Top2α and Top2β) produce transient double-stranded DNA breaks. Both nuclear Top1 and Top2 are important targets for cancer chemotherapy, and Top inhibitors are used in therapeutic protocols [65][66][67]. Top inhibitors are classified into two groups: poisons and catalytic inhibitors. Top poisons (or interfacial poisons) stabilize the reversible cleavage complex formed between Top and DNA and form a ternary complex. Top2 catalytic inhibitors can prevent DNA strands cleavage through inhibition of the ATPase activity (novobiocin, merbarone), by impeding ATP hydrolysis to block Top dissociation from the DNA (ICRF-193), or by DNA intercalation at the Top fixation site (aclarubicinet) see [68]. In all cases, inhibitors convert the indispensable nuclear Top enzyme into a killing tool.

Top inhibitors' activity increases upon complexation with copper ion. Top1, Top2, or Top1/2 inhibitors synthesized in the form of copper complexes (CuC) are mostly mononuclear Cu(II) complexes associated with a variety of ligands (Table 1). Different strategies are currently proposed to design and develop Top inhibitory agents based on ligands' properties [69]. If both Top1 and Top2 inhibitors CuC primarily target DNA by a direct interaction through intercalation or cleavage, their antiproliferative activity is reinforced by ROS production and other molecular targets (Table 1) [25,52].

CuC Top1 Inhibitors

All the structures of CuC Top1 inhibitors are reported in Figure 2 and the main characteristics in Table 1. Oxindolimine-Cu(II) Top1 inhibitors such as 1 are planar copper compounds [70] that do not permit enzyme-DNA complex formation [71][72][73]. Besides, they produce ROS [70]. Cu(II) derivative complexes of the hydrazone ligand with triphenylphosphonium moiety 2 can bind DNA and the Top enzyme [74]. Plumbagin-Cu(II) 3 selectively intercalates into DNA [75]. The latter compound [75] and the phenanthroline-Cu(II) complexes modulated by amino acids 4 [76] can induce cancer cell apoptosis via mitochondrial signaling. Copper pyrophosphate-bridged binuclear complex 5 interacts with DNA, and based on the redox chemistry of copper, induces significant oxidative stress in cancer cell lines [77].

Cancers 2020, 12, x 7 of 26

CuC Top1 Inhibitors

All the structures of CuC Top1 inhibitors are reported in Figure 2 and the main characteristics in Table 1. Oxindolimine-Cu(II) Top1 inhibitors such as 1 are planar copper compounds [70] that do not permit enzyme-DNA complex formation [71][72][73]. Besides, they produce ROS [70]. Cu(II) derivative complexes of the hydrazone ligand with triphenylphosphonium moiety 2 can bind DNA and the Top enzyme [74]. Plumbagin-Cu(II) 3 selectively intercalates into DNA [75]. The latter compound [75] and the phenanthroline-Cu(II) complexes modulated by amino acids 4 [76] can induce cancer cell apoptosis via mitochondrial signaling. Copper pyrophosphate-bridged binuclear complex 5 interacts with DNA, and based on the redox chemistry of copper, induces significant oxidative stress in cancer cell lines [77]. In the heterobimetallic Cu(II)-Sn2(IV) (copper/tin) complex 6, the planar phenanthroline heterocyclic ring approaches the Top-DNA complex Cu(II)-Sn2(IV) toward the DNA cleavage site and forms a stable complex with Top1 [78,79]. Other Cu(II)-Sn2(IV) analogs induce apoptosis [80]. Chiral monometallic or heterobimetallic complexes 7 and 8 with tridentate chiral Schiff base-ONOligand are DNA groove binders and produce ROS [81,82].

Salicylidene-Cu(II) derivative 9 of 2-[2-bromoethyliminomethyl] phenol [83,84] is a bifunctional drug that inhibits both cancer cell growth and metastasis.

Chalcone-derived thiosemicarbazone (TSC) Cu(II) complex 10 prevents the DNA cleavage step of the Top1 catalytic cycle and DNA relegation [85].

Tetrazolo [1,5-a]pyrimidine-based Cu(II) complexes 11 have a square planar geometry, and despite their high capability to inhibit Top1, interact with CDK for 11 [86] and VEGF receptors for an analog of 11 [87]. Binuclear Cu(II) dipeptide piperazine-bridged complex 12 recognizes specific sequences in the DNA, oxidatively cleaves DNA, and displays superoxide dismutase (SOD) activity [88].

Derived from elesclomol (in clinical trials: phase 3 against melanoma and randomized phases 2 and 3 for the treatment of a variety of other cancers), the elesclomol-Cu(II) complex 13 inhibits Top1 and induces apoptosis in cancer cells [89].

As recently studied, Cu(II)(SBCM)2 14 derived from S-benzyldithiocarbazate and 3acetylcoumarin intercalates into DNA, induces ROS production, and has an antiproliferative activity in breast cancer lines [90,91]. In the heterobimetallic Cu(II)-Sn 2 (IV) (copper/tin) complex 6, the planar phenanthroline heterocyclic ring approaches the Top-DNA complex Cu(II)-Sn 2 (IV) toward the DNA cleavage site and forms a stable complex with Top1 [78,79]. Other Cu(II)-Sn 2 (IV) analogs induce apoptosis [80]. Chiral monometallic or heterobimetallic complexes 7 and 8 with tridentate chiral Schiff base-ONO-ligand are DNA groove binders and produce ROS [81,82].

Salicylidene-Cu(II) derivative 9 of 2-[2-bromoethyliminomethyl] phenol [83,84] is a bifunctional drug that inhibits both cancer cell growth and metastasis.

Chalcone-derived thiosemicarbazone (TSC) Cu(II) complex 10 prevents the DNA cleavage step of the Top1 catalytic cycle and DNA relegation [85].

Tetrazolo [1,5-a]pyrimidine-based Cu(II) complexes 11 have a square planar geometry, and despite their high capability to inhibit Top1, interact with CDK for 11 [86] and VEGF receptors for an analog of 11 [87]. Binuclear Cu(II) dipeptide piperazine-bridged complex 12 recognizes specific sequences in the DNA, oxidatively cleaves DNA, and displays superoxide dismutase (SOD) activity [88].

Derived from elesclomol (in clinical trials: phase 3 against melanoma and randomized phases 2 and 3 for the treatment of a variety of other cancers), the elesclomol-Cu(II) complex 13 inhibits Top1 and induces apoptosis in cancer cells [89].

As recently studied, Cu(II)(SBCM) 2 14 derived from S-benzyldithiocarbazate and 3-acetylcoumarin intercalates into DNA, induces ROS production, and has an antiproliferative activity in breast cancer lines [90,91].

Cancers 2020, 12, 2863 9 of 27

CuC Top2α Inhibitors

Due to its cell cycle phase dependence and its high expression in proliferating cells, the Top2α isoform is primarily targeted by copper complexes (CuC), whereas Top2β remains unchanged during the course of the cell cycle [66]. Another reason to limit the clinical application of Top2β inhibitors is the strong unwanted side effects produced (secondary leukemia, myelodysplastic syndrome (MDS), and cardiac toxicity [92,93]).

The main characteristics and structures of CuC Top2 inhibitors are reported in Figure 3 and Table 1. Several α-(N)-heterocyclic thiosemicarbazone (TSC) CuC [94,95] present a greater inhibitory effect on Top2α than corresponding TSC ligands alone [96,97] due to a square planar structure around the Cu(II) ion. A specific subset of pyridine-TSC CuC 15 inhibits Top2α [98] acting as ATP hydrolysis inhibitors in a non-competitive mode [94,99,100]

CuC Top2α Inhibitors

Due to its cell cycle phase dependence and its high expression in proliferating cells, the Top2α isoform is primarily targeted by copper complexes (CuC), whereas Top2β remains unchanged during the course of the cell cycle [66]. Another reason to limit the clinical application of Top2β inhibitors is the strong unwanted side effects produced (secondary leukemia, myelodysplastic syndrome (MDS), and cardiac toxicity [92,93]).

The main characteristics and structures of CuC Top2 inhibitors are reported in Figure 3 and Table 1. Several α-(N)-heterocyclic thiosemicarbazone (TSC) CuC [94,95] present a greater inhibitory effect on Top2α than corresponding TSC ligands alone [96,97] due to a square planar structure around the Cu(II) ion. A specific subset of pyridine-TSC CuC 15 inhibits Top2α [98] acting as ATP hydrolysis inhibitors in a non-competitive mode [94,99,100]

Cell Cycle Regulation by Copper Complexes and Top Inhibitors

CuC inhibitors targeting Top1 [72,90] or Top2 [109] as DNA-damaging drugs or poisons arrest cancer cells in G2/M (Table 1). This common G2/M arrest involves the activation of two different cell cycle pathways: the DNA damage response (DDR) and the decatenation checkpoint.

Both Top1 and Top2 CuC inhibitors produce DNA damages. Top2 poisons prevent DNA relegation and stabilize an enzyme-DNA complex with the double-stranded cleaved DNA [114]. Top1 poisons induce single-stranded DNA breaks and associated signaling cascades. The collision between the Top1 cleavage complexes and the DNA replication forks ends up generating doublestrand breaks [115] (Figure 5A). Top1-and Top2-induced DNA breaks trigger a DDR executed by ATM-, ATR-, and DNA-PK-related kinases, and an arrest of the cell cycle machinery [116][117][118]. ATMand ATR-dependent phosphorylations of p53, Chk1, and Chk2 regulate the G1/S, S, or G2/M cell cycle checkpoints. Chk1 and Chk2 inhibit Cdc25 phosphatases (A,B,C) required for Cdks activation. Phosphorylated and ubiquitinated Cdc25A (Ser123) is degraded, leading to the absence of activation of the Cdk2/Cyclin E and the Cdk4/cyclin D complexes and followed by an arrest in G1/S. Phosphorylated Cdc25C (Ser216) binds to 14-3-3, prevents Cdk1/Cyclin B (MPF) activation, and induces a G2/M arrest (Figure 5A). Cdc25B inactivation also results in a G2 arrest [119,120]. The DNA damage-induced cell cycle arrest in G1 is dependent on p53 phosphorylation by ATM (Ser15) and Chk2 (Ser20) but arrest in S and G2 phases is p53-independent [121][122][123][124]. Phosphorylated p53 dissociates from MDM2 and activates the transcription of Cdk inhibitor p21WAF1 [125,126]. In several CuC (Top1 DNA binding CuC inhibitors [72,82,88]

Cell Cycle Regulation by Copper Complexes and Top Inhibitors

CuC inhibitors targeting Top1 [72,90] or Top2 [109] as DNA-damaging drugs or poisons arrest cancer cells in G2/M (Table 1). This common G2/M arrest involves the activation of two different cell cycle pathways: the DNA damage response (DDR) and the decatenation checkpoint.

Both Top1 and Top2 CuC inhibitors produce DNA damages. Top2 poisons prevent DNA relegation and stabilize an enzyme-DNA complex with the double-stranded cleaved DNA [114]. Top1 poisons induce single-stranded DNA breaks and associated signaling cascades. The collision between the Top1 cleavage complexes and the DNA replication forks ends up generating double-strand breaks [115] (Figure 5A). Top1-and Top2-induced DNA breaks trigger a DDR executed by ATM-, ATR-, and DNA-PK-related kinases, and an arrest of the cell cycle machinery [116][117][118]. ATMand ATR-dependent phosphorylations of p53, Chk1, and Chk2 regulate the G1/S, S, or G2/M cell cycle checkpoints. Chk1 and Chk2 inhibit Cdc25 phosphatases (A,B,C) required for Cdks activation. Phosphorylated and ubiquitinated Cdc25A (Ser123) is degraded, leading to the absence of activation of the Cdk2/Cyclin E and the Cdk4/cyclin D complexes and followed by an arrest in G1/S. Phosphorylated Cdc25C (Ser216) binds to 14-3-3, prevents Cdk1/Cyclin B (MPF) activation, and induces a G2/M arrest (Figure 5A). Cdc25B inactivation also results in a G2 arrest [119,120]. The DNA damage-induced cell cycle arrest in G1 is dependent on p53 phosphorylation by ATM (Ser15) and Chk2 (Ser20) but arrest in S and G2 phases is p53-independent [121][122][123][124]. Phosphorylated p53 dissociates from MDM2 and activates the transcription of Cdk inhibitor p21WAF1 [125,126]. In several CuC (Top1 DNA binding CuC inhibitors [72,82,88] By contrast to poisons, Top2 catalytic inhibitors do not form cleavable complexes. They function by enzymatic activity deprivation and cell cycle arrest in G2 through a decatenation checkpoint distinct from the DNA damage checkpoint. To delay the mitotic entry, an insufficient decatenation engages molecular components from the DDR and the spindle assembly checkpoint (SAC) (Rad9a, ATR, and BRCA1), SUMOylation and phosphorylation of Top2, the p38 and the MAPK pathways, and several decatenation checkpoint effectors but not p53 [66,[132][133][134][135][136] (Figure 5A).

