Contact

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Jürg P.F. Nüesch, Ph D                           

Tel. +49 6221 424982
FAX +49 6221 424962

Summary

Parvoviruses are known for their natural oncotropism and oncolytic activities. This, together with the low pathogenicity for humans and the capability to specifically kill cancer cells irrespective of their acquired resistance towards conventional therapeutics, led to a first phase I/IIa clinical trial treating glioblastoma multiforma with self-propagating H-1 parvovirus. My Group studies parvovirus host cell interactions on the molecular level to pursue two major goals in the fight against cancer:
a.    Identification of potential marker/functions promoting neoplastic transformation. In naturally permissive cells, rodent PVs stimulate the PDK1/PKB/PKC signalling cascade to counteract stress responses and to ensure productive infection and spreading. Upon host switch to human cells, PVs strongly depend on this signalling pathway to be activated. This makes cancer cells ideal hosts for PV. Complementarities between PV interference in natural host cells and factors promoting permissiveness in human cancer cells are thought to serve as markers for neoplastic transformation.
b.    By studying molecular mechanisms of PV induced oncolysis we intend to design novel PV-derived oncotoxins to arm heterologous viruses for Virotherapy of Cancer.
c.    To generate and validate a portfolio of self-propagating oncolytic (parvo)viruses for a virotherapy of cancer aiming to provide a panel of therapeutics for patient-tailored.

Current and future projects

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PV Interference with intracellular signalling cascades
Regulation of the PV non-structural protein NS1 is strongly dependent on the PDK1/PKC/PKB signalling cascade. Conversely PVs, particularly MVM was shown to interfere with this pathway, leading to activation of PDK1 and its downstream targets PKCλ the short-lived PKCη and PKB/Akt1 (Lachmann et al., 2008). In addition we found that ERM family proteins (mediators between actin cytoskeleton and membranes) play essential roles in the virus cycle. Particularly radixin, in conjunction with PKCη was shown to modulate NS1 and capsid phosphorylation (Nüesch et al., 2009). Recently, we were able to show that Rdx/PKCη-complex targets PDK1 for phosphorylation and activation not only in MVM-infected A9 cells but constitutes an internal loop-back activation mechanisms in highly aggressive cancer cells such as glioblastoma multiforma, rendering PDK1 activity independent of its cofactor PIP3 and, hence growth factor signalling through PI3-kinase (Bär et al., submitted). Currently we are determining whether Rdx/PKCη-mediated PDK1 phosphorylation could serve as a tumor promoting/prognostic marker in various cancer entities (Patent application). Besides stimulation of NS1-regulating kinases, activation of the PDK1/PKB signalling cascade is thought to counteract premature cell death through cellular anti-viral and stress responses eventually leading to translational shut-off and premature cell death prior to progeny production and spreading. Future research aims to identify additional PV targets involved in this process co-inciding with permissiveness in human cancer cell lines. Such interfaces will then be evaluated as potential tumor promoting/prognostic markers in cancer tissues. 

Cytotoxicity, Vesicular egress of progeny virions and oncolytic activity

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In past, most oncotoxic/oncolytic functions could be assigned to the large parvoviral multifunctional NS1 protein. This regulatory protein targets a multitude of cellular components and pathways leading to cell death independent of previously acquired resistance towards known death inducers such as cisplatin or TRAIL (reviewed in Nüesch et al., 2012, Nüesch and Rommelaere, 2014). A major oncotoxic function of NS1 was identified as a complex with the catalytic subunit of casein kinase II (CKIIα) targeting a variety of illegitimate substrates with the result of cytoskeleton collapse and necrosis (Nüesch et al., 2005; Nüesch and Rommelaere, 2006). This oncotoxicity could be mimicked by composing CKIIα (binding) with the NS1-targeting domain in novel polypeptides leading to the specific death of permissive cells (Nüesch and Rommelaere, 2007). Such toxins might serve to arm heterologous oncolytic viruses in order to improve virotherapy of cancer.
Egress of progeny virions from the nuclear periphery to the plasma membrane through ER and Golgi is a regulated process involving COPII vesicles (Bär et al., 2008). This pathway is not only needed to induce post-assembly modification to mature progeny particles, but also for the coordinated release of virions by induction of cytolysis (Bär et al., 2013). Ongoing research focuses on the mechanisms of vesicle loading and assembly, as well the release at the plasma membrane through induction of cytolysis. This is of interest, since intracellular tumor-associated antigens (TAAs) are transported as “co-cargoes” to the cellular periphery. Through this process PV-infected tumor cells could be unmasked to the host immune system and may represent a new facet of PV-induced immunogenic death. This further underlines the importance to use propagation-competent PVs for a virotherapy of cancer.

(Parvo)Virotherapy of Cancer

New H-1PV variants with enhanced anti-tumor activities: We isolated propagation-competent H-1PV variants through serial passaging in human glioma(GBM)-derived cell lines. These isolates surpassed the “wild type” strain for multiplication and spread not only in GBM, but also human melanoma cell lines. All isolates shared a deletion similar but not identical to the one of Del H-1PV characterized in C. Dinsart’s Group, suggesting this region in H-1PV to be a hotspot for host range adaptation. Besides these NS deletions we observed coding changes in the capsids improving infectivity of the GBM-variants in human cancer cell(line)s. Reference to the crystal structure of H-1PV could provide information about the impact of such changes for the viral host range. Propagation-competent PV variants are of particular interest, since they are expected to achieve multiple rounds of infection in human tumors. The impact of distinct variations for permissive infections in human cancer cells is currently investigated on the molecular level. For the future we aim to establish methods to measure PV-permissiveness and efficacy in patient-derived tumor slices. This technology is essential to determine the suitability of parvo treatment and could enable to assess the most efficient PV-isolate of our portfolio. In fact, it constitutes a first step towords patient-tailored medicine.
Arming heterologous viral vectors with PV-derived oncotoxins. Aside of generating a portfolio of propagation competent PVs, future investigations aim to design new oncotoxins mimicking PV-interference with cancer cell growth. This will include the potential induction of an anti-tumor immune response through exposure of intracellular TAAs together with PV proteins at the cell surface. To achieve this goal, additional efforts will be made to dissect PV(NS1) toxicity on the molecular level. In collaboration with Lars Redecke (University Hamburg), we currently pursue the determination of the crystal structre of NS1 to identify new interfaces with cellular components, which will then be the subject for further in cell analyses using recombinant Adeno-associated virus vector transfer for their immediate impact, later on to arm other oncolytic viruses considered as cancer therapeutics.

Significant Accomplishments

•    Identification of activated PDK1 as a mediator of PV (onco) tropism
•    Identification of phosphoPDK1 as a diagnostic/prognostic marker in human glioma
•    Generation of new oncotoxins on the base of NS1 induced cytolysis
•    Characterization of vesicular egress of PV through ER and Golgi
•    Generation of H-1PV variants surpassing the wild type virus in anti-cancer activity

External Collaborations

Peter Tatterall, Yale University, New Haven (USA): “Parvovirus small non-structural protein functioning”

Claytus Davis, Ben Gurion University of the Negev, Beer Sheva (Israel): “PV interaction with the host innate immune and stress responses”

Lars Redecke, University of Hamburg, Hamburg (Germany): “Determination of the NS1 crystal structure”

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