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
Most oncotoxic/oncolytic functions are assigned to the large parvoviral multifunctional protein NS1. 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, such as VP1 and SAT (Bretscher, C Ph D Thesis).
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.). This is of interest, since intracellular danger signals and 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. Indeed, we observed the active exposure of NS1 and VP1 together a variety of cell proteins at the cell surface of infected cells, potentially serving for the induction of an anti-tumor immune-response. 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
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 generated 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.