Cell cycle checkpoint effectors arrest DNA-damaged cells and induce their death providing that cell cycle regulatory networks are effective. Cell cycle checkpoint effectors integrity influences responses to Top2 inhibitors [137]. Besides, cancer disease is associated with multiple overexpression and mutations [138] in Cdc25 [139,140] and p53 [141,142], to a loss of Cdk inhibitors expression and/or overexpression of cell cycle-regulated protein [143,144], Top deregulation, and multidrug resistance [145][146][147]. Moreover, cell cycle variation of Top2α is regulated by post-translational modifications that represent potential targets.

These alterations include ubiquitination by Cdk- 1 [148], sumoylation [149], phosphorylation by polo-like kinase 1, Cdc7 [150], protein kinase C, Ca/calmodulin-dependent kinase II, and casein kinase [151], and the association with 14-3-3 [152]. Rewiring cellular pathways leading to cell death is a challenge that requires targeting specific molecular checkpoint effectors [153]. For example, a mutated p53 pathway arrests the cell cycle but avoids DDR-induced cell death [154]. Some anticancer therapeutic strategies (e.g., Chk1/2 pathways targeting drugs associated with DNA-damaging drugs) can force cancer cells to bypass S and G2/M arrest, enter mitosis with damaged DNA, and finally undergo a mitotic catastrophe and death [155]. ATR inhibition is another strategy to overcome the resistance of BRCA-deficient cancers [156].

Programmed Cell Death Engaged by Copper Complexes and Top Inhibitors

Multiple stress factors ranging from various cell damages, ATP levels, and specific pathways (e.g., caspases) determine the type of cell death [157]. Most Top1 CuC inhibitors that interact with DNA [70,76,79,86,87,90], Top1 poison [89], Top2α CuC poison [109], or dual Top1/Top2 inhibitor [113] trigger apoptotic programmed cell death. Genetic damages and oxidative stress activate an intrinsic mitochondrial response [158]. Pro-apoptotic members of the Bcl-2 family (Bid, Noxa, Puma, BAX, BAK) neutralize the anti-apoptotic members , disrupt the mitochondrial outer membrane, and allow cytoplasmic cytochrome-c release. The binding of cytochrome c to the apoptotic protease activating factor-1 (Apaf-1), ATP, and the pro-caspase-9 create the apoptosome protein complex. Pro-caspase 9 is cleaved into its active caspase-9 form, which in turn cleaves pro-caspase-3 into caspase-3 effector, and the downstream executor caspase-7. SMAC (second mitochondria-derived activator of caspases), and Omi/HtrA2 (high-temperature requirement protein A2) are simultaneously released from mitochondria and deactivate the IAPs factors (inhibitors of apoptosis proteins). p53, activated by the DNA damage, contributes to apoptosis through the translation of several pro-apoptotic members of the Bcl-2 family (Bid, Puma) that inhibit the pro-survival action of Bcl-2 on BAX (Figure 5A). Most cancer cells evade apoptosis through caspase inhibition, upregulation of Bcl-2 (in more than 50% of all types of cancers), and loss of BAX/BAK and become resistant to anticancer drugs [159].

A Top1 DNA-damaging CuC inhibitor induces necrotic cell death. To facilitate cell destruction, necrosis is activated by ROS or ATP metabolic stresses in crosstalk with apoptosis [160]. When the intracellular energy/ATP level is low, the apoptotic cell death is converted into necrosis [161] (Figure 5A). However, necrosis releases pro-inflammatory and tumor-promoting cytokine HMGB1 [162] into the extracellular space reported to stimulate inflammation and angiogenesis, and promote tumor progression [163].

Apoptosis and necrosis often co-exist with another cell death with controversial pro-death and pro-survival functions: autophagy [164]. Up to the current study, no CuC Top inhibitors are involved in autophagic or necroptotic programmed cell death (Table 1). However, some CuC trigger stress-mediated protective autophagy in response to ROS that impedes apoptosis and creates survival of malignant cells [165]. Moreover, topoisomerase inhibition-induced autophagy is associated with cancer resistance [166].

Future Strategies for Copper Complexes as Top Inhibitors in Cancer Cell Treatments

The development of new effective anticancer drugs is a major research area against the continuing increase in cancers worldwide. Top inhibitors used in chemotherapy are limited in number [61,167,168]. Top1 inhibitors' camptothecin derivatives used are irinotecan (colorectal [169], pancreatic (in combination) [170], and small cell lung cancers (in clinical trials and in combination) [171,172]), and topotecan (ovarian [173,174], cervical [175], and small cell lung cancers [176]). Top2 anticancer drugs commonly used are from the anthracycline group such as doxorubicin (acute leukemia [177], lymphomas [178], sarcomas [179,180], and solid tumors [181]), epirubicin (breast cancer [182]), valrubicin (bladder cancer [183]), and idarubicin (acute myeloid leukemia [184]), from the anthracenedione classes: mitoxantron and pixantron (lymphoma, [185-187]), and from the epipodopodophyllotoxins group such as etoposide (testicular [188] and small cell lung cancers [189]) and teniposide (brain [190] and small cell lung [191] cancers, acute lymphocytic leukemia [192]). Only a few numbers of Top1 inhibitors are in clinical trials including the promising indenoisoquinoline derivatives LMP400 (Indotecan), LMP776 (Indimitecan) (phase I), and LMP744 examined in a phase I study on lymphoma in dogs [193]. In addition to better stability, and milder side effects, they can escape ABC transporter efflux and the drug resistance mechanism, as Elesclomol-CuC Top complexes 13 [89] or Quinolinone-CuC 25 [112]. Perspectives to use CuC of Top inhibitors in clinical trials are summarized in Figure 5B. Development and optimization in CuC of Top inhibitors imply structure modifications that must encompass several specific strategies [194], such as scaffold hopping [195], pharmacophore hybridization [196], bioisosteric replacement [197], and conformational restrictions. Generally, a rigidification of the ligand heterocycle structure with a copper metal [78] provides a planar configuration that facilitates DNA intercalation and Top-DNA ternary complex formation compared to the molecular backbone alone.

Top inhibitors in clinical use and particularly Top poison display unwanted drawbacks, such as cumulative cardiotoxicity in long-term protocols, secondary malignancies, and drug resistance [198]. A therapeutic option would be to use preferentially catalytic Top agents that disturb the catalytic cycle without the formation of a ternary complex. CuC Top catalytic inhibitors, listed in Table 1, exhibit high antitumor effects on cancer cell lines and for some compounds on tumor growth in animal models, compared to their respective ligands (see Table 1). They constitute a reservoir of anticancer drugs. For example, TSC-based CuC Top2 inhibitors (Figure 3) [98,102,103,105,107] have demonstrated strong inhibition of tumor growth compared to TSC derivatives currently used in cancer chemotherapies [199].

Considering that cancer is a multigenetic and multifactorial disease that recruits numerous molecular effectors, monotherapies (based on Top inhibitors) do not provide the optimal curative effects. Combination therapy with a few numbers of therapeutics against two or more biotargets is the base of promising treatments such as the association of a Top 2 inhibitor (vosaroxin) with a DNA methyltransferase inhibitor (decitabine) in AML [200,201]. Inhibitors of Top1 and Top2, currently developed, also exert their effect against other cancer-related targets [202]. Dual Top inhibitors, e.g., Top 1/2 [203], Top2/microtubule [204], or Top2/histone deacetylase [205], may exert improved efficacy. Besides, Top1 inhibitors are nonspecific RNA polymerase inhibitors. An RNA Pol1-mediated ribosomal RNA gene increase is involved in cancer progression, through the control of cellular checkpoints and chromatin structure and is, therefore, an interesting co-target [206]. CuC dual Top inhibitors display a high antiproliferative activity. Particularly, some CuC and non-CuC are dual inhibitors of Top1 and superoxide dismutase agonist [88,207,208] or Cdk receptor, like VEGF inhibitors, involved in cancer cells proliferation [86,87,209,210] (Figure 5B). Another strategy to improve therapies is the association of a CuC with a TDP1/2 (tyrosyl-DNA-phosphodiesterase 1/2) inhibitor. TDP1/2 are enzymes responsible for the reparation of DNA breaks induced by topoisomerase poisons [57,211,212]. TDP1/2 inhibitors are capable of improving cancer cells' sensitivity to these poisons [213].

Autophagy, an essential mechanism for cell integrity and survival, is stimulated in cancer cells under several chemotherapeutic drugs and acts as an unwanted protective system towards tumor cells. Association of specific autophagic inhibitors with Cu-C treatment (disulfiram) in non-small cell lung cancer [214] has proven to be a novel efficient strategy to enhance apoptosis in cancer therapy.

Immunogenic cell death is an important mechanism used in chemotherapy. Association of CuC with immune checkpoint therapies is certainly a new avenue in cancer treatment. CuC and non-CuC Top inhibitors induce DNA damages and are linked to adaptive and innate immunities [215]. Top poisons promote immunogenicity in various ways [216]. Top1 poison camptothecin enhances the adaptive immune response [217]. Top inhibitors also increase chromosomal instability and mutations accumulated by cancer cells [59,218]. Consequently, due to their high number of mutations, tumors display more neoantigens presented at their surface by the major histocompatibility complex class I (MHCI) and recruit lymphocytes T harboring TCR (T cell receptor) and CD8 co-receptor (adaptive immunity). This response is counterbalanced by the overexpression of immune checkpoint modulators, such as the immune-suppressive ligand PD-L1 (programmed death-ligand 1) targeted in immune therapies [219] (Figure 5B). DNA-damaging agents such as Top2 poison anthracycline also interfere with the innate immune response. They enhance the malignant formation of cytosolic bicatenated DNA fragments that activate the cyclic GMP-AMP synthase-stimulator of the interferon (IFN) gene pathway (cGAS-STING) and initiate innate anti-cancer immunity. cGAS-STING agonist serves as a sensitizer in immunotherapies [220]. Top1-DNA covalent cleavage complex enables cGAS-mediated cytoplasmic chromatin recognition and immune checkpoint response [221] (Figure 5B). Top2 inhibitors teniposide and doxorubicin potentiate the therapeutic immune checkpoint blockade therapies based on anti-PD-1 (programmed cell death 1) in multiple types of mouse tumor models [222,223]. Besides, ROS produced by Top inhibitors alter the molecular pattern recognized as immunogenic structures and enhance apoptosis [224] (Figure 5B).

As DDR gene mutations exist in a large range of tumor types, the determination of tumor-specific mutations is another accurate strategy to generate chemotypes with beneficial efficacies superior to adverse effects [225,226]. In each tumor, the signaling components of the DDR exhibit numerous defects that result in a unique mutational signature [227]. Cancer cells with defects in their homologous recombination mechanism are more sensitive to Top2 inhibitory therapies that generate DNA double-strand breaks [228]. Moreover, the prediction of anticancer treatments determined by the clinical stage and the pathological features of the tumor does not always ascertain a cancer death response. Cellular biomarkers that may predict sensitivity or resistance to therapy based on DNA damage induced by Top inhibitors would be useful. Insights into the Top2 regulatory mechanisms have identified genetic markers to allow the prediction of an overcome treatment with a Top inhibitor. γ-H2AX is a DNA-damaged marker, recruited on DNA breaks after Top poison action, currently evaluated [229]. Schlaffen is also a promising marker for an accurate response to Top1 and Top2 inhibitors, especially for colon and ovarian adenocarcinomas [56,230] (Figure 5B).

Recently, cancer cells were targeted specifically by a Top2 inhibitor, etoposide, attached to a single-stranded oligonucleotide with a complementary sequence to a DNA cleavage hotspot corresponding to a translocated region only present in promyelocytic leukemia cells [231].

Finally, to overcome toxicity to normal cells, Top drugs could be attached to vehicles. Top2 inhibitors delivery has been optimized using liposomes [232], micelles [233], or functionalized nanoparticles [234] (Figure 5B).

Topoisomerases are present in mitochondria where they participate in mitochondrial DNA replication and transcription. Mitochondrial Top1 isoform (Top1mt) is involved in the metabolism of cancer cells providing energy to tumors surrounded by a nutrient-low microenvironment. Exposures to a Top1 inhibitor (lamellarin D) or Top2 inhibitors (doxorubicin or fluoroquinolones) exert mitochondrial toxicity [235]. However, the loss of Top1mt in liver cancers correlates with increased survival of hepatocellular carcinoma patients, showing that co-targeting Top1mt in addition to nuclear topoisomerases is another option for anticancer therapies [236].

P a g e | 5 Résumé:

 5 Mécanismes d'action d'un nouveau composé organométallique, WN197, inhibiteur des topoisomérases, inducteur d'arrêt dans le cycle cellulaire et de mort cellulaire.

