Characterisation of Oncolytic H-1 Parvovirus Cell Entry Pathways

Abstract

H-1 protoparvovirus (H-1PV) is a self-propagating virus, non-pathogenic in humans, \nendowed with oncolytic and oncosuppressive activities. H-1PV is the only member of \nthe Parvoviridae family to be tested as an anticancer agent in a clinical setting. Results \nfrom clinical trials in patients with glioblastoma or pancreatic carcinoma showed that \nvirus treatment is safe and well-tolerated. Virus treatment was associated with first \nsigns of efficacy, including immune conversion of tumour microenvironment, good \nvirus distribution in the tumour bed, as well as improved patient overall survival \ncompared with historical controls. However, monotherapeutic use of the virus was not \nsufficient to eradicate the tumours. In this manner, my approach consists of further \nunderstanding the virus life cycle in order to improve H-1PV-based anticancer \ntherapies. This knowledge can provide hints on which drugs or treatment modalities \ncould be combined with the virus in order to enhance its oncotoxicity. In addition, a \ndeeper understanding of H-1PV life cycle can help to identify biomarkers capable of \npredicting which patients would most likely benefit from virus treatment. To achieve \nthis goal, previous members of the laboratory performed a druggable genome-wide \nsiRNA library screening to identify putative modulators of H-1PV infection. Focusing \non cellular factors potentially involved at the early steps of H-1PV infection, three top \nactivators were identified: LAMC1, LGALS1 and AP2M1. (i) Laminin containing the \nγ1 chain, encoded by LAMC1, had previously been demonstrated to play a pivotal role \nat the level of binding and entry into cancer cells. Building on these results, I provide \ndirect evidence that H-1PV binds to laminins through the sialic acid moieties present \nin these molecules. (ii) Galectin-1, encoded by LGALS1, is here shown to interact \ndirectly with H-1PV at the cell surface and promote the efficient virus internalisation \ninto cancer cells. Knock-down/out of LGALS1 strongly decreases the ability of H-1PV \nto infect and kill cancer cells. These properties are rescued by the re-introduction of \nLGALS1 into cancer cells. The in silico analysis reveals that LGALS1 is overexpressed \nin glioblastoma and pancreatic carcinoma. In collaboration with Dr. Miletic \n(University of Bergen, Norway), we also show by immunohistochemistry analysis on \n122 glioblastoma biopsies that galectin-1 protein levels vary across the different \ntumours with higher levels detected in glioblastoma than normal tissues, and higher in \nrecurrent in comparison with primary glioblastoma tumours. We also found a direct \ncorrelation between LGALS1 transcript levels and H-1PV oncolytic activity in 59 \ncancer cell lines from different tumour origins. Together these results suggest that \ntumours with higher galectin-1 content may be a more suitable target for H-1PV. \nStrikingly, the addition of purified galectin-1 sensitises poorly susceptible glioma cell \nlines to H-1PV killing activity by rescuing virus cell entry. (iii) AP2μ1, encoded by \nAP2M1, is a subunit from the adaptor 2, a key regulator of clathrin-mediated \nendocytosis. Indeed, siRNA-mediated knockdown of AP2M1 or chemical inhibition \nof clathrin-mediated endocytosis strongly decreased H-1PV entry. Using electron and \nconfocal microscopy, H-1PV particles were detected within clathrin-coated pits and \nvesicles, further corroborating that H-1PV uses clathrin-mediated endocytosis for cell \n \nx \nentry. In contrast, I observed no evidence of viral entry through caveolae-mediated \nendocytosis. I also show that H-1PV internalisation depends on dynamin activity, and \nthat viral trafficking occurs from early to late endosomes, with low endosomal pH \nrequired for a successful infection. Based on the body of evidence gathered during this \ndissertation, I propose a model where H-1PV binds to the sialic acid present in laminins containing γ1 chains, and then galectin-1 promotes the efficient internalisation of virus particles through clathrin-mediated endocytosis. For the first \ntime, this dissertation describes the cell entry pathways of oncolytic H-1PV.