The DNA damage response to adeno-associated virus : centrosome overduplication and induction of cell death independently of the spindle checkpoint

Summary: Adeno-associated virus type 2 (AAV2) is a small virus containing single-stranded DNA of approximately 4.7kb in size. Both ends of the viral genome are flanked with inverted terminal repeat sequences (ITRs), which serve as primers for viral replication. Previous work in our laboratory has shown that AAV2 DNA with ultraviolet radiation-generated crosslinks (UV-AAV2) provokes a DNA damage response in the host cell by mimicking a stalled replication fork. Infection of cells with UV-AAV2 leads to a p53-and Chk1-mediated cell cycle arrest at the G2/M border of the cell cycle. However, tumour cells lacking the tumour suppressor protein p53 cannot sustain this arrest and enter a prolonged impaired mitosis, the outcome of which is cell death. The aim of my thesis was to investigate how UV-inactivated AAV2 kilts p53-deficient cancer cells. I found that the UV-AAV2-induced DNA damage signalling induces centriole overduplication in infected cells. The virus is able to uncouple the centriole duplication cycle from the cell cycle, leading to amplified centrosome numbers. Chk1 colocalises with centrosomes in the infected cells and the centrosome overduplication is dependent on the presence of Chk1, as well as on the activities of ATR and Cdk kinases and on the G2 arrest. The UV-AAV2-induced DNA damage signalling inhibits the degradation of cyclin B 1 and securin by the anaphase promoting complex, suggesting that the spindle checkpoint is activated in these mitotic cells. Interference with the spindle checkpoint components Mad2 and BubR1 revealed that the UV-AAV2-provoked mitotic catastrophe occurs independently of spindle checkpoint function, This work shows that, in the p53 deficient cells, UV-AAV2 triggers mitotic catastrophe associated with a dramatic Chk1-dependent overduplication of centrioles and the consequent formation of multiple spindle poles in mitosis. Résumé Le virus associé à l'adénovirus type 2 (AAV2) est un petit virus contenant un simple brin d'ADN d'environ 4.7kb. Des expériences antérieures dans notre laboratoire ont montré que les liens intramoléculaires sur l'ADN de AAV2 provoqués paz l'irradiation aux ultraviolets (UV) ressemblent à une fourche de réplication bloquée, ce qui provoque une réponse aux dommages à l'ADN dans la cellule hôte. L'infection des cellules avec UV-AAV2 résulte en un arrêt du cycle cellulaire à la transition G2/M entraîné par les protéines ATR et Chk1. Cependant, les cellules tumorales auxquelles il manque le suppresseur de tumeur p53 ne peuvent pas tenir cet arrêt et entrent dans une mitose anormale et prolongée qui se terminera par la mort cellulaire. Le but de ma thèse était d'étudier comment l'AAV2 inactivé par l'irradiation UV tue les cellules cancéreuses n'ayant pas p53. Je montre ici que le signal de dommages à l'ADN induit par UV-AAV2 génère une surduplication des centrioles dans les cellules infectées. Le virus est capable de dissocier le cycle de duplication du centriole du cycle cellulaire ce qui crée un nombre amplifié de centrosomes. Chk1 est co-localisé avec le centrosome dans les cellules infectées et la swduplication du centrosome est dépendante de la présence de Chk1, de l'activité des kinases ATR et Cdk et de l'arrêt en G2 de la cellule. Le signal d'ADN endommagé induit par UV-AAV2 réprime la dégradation des protéines cycline B1 et securine par le complexe promoteur de l'anaphase (APC), ce qui suggère que le point de contrôle du fuseau mitotique est activé dans ces cellules en mitose. L'étude d'interférence avec des éléments du point de contrôle du fuseau mitotique, Mad2 et BubR1, a révélé que la catastrophe mitotique provoquée paz UV-AAV2 survient indépendamment du point de contrôle du fuseau mitotique. Ce travail montre que dans les cellules déficientes en p53, UV-AAV2 induit une catastrophe mitotique associée à une surduplication des centrioles dépendant de Chk1 et ayant pour conséquence dramatique la formation de multiples fuseaux mitotiques dans la cellule en mitose

Pharmacological inhibition of 3 integrin reduces the inflammatory toxicities caused by oncolytic adenovirus without compromising anticancer activity

Adenoviruses have been clinically tested as anticancer therapies but their utility has been severely limited by rapid, systemic cytokine release and consequent inflammatory toxicity. Here, we describe a new approach to tackling these dangerous side effects. Using human ovarian cancer cell lines as well as malignant epithelial cells harvested from the ascites of women with ovarian cancer, we show that tumor cells do not produce cytokines in the first 24 hours following in vitro infection with the oncolytic adenovirus dl922-947. In contrast, dl922-947 does induce inflammatory cytokines at early time points following intraperitoneal delivery in mice with human ovarian cancer intraperitoneal xenografts. In these animals, cytokines originate predominantly in murine tissues, especially in macrophage-rich organs such as the spleen. We use a nonreplicating adenovirus to confirm that early cytokine production is independent of adenoviral replication. Using β3 integrin knockout mice injected intraperitoneally with dl922-947 and β3 null murine peritoneal macrophages, we confirm a role for macrophage cell surface β3 integrin in this dl922-947–induced inflammation. We present new evidence that co-administration of a cyclic RGD-mimetic–specific inhibitor of β3 integrin significantly attenuates the cytokine release and inflammatory hepatic toxicity induced by dl922-947 in an intraperitoneal murine model of ovarian cancer. Importantly, we find no evidence that β3 inhibition compromises viral infectivity and oncolysis in vitro or anticancer efficacy in vivo. By enabling safe, systemic delivery of replicating adenoviruses, this novel approach could have a major impact on the future development of these effective anticancer agents