Figure I :

 I Figure I: number of worldwide cancer new cases by gender, and distribution of new cases and new deaths by gender. …………………………………………………………………………………………………………………… page 21 Figure II: female breast anatomy. …………………………………………………………………………………………… page 22 Figure III: cervix anatomy. ………………………………………………………………………………………………………. page 23 Figure IV: cancer immunotherapy approaches: checkpoint inhibitors, T-cell transfer therapy, vaccination, and cytokine immunotherapy. ……………………………………………………………………………. page 27 Figure V: women hormonotherapies: anti-estrogens, LHRH analogs and antagonists, and antiaromatases. ……………………………………………………………………………………………………………………………. page 28 Figure VI: major pathways targeted by antibodies or small molecules in anti-cancer strategies. ……………………………………………………………………………………………………………………………………………….. page 30 Figure VII: various activated DNA damage response pathways. ………………………………………………. page 34 Figure VIII: topoisomerases 1 and 2 mechanism of action. ……………………………………………………… page 37 Figure IX: human somatic cells topoisomerases repartition. …………………………………………………… page 38 Figure X: human topoisomerases and their protein domains. …………………………………………………. page 39 Figure XI: overall structure and domains of the sub-family types of two human topoisomerases. ……………………………………………………………………………..………………………………………………………………… page 40 Figure XII: mechanism of action of toposiomerases' poisons. …………………………………………………. page 44 Figure XIII: action mechanism of catalytic and poisons inhibitors. ………………………………….………. page 45 Figure XIV: indenoisoquinoline organometallic derivative compounds skeleton. ……………………. page 55 Figure XV: IC50 (in µM) of organometallic indenoisoquinoline derivatives on 5 cancer cell lines. page 56 Figure XVI: structure of WN197 and its precursor WN170. ……………………………………………………… page 57 Table I: breast, cervical, and colorectal cancers and their cell types, incidences, associated deaths, risk factors, and treatments. …………………………………………………………………………………………………………. page 25

Figure A :Figure 1 :Figure 2 :Figure 3 :Figure 4 :Figure 5 :Figure 6 :Figure 8 :Figure 9 :

 A12345689 Figure A: schematic representation of the MTS assay. …………………………………………………………….. page Figure B: principle of topoisomerase 1 (Top1) in vitro activity test. …………………………………………. page Figure C: principle of topoisomerase 2 (Top2) in vitro activity test. …………………………………………. page Figure D: representative scheme of indirect immunofluorescence used to detect γH2AX. ………. page Figure E: schematic representation of cell cycle analysis by flow cytometry. …………………………… page Figure F: principle of annexin V-propidium iodide (PI) analysis by flow cytometry. …………………. page Figure G: DNA damage and cell cycle regulation. ……………………………………………………………………. page Figure H: schematic representation of Cdc25C detection by immunoprecipitation and Western blot. ……………………………………………………………………………………………………………………………………………….. page Figure I: analysed apoptosis and autophagy pathway effectors. ……………………………………………… page Figure J: developmental stages of Xenopus laevis embryos. …………………………………………………… page Results Figure 1: γH2AX detection by immunofluorescence in MDA-MB-231. ……………………………………… page Figure 2: γH2AX detection by immunofluorescence in HeLa. …………………………………………………… page Figure 3: γH2AX detection by immunofluorescence in HT-29. ………………………………………………… page Figure 4: γH2AX foci quantification and Western blot analysis in three adenocarcinomas. ……… page Figure 5: WN197 inhibits Top1 and Top2α/β in a dose-dependent manner. …………………………… page Figure 6: DNA Damage Response (DDR) pathway activation. …………………………………………………… page Figure 7: cell cycle repartition analysis of MDA-MB-231, HeLa, and HT-29 cells. ……………………… page Figure 8: Western blot analysis of cell cycle markers in MDA-MB-231, HeLa, and HT-29 cells. … page Figure 9: apoptosis analysis. …………………………………………………………………………………………………… page Figure 10: autophagy analysis by Western blots. ……………………………………………………………………. page Figure 11: autophagy analysis by immunofluorescence. …………………………………………………………. page Figure 12: effect of WN197 on Xenopus laevis embryos oocytes. …………………………………………… pageTable 1: half maximal inhibitory concentrations (IC50 in µM) for cell survival of MCF-10A……….. page Table 2: half maximal inhibitory concentrations (IC50 in µM) for cell survival of BT-549 and SUM-159. …………………………………………………………………………………………………………………………………………….…. page Table 3: half maximal inhibitory concentrations (IC50 in µM) for cell survival of H69AR. …………. page

Figure I .

 I Figure I. Number of worldwide cancer new cases by gender (upper left), and distribution of new cases (upper middle and right) and new deaths (bellow) by gender (other cancers are listed in Appendice 1).

  new cases of cervical cancers are diagnosed every year in France (INCa: France cancers panorama 2022). The cervix is covered by squamous cells on the outer surface (endocervix) and glandular cells on the inner surface (ectocervix) (Figure III).

Figure

  Figure III. Cervix anatomy.

Figure

  Figure IV. Four cancer immunotherapy approaches: checkpoint inhibitors, T-cell transfer therapy, vaccination, and cytokine immunotherapy. CAR: chimeric antigen receptor; IL: interleukines; NK: natural killer.

Figure V .

 V Figure V. Women hormonotherapies: anti-estrogens, LHRH analogs and antagonists, and antiaromatases. ER: Estrogen Receptor; LH: Luteinizing Hormone; LHRH: Luteinizing Hormone Releasing Hormone; SERD: Selective Estrogen Receptor Degradation; SERM: Selective Estrogen Receptor Modulator.

Figure

  Figure VI. Major pathways targeted by antibodies or small molecules in anti-cancer strategies. EGF/R: Epidermal Growth Factor/Receptor; TK: tyrosine kinases; VEGF/R: Vascular Endothelial Growth Factor/Receptor.

  (Figure VII).

  P a g e | 34

Figure

  Figure VII. DNA damage response pathways.

  (Figure VIII). Type one topoisomerases (Top1) cut one strand of the DNA, while type two topoisomerases (Top2) cut both strands of the DNA. P a g e | 37

Figure

  Figure VIII. Topoisomerases 1 and 2 mechanism of action. Top1 (left) or Top2 (right) act as monomer or dimer respectively, and bind to supercoiled DNA (SCDNA). They cut one (Top1) or two strands (Top2) of the DNA via transesterification between the active tyrosine site of the enzyme and the phosphate group of the DNA, either on the 5' side (Top1) or 3' side (Top2). This creates transient single-strand breaks (SSB) or double-strand breaks (DSB), and allows the passage of one strand through the other to relax DNA (RDNA). Topoisomerases rejoin the DNA strands by reverse transesterification, and finally detach from the DNA. 1.1.4.1 Topoisomerase classification

Figure

  Figure IX. Human somatic cells topoisomerases repartition.

Figure

  Figure X. Human topoisomerases and their protein domains. aa: amino acids. Modified, from McKinnon, 2016.

Figure

  Figure XI. Overall structure and domains of human topoisomerases 1 and 2β. Left: structure of human Top1 (70 kDa; blue) in complex with a 22 base pairs DNA (green and brown) (PDB ID: 1A35; Redinbo et al., 1998). Right: structure of human Top2β dimer (183 kDa; chain A of Top2β in orange; chain B of Top2β in green) in complex with DNA (pink/green and purple/orange) (PDB ID: 4J3N; Wu et al., 2013).

Figure

  Figure XII. Mechanism of action of topoisomerases' poisons. After binding of the topoisomerase to the DNA and its cleavage, the poison interacts with the DNA and the topoisomerase, forming the ternary cleavage complex (TCC). This interaction generates single-and double-strand breaks (SSB and DSB), which induce the DNA damage response pathway (DDR). Three PI3K kinases, ATM, ATR, and DNA-PK, are activated, and trigger a signaling cascade. Some effectors are involved in cell cycle arrest to allow DNA repair, or cell death if the damage is too extended.

Figure

  Figure XIII. Action mechanism of Top2 catalytic and poison inhibitors. (1): Top2 enzymes bind to two dsDNA segments, the G-and T-segments. (2): two ATP molecules bind to the ATPase domains of Top2, which induce their dimerization. (3): the addition of Mg 2+ allows Top2 to cut both strands of the G-segment. (4): one molecule of ATP is hydrolyzed to allow the passage of the T-segment through the break of the G-segment. (5): the second ATP molecule is hydrolyzed to religate the DSB of the G-segment. (6): finally, the two ADP molecules dissociate, and the two dsDNA are released through the Top2 C-terminal end before the ATPase domain reopens to enable a new cycle of activity. Catalytic inhibitors (in sand pink) can (i) intercalate in the DNA, (ii) act as ATP competitors, or (iii) inhibit ATP hydrolysis. Poison inhibitors (iv) create DNA breaks accumulation by complexation with the broken DNA and Top2.

Figure

  Figure XIV. Indenoisoquinoline organometallic derivative compounds skeleton. The derivatives are composed of the indenoisoquinoline backbone, a spacer (three or four carbons: 3C or 4C), and a metal (ruthenium: Ru, zinc: Zn or copper: Cu). Parameters including length of the carbon spacer and nature of the metal bound were tested for toxicity on cancer cells by MTS to classify the compounds according to their efficiency.

Figure

  Figure XV. IC50 (in µM) of organometallic indenoisoquinoline derivatives on five cancer cell lines. L1 to L4 are derivative ligands without a metal atom. Roman numerals (II) correspond to the form of copper and zinc atoms (Cu(II) and Zn(II)), and Arabic numerals are their serial numbers.

Figure

  Figure XVI. Structure of WN197 and its precursor WN170.

Figure A .

 A Figure A. Schematic representation of the MTS assay. Cells are treated or not with the compound of interest in a 96-well plate. After treatments, the yellow MTS compound is added to the wells, and incubation proceeded for 1 to 4 hours. The mitochondria of viable cells transform the yellow MTS into brown colored formazan quantified by spectrophotometry at 490 nm.

Figure B .

 B Figure B. Principle of topoisomerase 1 (Top1) in vitro activity test. One strand of the supercoiled DNA (in blue) is cleaved by Top1, leading to a transient open circular DNA (or nicked DNA) (in pink), and religated to form a relaxed DNA (in yellow). In the presence of a Top1 poison or a catalytic inhibitor, DNA stays supercoiled. More specifically, a Top1 poison intercalates between the topoisomerase and the cleaved DNA, stabilizing the DNA in a nicked form, detected on a 1% agarose gel.

Figure

  Figure C. Principle of topoisomerase 2 (Top2) in vitro activity test. Both DNA strands of supercoiled DNA (in blue) are cleaved by Top2, forming a transient double-strand broken DNA (in pink), and religated to form a relaxed DNA (in yellow). In the presence of a Top2 poison or a catalytic inhibitor, the DNA stays supercoiled. More specifically, a Top2 poison intercalates between the topoisomerase and the cleaved DNA, stabilizing the DNA in a double-strand break form. The addition of proteinase K in the assay allows the separation of the topoisomerase enzyme from the broken DNA and the liberation of a linear DNA (in green), visible after migration in a 1% agarose gel.

Figure D .

 D Figure D. Representative scheme of indirect immunofluorescence used to detect γH2AX. H2AX is phosphorylated on the DNA damaged sites, and recognized by the primary mouse antibody directed against the phosphorylated S139 form of H2AX. The secondary antibody coupled to Alexa 488 fluorophore emits a green fluorescence (519 nm) after excitation at 488 nm.

Flow

  cytometry works on the principle of light scattering and fluorescence emission by specific fluorescent probe-labeled cells as they pass one by one through a laser beam. The cell cycle analysis by flow cytometry consists of the detection of the quantity of fluorescent propidium iodide (PI) intercalated in the cell nucleus and allows a distinction between the different cell cycle phases. PI intercalates in the DNA, with an arbitrary quantity of n=1 in P a g e | 65G0/G1 phases leading to the emission of a low fluorescent signal, a quantity of n=2 in G2/M phases as a result of DNA duplication with a higher fluorescent signal emission, and with an intermediate quantity when DNA duplicates in S phase resulting in an intermediate emission of fluorescence between n=1 and n=2.

Figure E .

 E Figure E. Schematic representation of cell cycle analysis by flow cytometry. Labeled cells are flowing one by one in a laser beam that excites a fluorochrome. Each cell is sorted and its fluorescent emission is detected and quantified. Left: debris (low density (SSC) and height (FSC)) and cell doublets (high density and height) are excluded from the gate of interest (P1). Right: cells from the gate P1 are distributed according to the intensity of their fluorescent signal, and divided into three different pools corresponding to cell cycle phases (representation of the control distribution of a human proliferating cell population with cell cycle phases repartition as follow: ~45% in G0/G1, ~20% in S, ~35% in G2/M).

  P a g e | 66

Figure F .

