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Parvovirus Host Cell Interactions

Contact:

Jürg P.F. Nüesch, PhD                                                                               

Tel. +49 6221 424982

FAX +49 6221 424971

Email

 

Summary

© dkfz.de

Protoparvoviruses were originally detected as contaminants in human tumor cell lines grown in nude rats and nude mice. This became the basis for their natural oncotropism and oncolytic activities. One of these agents, H-1PV is validated as a potential anti-cancer agent in clinical trials of patients suffering of currently untreatable cancers. My Group is interested to study parvovirus host cell interactions on the molecular level to fund the basis for the improvement of safety and efficacy of these viruses in cancer therapy. For this we are addressing the following issues:
  1. 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.
  2. By studying molecular mechanisms of PV induced egress and oncolysis we intend to design novel PV-derived oncotoxins to arm heterologous viruses for Virotherapy of Cancer and to improve the induction of a bystander effect by inducing an anti-tumor immune response.

To generate a portfolio of self-propagating oncolytic (parvo)viruses for a virotherapy of cancer aiming to provide a panel of therapeutics for patient-tailored and to establish pre-treatment diagnostics.

Current and future projects

© dkfz.de

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 [1]. In addition we found that ERM family proteins (mediators between actin cytoskeleton and membranes) play essential roles in the virus cycle, particularly at late stages of infection ensuring virus egress and cytolysis. Thus, radixin, in conjunction with PKCη was shown to modulate NS1 and capsid phosphorylation [2]. Moreover, 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 [3]. Currently we are aiming to determine PV-induced activation of the PDK1-downstream survival-kinase PKB/Akt1 and its action on PV-late functioning for progeny particle egress and spreading.

Cytotoxicity, Vesicular egress of progeny virions and oncolytic activity

In the past, most oncotoxic/oncolytic functions were 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 [4,5]). 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 [6,7]. 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 [8]. However, although NS1 is capable to kill target tumor cells alone, this is a rather inefficient process, which is significantly improved in combination with other viral polypeptides, including VP1 and SAT (Bretscher et al., in prep.)

During PV-infection oncolysis appears to be a tightly regulated process associated with the transport of progeny virions from the nuclear periphery to the plasma membrane. This pathway through ER and Golgi involves COPII vesicles and is necessary to introduce post-assembly modification leading to the maturation of progeny particles and induction of cell lysis ([9,10]). Besides progeny particles, intracellular vesicles contain viral and cellular polypeptides, which become exposed at the cell surface during the budding process (Steinfass et al., in prep.) and might be involved in cell lysis (Bretscher et al., in preparation). This is of interest, since intracellular tumor-associated antigens (TAAs) are transported as “co-cargoes” to the cellular periphery and might serve, together with viral antigens as DAMPs and PAMPs in order to attract the host immune system after oncolysis. In collaboration with Prof. Dr. Jörg Huwyler (Pharmaceutical Institute University Basel) these findings will be applied for the generation of PV-derived oncotoxins, allowing to mimic PV-induced oncolysis/induction of anti-tumor immune responses, independent of viral infections [11]. In order to obtain more insight into the mechanisms of PV-induced immune-activation, we are in close cooperation with Prof. Dr. Nathalia Giese, Uni HD [12], Dr. Laurent Daeffler (INSERM) and Prof. Markus Möhler, Uni Mainz to improve PV-associated cancer therapies through anti-tumor immune-activation.

(Parvo)Virotherapy of Cancer

© dkfz.de

Improving Efficacy:  To improve a virotherapy of cancer with self-propagating oncolytic protoparvoviruses, we are analysing, besides H-1PV and variants thereof, the properties of naturally occurring isolates of other potential oncolytic PVs, including LuIII, TVX, H3, X14, and KRV. This will not only allow to characterize the genetic drift of the different virus entities (Thomas et al., in prep), but also to identify new variants with enhanced tumor-suppressive potential as well as candidates targeting so far resistant tumor entities. Moreover, by increasing the portfolio of oncolytic agents, it will be possible to adopt to the needs of individual patients and to escape neutralization in order to allow multiple treatments. To allow successful characterization of these variants, we generate replication-competent molecular clones and a panel of monoclonal antibodies in collaboration with the in house core-facility for mAbs to monitor infections and efficacy in various cancer cell models [13]. Finally, we intend to establish pre- and post-treatment diagnostics to pave the way for future clinical trials with oncolytic PVs.  

Tackling Safety Issues: Recently, genetic screens have identified human Protoparvoviruses, close relatives to the rodent species currently under evaluation for cancer therapies. So far, little is known about these viruses. However, we might obtain valuable information from these potential human pathogens about their life cycle and persistence in humans. Investigations about the properties of these viruses will be achieved in collaboration with Prof. Dr. Maria Söderlund-Venermo (University of Helsinki), allowing us to identify potential pitfalls, but also new avenues for targeting specific diseased tissues.

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
  • Establishment of replication-competent molecular clones for KRV, H3, TVX, and X14.

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