Passaro, Carmela (2013) PARP inhibition as new approach to increase the oncolytic activity of the adenoviral mutant dl922-947 against Anaplastic Thyroid Carcinoma. [Tesi di dottorato]

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Abstract

Anaplastic Thyroid Carcinoma (ATC) is one of the most aggressive tumor characterized by an average survival time of 3-6 months after diagnosis. Multimodality therapy, which includes surgical debulking, external radiation therapy and chemotherapy, has failed to show any improvements in survival. Therefore, novel therapies with different mechanisms of action are required. Oncolytic viruses (OVs) represent a novel therapeutic tool for the treatment of aggressive tumors. OVs as single agents have demonstrated limited efficacy in a clinical setting, thus highlighting the need of novel combinatorial therapies (i.e. OVs plus a rationally selected drug) with a potential great impact on clinical use. In this study, we showed that the oncolytic adenovirus dl922-947 induces massive S-phase entry in ATC cell lines, followed by an extensive DNA damage, quantified by γH2AX staining. Moreover, we demonstrated that dl922-947 triggered a DNA damage response, characterized by the activation of the checkpoint kinases ATM and Chk1, and at the same time impaired the ability of the DNA repair, by affecting MRN complex protein levels. The virus-induced single-strand DNA breaks (SSBs) activate the Poly-ADP-Ribose Polymerase (PARP) to signal SSBs to the enzymatic machinery involved in their repair. Here, we demonstrate that the pharmacological inhibition of PARP increases dl922-947 cytotoxicity against Anaplastic Thyroid Carcinoma both in vitro and in vivo. We also show that PARP inhibitor AZD2281 synergizes with dl922-947 increasing viral replication and accelerating cell death pathways. Furthermore, we showed that the virus as single treatment induced an apoptotic-like cell death, as indicated by the presence of Caspase 3 activation, PARP cleavage, Annexin V positivity and Cytochrome C release from mitochondria, although lacking the morphological features of apoptosis. In this regard we also proved that dl922-947 acts on the pathway involved in membrane blebbing by inhibiting Myosin Light Chain 2 (MLC2) phosphorylation. Using a pan caspase inhibitor, both caspase-dependent and -independent cell death pathways are found activated upon dl922-947 infection. Our data clearly suggest that the combination of the oncolytic adenovirus dl922-947 with PARP inhibitor could represent a novel and effective therapeutic approach for the treatment of Anaplastic Thyroid Carcinoma.

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