 F Figure F. Principle of annexin V-propidium iodide (PI) analysis by flow cytometry. (Upper) In normal cells, the phosphatidylserine (PS) sets on the inner side of the plasma membrane and the addition of annexin V and PI results in an absence of staining. In a cultured cell population, a basal level of apoptosis is often detected. (Middle) In early apoptotic cells, PS is present on the outer side of the cell plasma membranes, allowing the fixation of annexin V, and an annexin V positive staining is obtained. (Bottom) In late apoptotic cells, PS is detected on the outer side of the membrane, and the membrane is permeabilized, allowing PI entrance into the cells which results in positive staining for annexin V and PI.

  . ATM/ATR and Chk1/Chk2 interact with p53 and p21 to regulate their activity or transcription (Figure G). The phosphorylation state or quantity of seven proteins of the DDR pathway were analyzed by Western blots.

Figure

  Figure G. DNA damage and cell cycle regulation. After DNA double-or single-strand breaks (DSB and SSB) an activation cascade is triggered. The ATM/ATR transducers activate Chk1/Chk2 and p53/p21 effectors. ATM is mostly recruited following DSB while ATR is after SSB. Chk1 and Chk2 subsequently inhibit proteins of the Cdc25 family involved in the activation of Cyclin/Cdk complexes activation, regulators of the different cell cycle phases. Consequently, cell cycle inhibition and arrest are possible in different phases, according to the targeted couple of Cyclin/Cdk checkpoint regulator. Here is represented the G2/M checkpoint blockage.

Figure H .

 H Figure H. Schematic representation of Cdc25C detection by immunoprecipitation and Western blot.During the M phase, active Cdc25C is not phosphorylated on S216 (inhibitory phosphorylation), which allows MPF (Cyclin B/Cdk1) activation. When cells are accumulated or stopped in the M phase, the Cdk1/Cyclin B complex is activated and no 14-3-3 protein is found on Western blots after a Cdc25C immunoprecipitation. In case of an arrest in the other phases of the cell cycle, Cdc25C is inactivated by phosphorylation on S216, and recognized by 14-3-3 to which it binds, allowing a detection of both partners after immunoprecipitation and Western blots.

Figure I .

 I Figure I. Analyzed apoptosis and autophagy pathway effectors.

Figure

  Figure J. Developmental stages of Xenopus laevis embryos. NF: Nieuwkoop and Faber. From[START_REF] Nieuwkoop | Normal table of Xenopus laevis (Daudin): A systematical and chronological survey of the development from the fertilized egg till the end of metamorphosis[END_REF]

  developmental stages: gastrula (NF stage 12), neurula (NF stage 16), and tailbud (NF stage 30) (Figure L). Statistical analyses were performed with Graphpad Prism V6.0 software, and significances (mean ± SD) were determined by two-way ANOVA followed by Dunnett's multiple comparison test (***p<0.005, ****p<0.001). P a g e | 72 Results P a g e | 73

Figure 1 :

 1 Figure 1: γH2AX detection by immunofluorescence in MDA-MB-231. Cells were treated with DMSO (0.5%, solvent control), doxorubicin (5 µM, positive control), cisplatin (20 µM, positive control), WN170 (0.5 µM) or WN197 (0.5 µM). After 24 h of treatment, the antibody against γH2AX (green) revealed DNA breaks staining as foci (dots). Nuclei were stained with DAPI (blue). Images were obtained on a Leica fluorescent microscope (x100) and were representative of three independent experiments. Scale bar: 20 µm.

Figure 2 :

 2 Figure 2: γH2AX detection by immunofluorescence in HeLa. Cells were treated with DMSO (0.5%, solvent control), doxorubicin (5 µM, positive control), cisplatin (20 µM, positive control), WN170 (0.5 µM) or WN197 (0.5 µM). After 24 h of treatment, the antibody against γH2AX (green) revealed DNA breaks staining as foci (dots). Nuclei were stained with DAPI (blue). Images were obtained on a Leica fluorescent microscope (x100) and were representative of three independent experiments. Scale bar: 20 µm.

  P a g e | 76

Figure 3 :

 3 Figure 3: γH2AX detection by immunofluorescence in HT-29. Cells were treated with DMSO (0.5%, solvent control), doxorubicin (5 µM, positive control), cisplatin (20 µM, positive control), WN170 (0.5 µM) or WN197 (0.5 µM). After 24 h of treatment, the antibody against γH2AX (green) revealed DNA breaks staining as foci (dots). Nuclei were stained with DAPI (blue). Images were obtained on a Leica fluorescent microscope (x100) and were representative of three independent experiments. Scale bar: 20 µm.

Figure 4 :

 4 Figure 4: γH2AX foci quantification and Western blot analysis in three adenocarcinomas. MDA-MB-231, HeLa, and HT-29 cells were treated with DMSO (0.5%, solvent control), doxorubicin (5 µM, positive contro, Doxl), cisplatin (20 µM, positive control, Cis), WN170 (0.5 µM) or WN197 (0.5 µM). (A) Quantification of immunofluorescence γH2AX mean foci per cells after 24 h of treatment. (B) Western blot analysis of γH2AX after 24 h of treatments. β-actin was used as a loading control and relative γH2AX level was quantified by densitometry using Image J (Fiji Software, v1.52i). (C) Quantification of γH2AX mean foci per cells after 30 min of treatment based on immunofluorescence experiments. (A, C), data were expressed as the mean ± SD for 30 nuclei of three independent experiments. Statistical analyses were based on a two-way ANOVA followed by a Dunnett's test (*p<0.05, **p<0.01, ***p<0.005 and ****p<0.001).

  P a g e | 79

Figure 5 :

 5 Figure 5: WN197 inhibits Top1 and Top2α/β in a dose-dependent manner. (A) In vitro Top1 activity assay. Relaxed DNA (RDNA, lane 1) or supercoiled DNA (SCDNA, lane 2) were used as migration controls. SCDNA was used in all other reactions in presence of Top1. The Top1 activity control allowing the relaxation of SCDNA is in lane 3. The Top1 activity was assayed in presence of either DMSO (5%, solvent control, lane 4), WN197 at different concentrations (0.2, 0.5, 1 and 2 µM, lanes 5-8), etoposide (VP-16, 50 µM; Top2 poison, lane 9) the negative control of Top1 activity inhibition, or camptothecin (CPT, 10 µM; Top1 poison, lane 10) the positive control of Top1 activity inhibition. The addition of proteinase K allowed the detection of nicked DNA (NDNA), a witness of the single-strand broken DNA stabilization by a topoisomerase poison. (B) In vitro Top2α activity assay. The migration control of supercoiled DNA (SCDNA) was performed in lane 1. Top2α was present in all other reactions. The Top2α activity control for the relaxation of SCDNA is in lane 2, the first band corresponds to the transitional open circular DNA (OCDNA) and topoisomers correspond to the diverse states of relaxed DNA. The Top2α activity was assayed in the presence of either DMSO (5%, solvent control) in lane 3, WN197 (concentrations of 0.2, 0.5, 1, and 2 µM) in lanes 4-7, etoposide (VP-16, 50 µM; Top2 poison) in lane 8, and camptothecin (CPT, 10 µM; Top1 poison) in lane 9. (C) In vitro Top2β activity assay. Migration control of SCDNA was performed in lane 1. Top2β was present in all other reactions. The Top2β activity control for the relaxation of SCDNA is in lane 2, DMSO (5%, solvent control) in lane 3, WN197 (concentrations of 0.2, 0.5, 1, and 2 µM) in lanes 4-7, etoposide (VP-16, 50 µM; Top2 poison) in lane 8, and camptothecin (CPT, 10 µM; Top1 poison) in lane 9. In (A-C) after topoisomerase reactions, DNA was run in a 1% agarose gel, stained with ethidium bromide (0.5 µg/mL), and visualized under UV light. Each test is representative of three independent experiments.

  P a g e | 81

Figure 6 :

 6 Figure 6: DNA Damage Response (DDR) pathway activation. MDA-MB-231, HeLa, and HT-29were treated for 24 h with doxorubicin (5 µM, Dox), cisplatin (20 µM, Cis), WN170 (0.5 µM), or WN197 (0.5 µM). Western blots were performed to detect ATM, ATR, Chk1, Chk2, p53, and their active phosphorylated forms, and p21. β-actin was used as a loading control and relative protein levels were quantified by densitometry using Image J software (Fiji Software, v1.52i). Results are representative of three independent experiments.

Figure 7 :

 7 Figure 7: cell cycle repartition analysis of MDA-MB-231, HeLa, and HT-29 cells. (A) Flow cytometry analysis of MDA-MB-231, HeLa, and HT-29 cells repartition in the cell cycle 24 h after treatments with cisplatin (20 µM, S phase arrest control), nocodazole (84 nM, M phase arrest control), WN170 or WN197 (0.5 µM). Raw data available in Appendice 2. (B) Dose-response analysis by flow cytometry of the cell cycle repartition in the three cell lines 24 h after treatment with WN197 or untreated (control). (C) Time course analysis by flow cytometry of the cell cycle repartition in the three cell lines treated with WN197 (0.5 µM). Statistical analyses were based on two-way ANOVA followed by Dunnett's test (*p<0.05; **p<0.01; ***p<0.005; ****p<0.001) on three independent experiments.

Figure 8 :

 8 Figure 8: Western blot analysis of cell cycle markers in MDA-MB-231, HeLa, and HT-29 cells. (A)Cells were treated for 24 h with doxorubicin (5 µM), cisplatin (20 µM), WN170 (0.5 µM), WN197 (0.5 µM), or nocodazole (84 nM). β-actin was used as a loading control. For H3 S10 phosphorylation, respective H3 total levels were used as loading controls. Relative protein levels were expressed by densitometry using Image J software (Fiji Software, v1.52i). (B) 14-3-3 and Cdc25C immunoprecipitations were realized in cell lines treated for 24 h with cisplatin (20 µM) or WN197 (0.5 µM). Results are representative of three independent experiments.

Figure 9 :

 9 Figure9: apoptosis analysis. Cells were treated for 24 h with doxorubicin(5 µM, Dox), cisplatin(20 µM, Cis), WN170 (0.5 µM), WN197 (0.5 µM), nocodazole(84 nM, Noc), or camptothecin (20 µM). (A) Cleaved caspase 3 and PARP analysis were realized after 24 h of treatment or untreated in controls. (B) Cleaved PARP and (C) cytosolic cytochrome c analysis after 3, 16, 24 (cytochrome c only), 48, and 72 h of treatment with WN197 or doxorubicin for 24 and 48 h. βactin levels were used as a loading control. Relative protein levels were analyzed by densitometry using Image J software(Fiji Software, v1.52i). (D) Histogram representations of annexin V-Propidium iodide analysis by flow cytometry, showing the apoptotic cell repartition after 24 h of treatments. Raw data available inAppendice 3.

Figure 10 :

 10 Figure10: autophagy analysis by Western blots. MDA-MB-231, HeLa, and HT-29 cells were treated for 24 h with doxorubicin (5 µM, Dox), WN170 (0.5 µM), WN197 (0.5 µM), or rapamycin (0.5 µM, Rap). (A) p62, Beclin-1, and LC3 markers were analyzed, LC3 levels were expressed as the LC3-II/LC3-I ratio. β-actin levels were used as a loading control. (B) mTOR immunoprecipitations were realized in cell lines treated or not with doxorubicin (5 µM), rapamycin (0.5 µM) or WN197 (0.5 µM) for 24 h. Relative protein levels were expressed by densitometry using Image J software (Fiji Software, v1.52i).

Figure 11 :

 11 Figure 11: autophagic vacuoles detection by fluorescence. MDA-MB-231, HeLa, and HT-29 cells were treated for 24 h with DMSO (0.1%, solvent control), WN197 (0.5 µM), or rapamycin (0.5 µM). (A) After 24 h of treatment, the green dye revealed autophagic vacuoles (dots). Nuclei were stained with Hoechst 33342 (blue). Images were obtained on a Leica fluorescent microscope (x100) and were representative of three independent experiments. Scale bar: 20 µm. (B) Quantification of the mean vacuoles per cells were performed after 24 h of treatment.

Figure 12 :

 12 Figure12: effect of WN197 on Xenopus laevis embryos. Fertilized oocytes were treated or not with 0.5 or 5 µM of doxorubicin, or WN197 and scored at indicated time as follow: fertilized egg (T=0, control eggs), gastrula (NF12, 14 h), neurula (NF16, 19 h), tailbud (NF30, 25 h) and their viability determined by visual observation under a stereomicroscope. Ratio for treated embryo/control embryo were calculated at each developmental stage. Data were expressed as the mean ± SD for 20 embryos from three independent experiments. Statistical analyses were based on a two-way ANOVA followed by a Dunnett's test (***p<0.005 and ****p<0.001).

  P a g e | 98

Figure

  Figure ii. Furture research on the WN197 organometallic compound. Upper left: docking evaluation of WN197 with DNA and topoisomerase 1, and the use of Top1 siRNA in cancer cell lines could confirm that WN197 has no other target. ROS production and P-glycoproteins escape could be verified. Upper right: examples of inhibitors that could be used in combination with WN197 to optimize the cytotoxic effects on cancer cells. Bottom left: for further development of WN197 as a chemotherapeutic agent, WN197 will be tested on colon organoids, and studies on mice could be performed. Bottom right: WN197 possible structural modifications to improve its permeability, toxicity, and selectivity.

P a g e | 99 BIBLIOGRAPHYA

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  Abbreviations: ATM, ataxia telangiectasia mutated; ATR, ataxia telangiectasia related; BSA, bovine serum albumin; Cdc25, cell division cycle 25; Cdk1, cyclin dependent kinase 1; Chk1/2, checkpoint kinases 1/2; c-IAP1, cellular inhibitor of apoptosis protein 1; DDR, DNA damage response; DNA-PK, DNA-dependent protein kinase; DSB, double strand break; H2AX, H2A histone family member X; IC 50 , half maximal inhibitory concentration; Kapp, apparent dissociation constant; mTOR, mammalian target of rapamycin; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium; N DNA, nicked DNA; PBS, phosphate-buffered saline; RAPTOR, regulatory-associated protein of mTOR; R DNA, relaxed DNA; SC DNA, supercoiled DNA; SDS, sodium dodecyl sulfate; SSB, single strand break; t-AML, therapyrelated acute myeloid leukemia; TLC, thin-layer chromatography; Tm, temperature of melting; Top, Topoisomerase; etoposide.

FIGURE 2 |

 2 FIGURE 2 | The copper complex WN197 induced DNA damage in cancer cells. MDA-MB-231, HeLa and HT-29 cells were treated with DMSO (0.5%, solvent control), doxorubicin (5 µM, Top2 inhibitor inducing DNA breaks), cisplatin (20 µM, alkylating agent inducing DNA breaks), WN170 (0.5 µM, indenoisoquinoline without metal) or WN197 (0.5 µM). (A) Immunofluorescence of the DNA breaks marker gH2AX was visualized as green foci in nuclei stained with DAPI (blue) on a Leica fluorescent microscope 24 h after treatments. Images were representative of three independent experiments. Scale bar: 20 µm (B) Quantification of gH2AX foci number per cells. (C) Western blot analysis of gH2AX 24 h after treatments. b-actin was used as a loading control and relative gH2AX level was quantified by densitometry using Image J (Fiji Software, v1.52i). (D) Quantification of gH2AX foci number per cells 30 min after treatments, based on immunofluorescence experiments. In B and D, data were expressed as the mean ± SD for 30 nuclei of three independent experiments. Statistical analyses were based on a two-way ANOVA followed by a Dunnett's test (*p<0.05, **p<0,01, ***p<0,005 and ****p<0,001).

FIGURE 3 |

 3 FIGURE 3 | WN197 inhibited human topoisomerase activity in a dose-dependent manner. (A) Top1 activity was determined by in vitro assays after addition of either DMSO (5%, solvent control, lane 4), WN197 at different concentrations (0.2, 0.5, 1 and 2 µM, lanes 5-8), etoposide (VP-16, 50 µM; Top2 poison, lane 9) the negative control of Top1 activity inhibition, or camptothecin (CPT, 10 µM; Top1 poison, lane 10) the positive control of Top1 activity inhibition. Relaxed DNA ( R DNA, lane 1) or supercoiled DNA ( SC DNA, lane 2) were used as migration controls. SC DNA was used in all other reactions in presence of Top1. The Top1 activity control allowing the relaxation of SC DNA is in lane 3. The addition of proteinase K allowed detection of nicked DNA ( N DNA), a witness of the single-strand broken DNA stabilization by a topoisomerase poison. (B) Top2a activity inhibition assay. Migration control of supercoiled DNA ( SC DNA) was performed in lane 1. Top2a was present in all other reactions. The Top2a activity control for the relaxation of SC DNA is in lane 2, the first band corresponds to the transitional open circular DNA ( OC DNA) and topoisomers correspond to the relaxed DNA. DMSO (5%, solvent control) in lane 3, WN197 (concentrations of 0.2, 0.5, 1 and 2 µM) in lanes 4-7, etoposide (VP-16, 50 µM; Top2 poison) in lane 8, and camptothecin (CPT, 10 µM; Top1 poison) in lane 9. (C) Top2b activity inhibition assay. Migration control of SC DNA was performed in lane 1. Top2b was present in all other reactions. The Top2b activity control for the relaxation of SC DNA is in lane 2, DMSO (5%, solvent control) in lane 3, WN197 (concentrations of 0.2, 0.5, 1 and 2 µM) in lanes 4-7, etoposide (VP-16, 50 µM; Top2 poison) in lane 8, and camptothecin (CPT, 10 µM; Top1 poison) in lane 9. In (A-C) after topoisomerase reactions, DNA was run in a 1% agarose gel, stained with ethidium bromide (0.5 µg/mL), and visualized under UV light.

  Variations in melting temperature (DTm=Tm drug-DNA complex-Tm DNA alone) were performed at a ratio of 0.5. Apparent binding constant were measured by fluorescence using [EB]/ [DNA] = 1.26. Data were the mean of at least three independent experiments.

  different treatments (Figures5A, B). Untreated cells showed a classical cell cycle repartition in the 3 cell lines with averages of 50.52% cells in G0/G1 phases, 29.80% in the S phase and 19.68% in the G2/M phases. Cisplatin, known to promote the accumulation of cells in the S phase(57, 58), induced 79.79%, 59.61%, and 85.53% cells in S phase for MDA-MB-231, HeLa, and HT-29 cells, respectively. The mitotic spindle poison, nocodazole, led to an arrest in mitosis with 70.17%, 88.61%, and 39.68% cells in G2/M phase for MDA-MB-231, HeLa, and HT-29, respectively. WN170 did not modified the cell cycle repartition of MDA-MB-231 cells and induced a G2/M accumulation of HeLa and HT-29 cell lines. Treatments with WN197 triggered a G2/M phase accumulation. WN197 had the capacity to induce a higher percentage of cells accumulation in the G2/M phase compared to WN170 respectively with 51.29% and 21.08% for MDA-MB-231 cells, 70.51% and 54.19% for HeLa cells, and 74.4% and 48.06% for HT-29 cells. Sub-G1 peaks were not observed in WN197 treated cells, while they were

FIGURE 4 |FIGURE 5 |

 45 FIGURE 4 | Activation of the DNA Damage Response (DDR) pathway. Cells were treated for 24 h with doxorubicin (5 µM), cisplatin (20 µM), WN170 (0.5 µM), or WN197 (0.5 µM). Western blots were performed to detect ATM, ATR, Chk1, Chk2, p53 and their phosphorylated forms, and p21. b-actin was used as a loading control and relative protein levels were quantified by densitometry using Image J software (Fiji Software, v1.52i). Results were representative of three independent experiments.

FIGURE 7 |

 7 FIGURE 7 | WN197-induced autophagy. Cells were treated for 24 h with doxorubicin (5 µM), cisplatin (20 µM), WN170, WN197 (0.5 µM), nocodazole (84 nM) or rapamycin (0.5 µM). (A) Cleaved caspase 3 and PARP analysis by Western blots. Western blot analysis, after 3, 16, (24 or not), 48 and 72 h of treatment with WN197 or doxorubicin for 24 and 48 h, of (B) cleaved PARP or (C) cytosolic cytochrome (C, D) p62, Beclin-1, and LC3 markers analysis by Western blot. LC3 levels were expressed upon the LC3-II/LC3-I ratio. b-actin levels were used as a loading control. Relative protein levels were expressed by densitometry using Image J software(Fiji Software, v1.52i). (E) mTOR immunoprecipitations were realized in cell lines untreated or treated with doxorubicin (5 µM), rapamycin (0.5 µM) or WN197 (0.5 µM) for 24 h and followed by Western blots.

Figure 1 .

 1 Figure 1. Classification of proteins from the DNA repair processes according to the DNA damaging agents, the type of DNA damage in relation with recruited effectors, repair mechanisms, and cell cycle phases occurrence or induced arrests.

Figure 1 .

 1 Figure 1. Classification of proteins from the DNA repair processes according to the DNA damaging agents, the type of DNA damage in relation with recruited effectors, repair mechanisms, and cell cycle phases occurrence or induced arrests. DNA damaged proteins are classified as sensors (violet), transducers (orange), mediators (green), and effectors (black). Natural processes, showed in the upper blue part, display a non-canonical DDR. They inhibit effectors, restrain the DNA repair in mitosis and protects telomere ends (inhibited proteins in red). Canonical DDR pathways are shown in the pink lower part.

Figure 2 .

 2 Figure 2. Regulation of the DNA damage signaling network of proteins. In the yellow panel: after DNA damage, induced for example by topoisomerases misregulation, repair pathways are selected, and major sensors/effectors are recruited to the damaged lesions. These recruitments are accompanied by many post-translational modifications of proteins with major impacts on the cell response to DNA damage, e.g., O-GlcNAcylation of H2AX and NDC1 at the DNA damage repair site, ubiquitination and NEDDylation of Ku70/80 from the NHEJ and/or PARylation of NBS1 in the MRN complex from HR, and sumoylation of TIP60 in HR. The chromosomal state and the regions damaged mobilize different repair processes that are facilitated by chromatin decompaction through the action of HMGN1 on histone H3, histones

Figure 2 .

 2 Figure 2. Regulation of the DNA damage signaling network of proteins. In the yellow panel: after DNA damage, induced for example by topoisomerases misregulation, repair pathways are selected, and major sensors/effectors are recruited to the damaged lesions. These recruitments are accompanied by many post-translational modifications of proteins with major impacts on the cell response to DNA damage, e.g., O-GlcNAcylation of H2AX and NDC1 at the DNA damage repair site, ubiquitination and NEDDylation of Ku70/80 from the NHEJ and/or PARylation of NBS1 in the MRN complex from HR, and

Figure 3 .

 3 Figure3. DNA damage induces inflammation and immunity that connect to metabolism and aging, with the genesis of mutations leading to cancer formation. The DNA damage response is activated upon DNA damage, arresting the cell cycle by CDK inhibition, and triggering an inflammatory response. cGAS, a cytosolic DNA sensor, triggers an innate immune response that activates STING. In mitosis, the CDK1-cyclin B complex phosphorylates and inhibits cGAS, but upon mitotic exit, dephosphorylation of cGAS by PP1 enables DNA cytoplasmic sensing[163]. cGAS connects DNA damage to the immune system, and subsequent inflammation by the production of interferon (IFN) and cytokines, but also to senescence and cancer[164]. Chronic inflammation can lead to an impairment of the immune system and generate ROS that counteracts DNA repair mechanisms[165]. Immunity triggers an inflammatory process that increases blood pressure, stimulating organ and metabolism damage[166,167]. Metabolism shifts with increased oxygen consumption and the generation of reactive nitrogen and oxygen intermediates are associated with inflammatory and immune responses[168]. Dysbiosis produces inflammation and metabolism syndromes that contribute to senescence. Pathogens can induce DNA damage and trigger immune and inflammatory responses. Aging is associated with adaptative immune and inflammatory responses and cumulative DNA damage and genomic instability that increase mutations[169,170]. Cancer cells evade detection by the immune system using immune checkpoints PD-1/PD-L1 (programmed cell death protein 1/programmed cell death ligand 1) and CTLA-4 (anti-cytotoxic T lymphocyte-associated antigen-4), LAG-3 (lymphocyte activation gene-3), TIM-3 (T cell immunoglobulin and mucin domain containing-3), and TIGIT (T cell immunoglobulin and ITIM domain)[171].

Figure 4 .

 4 Figure 4. DNA damage strategies in cancer chemotherapies and possible combined therapies. Synthetic lethality is in red. Deficiency (-), dominant negative (dn), inhibitors (i), mutations (m).

Figure 4 .

 4 Figure 4. DNA damage strategies in cancer chemotherapies and possible combined therapies. Synthetic lethality is in red. Deficiency (-), dominant negative (dn), inhibitors (i), mutations (m).

Figure 1 .

 1 Figure 1. Platinum (II) complexes.

Figure 1 .

 1 Figure 1. Platinum (II) complexes.

Figure 2 .

 2 Figure 2. Structure of Cu(II) complexes as Top1 inhibitors.

Figure 2 .

 2 Figure 2. Structure of Cu(II) complexes as Top1 inhibitors.

  . Another pyridine-TSC CuC inhibits Top2β [100]. Molecular modeling supports the binding of the complexes near but outside the ATP binding pocket in communication with the DNA cleavage/ligation site of Top2. Piperazine-TSCs based CuC 16 inhibit Top2α [101,102] by a strong interaction with the ATP-binding pocket residues [99] without ROS production [102]. Thiazole-TSC CuC 17 and 18 are Top2α catalytic inhibitors [103, 104] or poisons [105]. The highly water-soluble proline-TSC CuC series 19 inhibit Top2α and cell proliferation [106]. Quinoline-TSC CuC 20 interact with the DNA phosphate group preventing relegation. The presence of two methyl groups on the terminal nitrogen is responsible for high activity and confers a cationic nature responsible for easier passive access into the cell [107]. Cancers 2020, 12, x 8 of 26

  . Another pyridine-TSC CuC inhibits Top2β [100]. Molecular modeling supports the binding of the complexes near but outside the ATP binding pocket in communication with the DNA cleavage/ligation site of Top2. Piperazine-TSCs based CuC 16 inhibit Top2α [101,102] by a strong interaction with the ATP-binding pocket residues [99] without ROS production [102]. Thiazole-TSC CuC 17 and 18 are Top2α catalytic inhibitors [103,104] or poisons [105]. The highly water-soluble proline-TSC CuC series 19 inhibit Top2α and cell proliferation [106]. Quinoline-TSC CuC 20 interact with the DNA phosphate group preventing relegation. The presence of two methyl groups on the terminal nitrogen is responsible for high activity and confers a cationic nature responsible for easier passive access into the cell [107].

Figure 3 .

 3 Figure 3. Structure of Cu(II) complexes as Top2 inhibitors.

Figure 3 .

 3 Figure 3. Structure of Cu(II) complexes as Top2 inhibitors. Non-heterocycle naphthoquinone-TSC CuC 21 [108] and bis-TSC CuC 22 [109] are Top2α inhibitors acting as poisons [109]; they induce apoptosis in various human cancer cell lines and delay colorectal growth of carcinoma xenografts in mice [109]. Carbohydrazone CuC 23 [110] is a Top2α inhibitor that binds DNA, induces apoptosis, and reduces mice xenograft (83% after a treatment of 2 mg/kg). Chiral chromone Cu(II)/Zn(II) 24 [111] revealed catalytic inhibition of Top2α with DNA binding in the major groove. Quinolinone CuC 25 [112] inhibit Top2α and DNA synthesis without DNA intercalation and are only minimized PGP (P-glycoprotein efflux transporter) substrates.

3 .

 3 CuC Dual Top1/Top2α Inhibitors Heteroleptic Cu(I) complexes of the bis-pyrazolyl carboxylate ligand with auxiliary phosphine 26 (Figure4) may inhibit Top1 by blocking the relegation step and inhibit Top2α by preventing ATP hydrolysis, as proposed by molecular docking analysis. They also perturb DNA replication, generate ROS, and induce apoptosis[113].Cancers 2020, 12, x 9 of 26 Heteroleptic Cu(I) complexes of the bis-pyrazolyl carboxylate ligand with auxiliary phosphine 26 (Figure4) may inhibit Top1 by blocking the relegation step and inhibit Top2α by preventing ATP hydrolysis, as proposed by molecular docking analysis. They also perturb DNA replication, generate ROS, and induce apoptosis[113].

Figure 4 .

 4 Figure 4. Structure of Cu(I) complex as a Top1/2α dual inhibitor.

  and a dual Top1/2 inhibitor with heteroleptic CuC [113]), Cu(II) exhibits a high redox potential and reinforces DDR activation by ROS production. ROS are also involved in a G2/M arrest through the decrease in Cdc25C [127] and Cdc25A levels [128], the activation of Chk1 [129] and Chk2 [130], and genomic instability through induced-DNA damages [131] (Figure 5A).

Figure 4 .

 4 Figure 4. Structure of Cu(I) complex as a Top1/2α dual inhibitor.

  and a dual Top1/2 inhibitor with heteroleptic CuC [113]), Cu(II) exhibits a high redox potential and reinforces DDR activation by ROS production. ROS are also involved in a G2/M arrest through the decrease in Cdc25C [127] and Cdc25A levels [128], the activation of Chk1 [129] and Chk2 [130], and genomic instability through induced-DNA damages [131] (Figure 5A).

Figure 5 .

 5 Figure 5. Cont.

Figure 5 .

 5 Figure 5. Molecular mechanisms and treatment perspectives for copper complexes (CuC) drugs. (A) Molecular checkpoints and networks involved in DNA damage (red), cell cycle regulation (yellow), and death response (violet) triggered by topoisomerase inhibitors (poison and catalytic), including CuC of topoisomerase inhibitors. (B) Treatment perspectives alone or in association with other chemotherapeutics (see text for more details).

Table VII :

 VII statistical comparison of synthesized organometallic derived from indenoisoquinoline. ……………………………………………………………………………………………………………………………………………….. page

	Materials & Methods

  .

		Cancer type Incidence	Risk factors	Treatments
			(new cases)
			and deaths
			(2020 WW)
	Breast	SCC, AC	2,261,419;	Alcohol, tobacco	Surgery (tumorectomy or
	cancer		684,996	smoking, obesity, lack of physical activity, age	mastectomy), extern radiotherapy, brachytherapy,
				(>50), menopause	chemotherapy (anthracyclines,
				hormonotherapy,	taxanes, cyclophosphamide, 5-
				radiation, genetic	FU, carboplatin…),
				predisposition/mutation	hormonotherapy (tamoxifen,
				(BRCA1/2…) familial or	anastrozole, letrozole,
				personal	exemestane…), targeted therapy
					(trastuzumab, lapatinib,
					palbociclib, everolimus, alpelisib,
					olaparib…), immunotherapy
					(pembrolizumab)
	Cervical	SCC, AC	604,127;	HPV, tobacco smoking,	Surgery (conization or
	cancer		341,831	extended use of hormonal	hysterectomy), extern radiotherapy, brachytherapy,
				contraceptives,	chemotherapy (carboplatin,
				immunosuppression	cisplatin, paclitaxel, topotecan),
					targeted therapy (bevacizumab),
					immunotherapy
					(pembrolizumab)
	Colorectal	AC	1,931,590;	Alcohol, tobacco	Surgery (hemicolectomy,
	cancer		935,173	smoking, obesity, lack of physical activity, age,	colectomy, polypectomy, proctectomy), extern
				alimentation (excessive	radiotherapy, brachytherapy,
				red meat and	chemotherapy (oxaliplatin,
				insufficient fibers),	capecitabine, irinotecan,
				familial background,	raltitrexed, 5-FU), targeted
				polyps, Lynch syndrome	therapy (bevacizumab,
				(HNPCC), inflammatory	ramucirumab, ziv-aflibercept,
				diseases	cetuximab, panitumumab,
					encorafenib, regorafenib)
					immunotherapy
					(pembrolizumab, nivolumab,
					ipilimumab)
					P a g e | 25

  against Top1, Top2α and Top2β. No Top3α/β or Top1mt inhibitors are yet known or developed at this point.

	➔ Poisons ligate to DNA and topoisomerases, forming the ternary cleavage complex (DNA-
	topoisomerase-inhibitor) with cleaved DNA, called interfacial inhibition (Figure XII). This
	ligation is covalent, and inhibits the DNA religation, inducing SSB (type I topoisomerase) or
	DSB (type II topoisomerase) in the DNA. SSB and DSB induce the DNA damage response
	(DDR) pathway associated with a cell cycle arrest and a cell death if DNA damage are too
	extended as explained previously ( §1.1.3, page 33)

Table V .

 V Main approved topoisomerase inhibitors as chemotherapy drugs.

	Family	Name	Target	Mechanism	Cancers
		Topotecan Irinotecan	Top1	Poison	Ovarian, cervical, SCLC Colorectal
		(Trastuzumab)	Top1 and	Poison and	Unresectable/metastasis
	Camptothecins	deruxtecan	(EGFR)	(EGFRi)	HER2 + breast cancer
		(Sacituzumab) govitecan	Top1 and (Trop-2)	Poison	Metastatic triple-negative breast cancer and urothelial cancer
					Various carcinomas,
	Anthracyclines	Doxorubicin Daunorubicin Epirubicin	Top2α/β	Poison (low doses) and Catalytic (intercalator; higher doses)	sarcomas, lymphoma, and leukemia AML, ALL, CML, Kaposi's sarcoma Carcinomas, sarcomas, lymphoma
		Idarubicin			AML, ALL, CML
	Anthracenedione	Mitoxantrone	Top2(α)/β	Poison, Catalytic (intercalator)	AML, prostate and breast cancers, NHL
					Lung, testicular, ovarian,
		Etoposide			neuroblastoma,
	Podophyllotoxins		Top2α/β	Poison	leukemia, lymphoma
		Teniposide			Lymphoma, leukemia, brain tumor
	EDTA derivative	Dexrazoxane	Top2	Catalytic inhibitor (ATP)	Following anthracycline treatment

Ligand class of Cu-C Targeted Top(s) Cancer cell lines IC50 (µM) Cell cycle arrest/Cell death Reference

	Oxindolimine		Neuroblastoma SH-SY5Y Promonocytic U937		G2/M arrest Apoptosis	Castelli et al., 2018 Cerchiaro et al., 2005
	Hydrazone with triphenylphosphonium		Lung A549 Prostatic PC-3	4.2 ± 0.8 3.2 ± 0.2		Chew et al., 2014
			Breast MCF-7	3.2 ± 1.1
			Colon HCT116	5.9 ± 1.4
			Hepatoma BEL7404	12.9 ± 3.6
	Plumbagin		Hepatoma HepG2	9.0 ± 0.7		Chen et al., 2009
			Kidney 786-O	2.5 ± 0.9
			Lung NCI-H460	2.0 ± 1.2
			Nasopharyngeal cancer CNE2	11.8 ± 5.9
	Phenanthroline with amino acids	Top1	Nasopharyngeal cancer HK1	2.2 -5.2	Apoptosis	Seng et al., 2012
	Pyrophosphate		Ovarian A2780/AD	0.64 ± 0.12		Ikotun et al., 2009
			Breast Zr-75-1
			Cervix SiHa
	Heterobimetallic Cu(II)-Sn2(IV) phenanthroline		Colon HCT15, SW620 Kidney 786-O, A498	< 10 (GI50)		Tabassum et al., 2012
			Lung Hop-62, A569
			Pancreatic MIA PaCa-2
			Neuroblastoma SH-SY5Y	2 -8	Apoptosis	Chauhan et al., 2007
	Tridentate		Hepatoma HuH7	25		Tbassum et al., 2013;
	chiral Schiff base		Hepatoma HepG2	6.2 ± 10		2015

Ligand class of Cu-C Targeted Top(s) Cancer cell lines IC50 (µM) Cell cycle arrest/Cell death Reference

			Prostatic PC-3	7.3 ± 0.2
			Breast MCF7	51.1 ± 1.6
			Colon HT29	16.6 ± 0.6
	Salicylidene		Hepatoma HepG2	2.3 ± 0.1		Lee et al., 2014; 2016
			Lung A549	16.8 ± 1.0
			Ovary A2780	14.6 ± 0.2
			Prostatic LNCaP	25.4 ± 0.8
	Chalcone-derived Thiosemicarbazone		Breast MCF-7 Leukemia THP-1	0.16 ± 0.06 0.20 ± 0.06		Vutey et al., 2016
	Pyridyl-substituted tetrazolopyrimidine	Top1	Cervix HeLa Lung A549 Colon HCT-15	0.565 ± 0.01 0.733 0.358	Apoptosis	Haleel et al., 2019
	Tetrazolopyrimidine diimine		Cervical HeLa Lung A549 Colon HCT-15	0.620 ± 0.0013 0.120 ± 0.002 0.540 ± 0.00015	Apoptosis	Haleel et al., 2016
	Piperazine					Tabassum et al., 2012
					Apoptosis
	Elesclomol		Erythroleukemic K562	0.0075	Necrosis Oxidative	Hasinoff et al., 2015
					stress
	Cu(SBCM)2		Breast MCF7 Breast MDA-MB-231	27 18.7 ±3.1	G2/M arrest Apoptosis	Foo et al., 2019
	Pyridine-TSC	Top2	Breast MDA-MB-231 Breast MCF7	1.01 0.0558		Conner et al., Wilson et al., Morris et al., 2019
		Top2β				Keck et al., 2019
	Piperazine-TSC		Breast MCF7 Breast SK-BR-3	4.7 ± 0.3 1.3 ± 0.3		Zeglis et al., 2011
			Breast MDA-MB-231 Breast MCF7	1.41 (EC50) 0.13 (EC50)		Lisic et al., 2018
			Breast
	Thiazole-TSC		HCC 70, HCC 1395,	1 to 20
		Top2	HCC 1500, and HCC 1806			Morris et al., 2019 Sandhaus et al., 2016
			Colon	0.83 to 41.2
			Caco-2, HCT-116 and HT-29
	L-and D-Proline-TSC		Ovarian carcinoma CH1	113 ± 16		Bacher et al.,
	Quinoline-TSC		Lymphoma U937	0.48-16.2		Bisceglie et al., 2015
	Naphthoquinone-TSC		Breast MCF7	3.98 ± 1.01	No apoptosis	Chen et al., 2004
	Table VI part 2. Copper-complexes inhibitors of human topoisomerases, toxicity on cancer cells
	(IC50), and induced cell cycle arrests and/or cell deaths when determined (Molinaro et al.,
	Table VI part 1. Copper-complexes inhibitors of human topoisomerases, toxicity on cancer cells 2020).
	(IC50), and induced cell cycle arrests and/or cell deaths when determined (Molinaro et al.,
	2020).
						P a g e | 52 P a g e | 53

Ligand class of Cu-C Targeted Top(s) Cancer cell lines IC50 (µM) Cell cycle arrest/Cell death Reference

			Breast MDA-MB-231	1.45 ± 0.07
	Bis-TSC		Colon HCT116 Keratinocyte HaCaT Colon HCT116	1.23 ± 0.27 0.65 ± 0.07 Delayed mice xenograft	G2/M arrest Apoptosis	Palanimuthu et al., 2013
			Breast MCF7	9.916
			Breast MDA-MB-231	7.557
			Breast HCC 1937	3.278
	Carbohydrazone		Breast MX1 Breast MDA-MB-436	4.534 5.249	Apoptosis	Nair et al., 2017
		Top2		Reducted mice
			Breast MX-1	xenograft
				(83%)
			Breast MCF7	18.6 (GI 50)
			Breast Zr-75-1	25.2 (GI 50)
			Colon HT29	>80 (GI 50)
	Chromone		Cervix SiHa	34.6 (GI 50)		Arjmand et al., 2012
			Kidney A498	73.3 (GI 50)
			Lung A549	31.7 (GI 50)
			Ovary A2780	17.4 (GI 50)
	Quinolinone Shiff Base		Hepatic HepG2	17.9 ± 3.8		Duff et al., 2012
	Bis-pyrazolyl Carboxylate	Dual Top1/Top2	Hepatic HepG2	3.3 ± 0.02	Apoptosis	Khan et al., 2017

  Table VI part 3.

Table VII .

 VII Statistical comparison of synthesized organometallic compounds derived from indenoisoquinoline. L1 to L4 are derivative ligands without a metal atom. Numbers refer to different compounds: Cu-1 to Cu-4 are copper-complexes number 1 to 4; Zn-1 to Zn-4 are zinccomplexes number 1 to 4; Ru-1 and Ru-2 are ruthenium-complexes number 1 and 2. The Cu(II)-2 (WN197) copper-complex is the most cytotoxic. Statistical analyses were realized using ANOVA on the mean IC50 of the five cancer cell lines (*p<0.05; **p<0.01; ****p<0.001).

		L2 (WN170)	L3 L4 Cu-1	Cu-2 (WN197)	Cu-3	Cu-4	Zn-1 Zn-2	Zn-3	Zn-4	Ru-1	Ru-2
	L1	ns	ns	ns	ns	**	ns	ns	ns	ns	ns	ns	****	ns
	L2 (WN170)		ns	*	ns	*	ns	*	ns	ns	ns	ns	****	ns
	L3			**	ns	ns	ns	**	ns	ns	*	ns	****	ns
	L4				*	****	ns	ns	*	ns	ns	ns	****	ns
	Cu-1					*	ns	*	ns	ns	ns	ns	****	ns
	Cu-2 (WN197)						**	****	*	**	****	**	****	ns
	Cu-3							ns	ns	ns	ns	ns	****	ns
	Cu-4								*	ns	ns	ns	****	ns
	Zn-1									ns	ns	ns	****	ns
	Zn-2										ns	ns	****	ns
	Zn-3											ns	****	ns
	Zn-4												****	ns
	Ru-1													****

P a g e | 57

Table 1 .

 1 Half maximal inhibitory concentrations (IC50 in µM) for cell survival of MCF-10A.

	Compound	IC50 (µM)
	WN197	1.080 ± 0.037
	Cisplatin	14.218 ± 7.157
	Statistical difference (WN197 on adenocarcinomas vs. on MCF-10A)	***

Data are expressed as the mean ± SD of three independent experiments. Statistics are based on Student's t-test of the difference between WN197 IC50 on adenocarcinomas and MCF-10A; ***p<0.001.

Table 2 .

 2 Half maximal inhibitory concentrations (IC50 in µM) for cell survival of

	Compound	BT-549	SUM-159
	WN197	0.676 ± 0.063	0.679 ± 0.178
	WN170	0.691 ± 0.308	0.689 ± 0.154
	Cisplatin	3.546 ± 0.924	13.96 ± 4.837
	Statistical difference (WN197/WN170)	ns	ns
	Data are expressed as the mean ± SD of three independent experiments. Statistics are based on
	Student's t-test of the difference between WN197 and WN170 IC50; ns: non-significative.

Table 3 .

 3 Half maximal inhibitory concentrations (IC50 in µM) for cell survival of H69AR.

	Compound	IC50 (µM)
	WN197	0.683
	WN170	4.966
	Cisplatin	15.10
	Data are expressed as the mean of a triplicate.

3 .

 3 Caroline Molinaro, Nathalie Wambang, Lydie Pélinski, Katia Cailliau and Alain Martoriati. Un nouveau composé organométallique inhibiteur de la croissance des adénocarcinomes : action sur le cycle cellulaire et sur l'activité des topoisomérases. Séminaire UGSF 2021. Talk. 4. Caroline Molinaro, Nathalie Wambang, Till Bousquet, Arlette Lescuyer, Katia Cailliau, Lydie Pelinski, Alain Martoriati. Un composé organométallique inhibiteur des topoisomérases : action sur le cycle et la mort cellulaire de lignées d'adénocarcinomes. Cancéropôle Nord-Ouest 2020. Poster. Caroline Molinaro 1 , Nathalie Wambang 2 , Till Bousquet 3 , Anne-Sophie Vercoutter-Edouart 1 , Lydie Pe ´linski 3 , Katia Cailliau 1 † and Alain Martoriati 1 * † 1 Univ. Lille, CNRS, UMR 8576-UGSF-Unite ´de Glycobiologie Structurale et Fonctionnelle, Lille, France, 2 AGAT Laboratories, Intertek, Montre ´al, QC, Canada, 3 Univ. Lille, CNRS, Centrale Lille, Univ. Artois, UMR 8181-UCCS-Unite ´de Catalyse et Chimie du Solide, Lille, France

	A Novel Copper(II) Indenoisoquinoline
	Complex Inhibits Topoisomerase I,
	Induces G2 Phase Arrest, and Autophagy
	in Three Adenocarcinomas
	Publication (research paper 1)
	A Novel Copper(II) Indenoisoquinoline Complex
	Inhibits Topoisomerase I, Induces G2 Phase
	Arrest, and Autophagy in Three Adenocarcinomas
	P a g e | 129

TABLE 1 |

 1 Half maximal inhibitory concentrations (IC 50 in mM) for cell survival.

	MDA-MB-231	HeLa	HT-29

TABLE 2 |

 2 Half maximal inhibitory concentrations (IC 50 in mM) for cell survival of MCF-10A.Data are expressed as the mean ± SD of three independent experiments. Statistics were based on Student's t-test of the difference between WN197 IC 50 on adenocarcinomas and MCF-10A; ***p<0.001.

	Compound	IC 50 (µM)
	WN197	1.080 ± 0.037
	Cisplatin	14.218 ± 7.157
	Statistical difference (WN197 on adenocarcinomas vs. on	***
	MCF-10A)

TABLE 3 |

 3 Melting curves and fluorescence measurements were determined for WN197 and WN170.

	Compound	DTm (°C)	Kapp (10^7 M-1)	EtBr displacement
	WN197	16.6	15.005 ± 0.290	90%
	WN170	16.1	2.436 ± 0.883	87%

Table 1 .

 1 DDR-proteins mutations associated to various cancers. ATPase (RAD50) to compose theMRN complex [212]. BRCA1, an E3 ubiquitin ligase, and BRCA2 facilitate response to DNA damage. PALB2/FANCN (partner and localizer of BRCA2) has a critical role through the recruitment of BRCA2 and RAD51 to DNA breaks[213]. FANCA and FANCB are associated with other FANC (Fanconi Anaemia) and FAAP (FA-associated proteins) (FANCC, FANCE, FANCF, FANCG, FANCL, FANCM and FAAP20, FAAP24, and FAAP100). They form the FA core complex carrying an E3 ligase activity to monoubiquitinateFANCI and FANCD2 [214], and initiate DNA repair by forming a platform to recruit additional proteins. RAD51C forms distinct complexes, one with a related DNA-dependent ATPase paralog, RAD51D, forming the BCDX2 complex (RAD51B-RAD51C-RAD51D-XRCC2), and one with the CX3 complex (RAD51C-XRCC3)[215]. BLM (Bloom syndrome protein) and WRN (Werner syndrome ATP-dependent helicase) are members of the RecQ helicase family. WRN also displays a 3 to 5 exonuclease activity[216]. In NER, ERCC1 has a

	DDR Proteins	DDR Signaling Pathways	Protein Activity/Function	Cancer-Associated Mutations *
	ATM			Breast, colon, endometrial, leukemia, lung, lymphoma, pancreatic, prostate
	ATR	Cell cycle regulators	Kinases	Breast, colon, endometrial, gastric, lung, lymphoma
	CHK2	Cell cycle	Phosphatases	Bladder, breast, colon, endometrial, lung
	APC-C/CDH1			Breast, gastric, lung
	PTEN		Phosphatase	Breast, endometrial, gastric
		Cell death
	P53		Transcription factor	Found in 39.52% of all cancers (AACR)
	DNA repair proteins	DNA repair pathways
	Ku70/Ku80	NHEJ	Helicases	Breast, colorectal, lung, melanoma
	DNA-PK		Kinase	Breast, colon, glioma, oesophagal, lung
	NBS1			Breast, colon, esophageal, head and neck, hepatoma, liver, lung, lymphoma, prostate
	MRE11		MRN complex	Breast
	RAD50			Colon, gastrointestinal, lung
	BRCA1		E3 ubiquitin-ligase	Breast, colon, gastrointestinal, haematological, lung, melanoma, ovarian,
	BRCA2		RAD51 binding to DNA	pancreatic, prostate
	PALB2/FANCN	HR	Recruitment of BRCA2 and RAD51	Breast, colon, head and neck, lung, ovarian
	FANCA, FANCB		FA repair complex	Breast, colon, leukemia, liver, lung
	FANCO/RAD51C		DNA-dependent ATPase	Breast, colon, lung, ovarian, prostate
	RAD51D			Breast, lung, ovarian
	BLM			Breast, colon, endometrial, leukemia, lung, lymphoma, melanoma
	WRN		Helicases	Colorectal, endometrial, lung, melanoma, pancreatic, thyroid
	ERCC1		Nuclease	Colorectal, glioma, lung, skin
	XPA, XPC		Scaffold protein
	XPD/ERCC2	NER	Helicase	Bladder, colon, lung, skin
	XPG/ERCC5		Endonuclease
	OGG1		Glycosylase	Breast, lung, renal
	PARP1	BER	ADP-ribosyltransferase	Breast, colon, endometrial, lung
	XRCC1		Scaffold protein	Non-small cell lung
	MLH1 MSH2, MSH6	MMR	ATPase Scaffold protein	Brain, breast, colorectal, endometrial, hepatobiliary, lung, ovarian, pancreatic, skin, stomach, upper urinary
	MGMT	DR	Methyltransferase	Gliomas
	Notes for Table 1: ATM, ATR, CHK1/2, and APC/C-CDH1 are involved in cell cycle regulation; PTEN and P53 in programmed cell death.
	In NHEJ, the ATP dependent helicases Ku70 and Ku80 form a heterocomplex with DNA-ends, and Ku80 C terminus recruits DNA-PK,
	a phosphatidylinositol 3-kinase related serine/threonine kinase [211]. In HR, two NBS1 subunits (phosphopeptide-binding Nijmegen
	breakage syndrome protein 1) are associated with two MRE11 subunits (meiotic recombination 11 homolog 1), and two ATP-binding
	cassette (ABC)-

Table 1 .

 1 Copper complexes inhibitors of topoisomerases: targeted top isoforms, cancer cell lines responses, and molecular mechanisms are summarized. * Tests were realized in vitro with human Top1 or Top2α/β unless specified. IC50: half-maximal inhibitory concentration. EC50: half-maximal effective concentration. GI50: half-average of growth inhibition.

				Inhibition of DNA
	Ligand Class of Cu-C	Compound Number	Targeted Top(s)	Relaxation Total (µM) (minimal	Inhibition Mecanism	Cancer Cell Lines	IC50 (µM)	Cell Cycle Arrest	Cell Death Type	Other Specificity	Reference Number
				(µM))
	Oxindolimine	1	Top1	(25) 50	Top1 binding site Fixation in the DNA	Promonocytic U937 Neuroblastoma SH-SY5Y		G2/M arrest	Apoptosis	ROS induction	[70-73]
	Hydrazone with triphenylphosphonium	2	Top1	40	DNA Binding Enzyme complex formation	Lung A549 Prostatic PC-3	4.2 ± 0.8 3.2 ± 0.2				[74]
			Top1	1.56	DNA intercalation	Breast MCF-7	3.2 ± 1.1				[75]
						Colon HCT116	5.9 ± 1.4
						Hepatoma BEL7404	12.9 ± 3.6
	Plumbagin	3				Hepatoma HepG2	9.0 ± 0.7
						Kidney 786-O	2.5 ± 0.9
						Lung NCI-H460	2.0 ± 1.2
						Nasopharyngeal cancer CNE2	11.8 ± 5.9
	Phenanthroline with amino acids	4	Top1	50 (10)	DNA intercalation	Nasopharyngeal cancer HK1	2.2-5.2		Apoptosis		[76]
	Pyrophosphate	5	Top1	500	DNA interaction	Ovarian A2780/AD	0.64 ± 0.12				[77]
			Top1	20	DNA intercalation	Breast Zr-75-1					[78]
					cleavage	Cervix SiHa
	Heterobimetallic Cu(II)-Sn2(IV) phenanthroline	6				Colon HCT15, SW620 Kidney 786-O, A498 Lung Hop-62, A569 Pancreatic MIA PaCa-2	<10 (GI50)
						Neuroblastoma SH-SY5Y	2-8		Apoptosis		[79]
	Analogs										[80]
			Top1	25	DNA binding	Hepatoma HuH7	25			ROS	[81,82]
	Tridentate chiral Schiff base	7, 8		(15)	major groove	Hepatoma HepG2	6.2 ± 10			Cytokine TGFb mRNA
										upregulation

Table 1 .

 1 Cont.

				Inhibition of DNA
	Ligand Class of Cu-C	Compound Number	Targeted Top(s)	Relaxation Total (µM) (minimal	Inhibition Mecanism	Cancer Cell Lines	IC50 (µM)	Cell Cycle Arrest	Cell Death Type	Other Specificity	Reference Number
				(µM))
			Top1	(E. coli) *	DNA binding	Prostatic PC-3	7.3 ± 0.2			antimetastasis	[83]
					DNA cleavage	Breast MCF7	51.1 ± 1.6				[84]
	Salicylidene	9				Colon HT29 Hepatoma HepG2	16.6 ± 0.6 2.3 ± 0.1
						Lung A549	16.8 ± 1.0
						Ovary A2780	14.6 ± 0.2
						Prostatic LNCaP	25.4 ± 0.8
	Chalcone-derived Thiosemicarbazone	10	Top1	3 (0.75)	DNA binding DNA cleavage Religation inhibition	Breast MCF-7 Leukemia THP-1	0.16 ± 0.06 0.20 ± 0.06				[85]
	Pyridyl-substituted tetrazolopyrimidie	11	Top1	(Molecular docking) *	DNA binding groove mode	Cervix HeLa Colon HCT-15 Lung A549	0.565 ± 0.01 0.358 0.733		Apoptosis	CDK receptor binding	[86]
	Tetrazolopyrimidine		Top1	102 ± 1.1	DNA binding	Cervical HeLa	0.620 ± 0.0013		Apoptosis	vEGF receptor	[87]
	Diimine				groove mode	Colon HCT-15	0.540 ± 0.00015			binding
						Lung A549	0.120 ± 0.002
	Piperazine	12	Top1	12.5 (5)	DNA binding minor groove					SOD mimic	[88]
			Top1	50	Poison	Erythroleukemic K562	0.0075		Apoptosis	Copper chelator	[89]
	Elesclomol	13							Necrosis	Not a substrat for
									Oxidative	ABC
									stress	transporters
	Cu(SBCM)2	14	Top1	* (Molecular docking)	DNA intercalation DNA binding	Breast MCF7 Breast MDA-MB-231	27 18.7 ± 3.1	G2/M arrest	Apoptosis	p53 increase No ROS	[90] [91]
	TSC and TSC CuC										[92-97]
				50		Breast MDA-MB-231	1.01				[98]
			Top2a	(10)		Breast MCF7	0.0558
	Pyridine-TSC	15		50	ATP hydrolysis inhibition						[99]
			Top2β	(5)	ATP hydrolysis inhibition						[100]

Table 1 .

 1 Cont.

				Inhibition of DNA
	Ligand Class of Cu-C	Compound Number	Targeted Top(s)	Relaxation Total (µM) (minimal	Inhibition Mecanism	Cancer Cell Lines	IC50 (µM)	Cell Cycle Arrest	Cell Death Type	Other Specificity	Reference Number
				(µM))
	Piperazine-TSC	16	Top2a	0.9 ± 0.7	Potentially catalytic	Breast MCF7 Breast SK-BR-3	4.7 ± 0.3 1.3 ± 0.3				[101,102] [99]
		17	Top2a	4 (2)		Breast MDA-MB-231 Breast MCF7	1.41 (EC50) 0.13 (EC50)				[103]
	Thiazole-TSC	17-18	Top2a	25 (10)	ATP hydrolysis inhibition + Poison	Breast HCC 70, HCC 1395, HCC 1500, and HCC 1806	1 to 20				[104,105]
						Colon	0.83 to 41.2
						Caco-2, HCT-116 and HT-29
	L-and D-Proline-TSC	19	Top2a	300		Ovarian carcinoma CH1	113 ± 16				[106]
	Quinoline-TSC	20	Top2a	0.48	Potentially catalytic	Lymphoma U937	0.48-16.2				[107]
	Naphthoquinone-TSC	21	Top2α	1 mM		Breast MCF7	3.98 ± 1.01		No apoptosis		[108]
			Top2a	100	Poison	Breast MDA-MB-231	1.45 ± 0.07	G2/M arrest	Apoptosis	DNA synthesis	[109]
				(5)		Colon HCT116	1.23 ± 0.27			inhibition
	Bis-TSC	22				Keratinocyte HaCaT	0.65 ± 0.07 Delayed			No ROS
						Colon HCT116	mice
							xenograft
			Top2α	250	DNA binding	Breast MCF7	9.916		Apoptosis		[110]
				(25)	major groove	Breast MDA-MB-231	7.557
						Breast HCC 1937	3.278
	Carbohydrazone	23				Breast MX1 Breast MDA-MB-436	4.534 5.249
							Reducted
						Breast MX-1	mice xenograft
							(83%)

Table 1 .

 1 Cont.

				Inhibition of DNA
	Ligand Class of Cu-C	Compound Number	Targeted Top(s)	Relaxation Total (µM) (minimal	Inhibition Mecanism	Cancer Cell Lines	IC50 (µM)	Cell Cycle Arrest	Cell Death Type	Other Specificity	Reference Number
				(µM))
			Top2a	25	DNA binding	Breast MCF7	18.6 (GI 50)				[111]
				(15)	major groove	Breast Zr-75-1	25.2 (GI 50)
						Colon HT29	>80 (GI 50)
	Chromone	24				Cervix SiHa	34.6 (GI 50)
						Kidney A498	73.3 (GI 50)
						Lung A549	31.7 (GI 50)
						Ovary A2780	17.4 (GI 50)
			Top2α	9	No intercalation	Hepatic HepG2	17.9 ± 3.8			DNA synthesis	[112]
	Quinolinone Shiff Base	25								inhibition Slight substrate
										for ABC
										transporter
	Bis-pyrazolyl Carboxylate	26	Dual Top1/Top2	(Molecular docking) *	ATP entry (potentially) DNA religation	Hepatic HepG2	3.3 ± 0.02		Apoptosis	DNA replication	[113]
					inhibition					ROS
					(potentially)

* * * * * * * * * * * * * * * * * * * * * * *

Frontiers in Oncology | www.frontiersin.org February 2022 | Volume 12 | Article 837373

ACKNOWLEDGMENTS

We are sincerely indebted to Arlette Lescuyer (UMR-CNRS 8576) for helpful discussions, and Corentin Spriet (Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, US 41 -UMS 2014 -PLBS, F-59000 Lille, France) for his help with microscopy.

We acknowledge C. Delabre for HR-MS analysis and micronanalysis and Nathalie Jouy (IRCL, Lille) for annexin V analysis.

Acknowledgments:

The authors are thankful to the "Centre National de la Recherche Scientifique", the "Université de Lille", and the "Ligue Contre le Cancer". C.M. is a recipient of a doctoral fellowship from the French ministry.

Conclusions

In a multifactorial disease such as cancer, Top inhibitors are efficient anticancer compounds used in monotherapy or polypharmacological strategies. They certainly have to target closely related modulators of the cellular checkpoints' networks. CuC Top inhibitors are particularly adapted to fulfill this role. A perspective in anticancer strategy is to increase and to enlarge this family of highly active anticancer drugs.

Acknowledgments:

The authors are thankful to the Research Federation FRABio (FR 3688, FRABio, Biochimie Structurale et Fonctionnelle des Assemblages Biomoléculaires) and to E. Germain (Inserm U1003) for reading the manuscript.

FUNDING

CM is a recipient of a doctoral fellowship from the French ministry. This work was supported by the CNRS, the University of Lille, and by grants from the "Ligue Contre le Cancer, Comiteś Nord et Aisne" (AM).

Funding: This review was funded by "Université de Lille" and grant from the "Ligue Contre le Cancer" to A.M. Funding: C.M. is a recipient from a doctoral fellowship from the French ministry. The scientific supports were provided by the "Centre National de la Recherche Scientifique ", "Université de Lille ", and the "Ligue Contre le Cancer".

DATA AVAILABILITY STATEMENT

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Appendices & Annexes

P a g e | 125 APPENDICES Appendix 1. Number of new worldwide cancer cases for both sexes in 2020: Non-Hodgkin lymphoma (544,352), Leukemia (474,519), Kidney (431,288), Lip/oral cavity (377,713), Melanoma of skin (324,635), Ovary (313,959), Brain/Central nervous system (308,102), Larynx (184,615), Multiple myeloma (176,404), Nasopharynx (133,354), Gallbladder (115,949), Oropharynx (98,412), Hypopharynx (84,254), Hodgkin lymphoma (83,087), Testis (74,458), Salivary gland (53,583), Vulva (45,240), Penis (36,068), Kaposi sarcoma (34,270), Mesothelioma (30,870), Vagina (17,908). Appendix 2. Raw data of cell cycle analysis by flow cytometry of MDA-MB-231, HeLa, and HT-29 after 24 h of treatments or not with cisplatin (20 µM), nocodazole (84 nM), WN170 and WN197 (0.5 µM). The first red peak represents the cell population in the G0/G1 phase, the second red peak the cell population in the G2/M phase, and the blue proportion the cell population in the S phase.

SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fonc.2022.837373/ full#supplementary-material Supplementary Figure 1 | Cytograms obtained after flow cytometry analysis of MDA-MB-231 cells 24 h after treatments or not with WN197 (0.5 µM) or apoptosis positive control doxorubicin (5 µM, showing sub-G1 accumulation).

Supplementary Figure 2 | Detection of apoptosis feature by annexin Vpropidium iodide (PI). MDA-MB-231, HeLa, and HT-29 cells were cultivated to 80% of confluence, incubated or not for 24 h with WN170 (0.5 µM), WN197 (0.5 µM), camptothecin (20 µM; CPT) or doxorubicin (5 µM; Doxo), trypsinized, and washed in ice-cold PBS. Cell suspensions were treated with PI and annexin V-FITC reagent (Apoptosis Detection Kit, BD) using the manufacturer's protocol before they were analysed by flow cytometry (CytoFLEX LX, Beckman Coulter) with Kaluza analysis software (v2.1.1). (A) Y-axis: number of PI-stained cells. X-axis: number of annexin V-FITC-stained cells. The lower left quadrant represents non-apoptotic cells (annexin V-FITC-negative and PI-negative cells; B-), the lower right quadrant represents early apoptotic cells (annexin V-FITC-positive and PI-negative cells; B+-), the upper right quadrant represents late apoptotic/necrotic cells (annexin V-FITC-positive and PI-positive cells; B++), and the upper left quadrant represents prenecrotic cells (annexin V-FITC-negative and PI-positive cells; B-+). (B) Representative histograms. Camptothecin and doxorubicin induced apoptosis in the three cancer cell lines, while WN170 and WN197 had no effect compared to the control.

Conflict of Interest: Author NW was employed by AGAT Laboratories, Intertek.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher's Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Author Contributions: C.M. and K.C. wrote the manuscript and generated all figures with additional support from A.M. All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest:

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Author Contributions: Writing-original draft preparation: C.M., K.C., A.M. and L.P. All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest:

The authors declare no conflict of interest.