MOLECULAR PATHWAYS: RODENT PARVOVIRUSES: …...Oncolytic parvoviruses Rodent parvoviruses (PV) are...

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MOLECULAR PATHWAYS: RODENT PARVOVIRUSES: MECHANISMS OF ONCOLYSIS AND PROSPECTS FOR CLINICAL CANCER TREATMENT

Jürg P.F. Nüesch, Jeannine Lacroix, Antonio Marchini, and Jean Rommelaere

Authors’ Affiliation: Program „Infection and Cancer“, Division F010, German Cancer

Research Center (DKFZ), Heidelberg, Germany.

Corresponding author: Jean Rommelaere, Program „Infection and Cancer“, Abt. F010,

Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 242, D-69120 Heidelberg,

Germany. Phone: +(49) 6221 424960 ; Fax: +(49) 6221 424962 ; E-mail:

[email protected]

Running Title: Parvovirus-tumor cell interplay

Key Words: rodent parvovirus, oncotropism, oncolysis, cancer virotherapy

Disclosure of Potential Conflicts of Interest: J. Rommelaere and J. Lacroix have

commercial research grants from Oryx GmbH & Co.KG. No potential conflicts of interest

were disclosed by other authors

Research. on August 9, 2020. © 2012 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on May 7, 2012; DOI: 10.1158/1078-0432.CCR-11-2325

http://clincancerres.aacrjournals.org/

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Abstract

Rodent parvoviruses (PVs) are recognized for their intrinsic oncotropism and oncolytic

activity contributing to their natural oncosuppressive effects. While PV uptake occurs in most

host cells, some of the subsequent steps leading to expression and amplification of the viral

genome and production of progeny particles are upregulated in malignantly transformed cells.

By usurping cellular processes such as DNA replication, DNA damage response and gene

expression, and/or by interfering with cellular signaling cascades involved in cytoskeleton

dynamics, vesicular integrity, cell survival and death, PVs can induce cytostasis and

cytotoxicity. While productive PV infections normally culminate in cytolysis, virus spread to

neighboring cells and secondary rounds of infection, even abortive infection or the sole

expression of the PV nonstructural protein NS1 is sufficient to cause significant tumor cell

death, either directly or indirectly (through activation of host immune responses). The present

review aims to highlight the molecular pathways involved in tumor cell targeting by PVs and

in PV-induced cell death. It concludes with a discussion of the relevance of these pathways to

the application of PVs in cancer therapy, linking basic knowledge of PV-host cell interactions

to preclinical assessment of PV oncosuppression.




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Background

Replication-competent oncolytic viruses

Over the last three decades, clinical research conducted with a view to optimizing multimodal

therapeutic strategies has led to increasing the 5-year survival rate of cancer patients by about

20% (from 49% survival for the 1975-1977 diagnosis period, to 68% survival for the period

2001-2007) (SEER Cancer Statistics Review 1975-2008). Yet to further improve the outcome

of cancer treatment, new therapeutic approaches are needed. In this perspective, replication-

competent oncolytic viruses (OV) appear to be promising. OVs are characterized by their

ability to infect, propagate in and kill tumor cells while sparing non-transformed cells, and

thereby prime an antitumor immune response. Some of these agents (e.g. vesicular stomatitis

virus, newcastle disease virus, reovirus, parvovirus) show inherent preference for replication

in cancer cells due to their dependence on neoplasia-associated features, including deficiency

of interferon response and aberrant activation of permissiveness factors. The oncotropism of

other OVs (e.g. adenovirus, vaccinia virus, measles virus, herpes simplex virus) has been

engineered by modifying/deleting viral genes or regulatory elements so as to make virus

expression/replication/entry conditional on the malignant phenotype. Furthermore, the

oncosuppressive potential of OVs can be boosted by arming them with transgenes/genetic

motifs that confer an additional therapeutic effector function (e.g. immune-stimulation,

prodrug activation) to recombinant viruses (1).

Oncolytic parvoviruses

Rodent parvoviruses (PV) are of particular interest in this regard, due to their natural

oncotropism, lack of pre-existing antiviral immunity in most of the human population, and

excellent safety profile (2). This suppressive activity of PVs against various rodent and human

cancers has been documented in both in vitro systems and animal models (3, 4). PVs

distinguish themselves by their dual oncolytic and immunostimulating activities, broad range

of target tumors, ability to circumvent tumor cell resistance to conventional death inducers

and suitability for both local and systemic applications. Furthermore, their cytopathic effects

can be recapitulated in vitro through the expression of a single parvoviral product, the non-

structural protein NS1, which functions as an oncogenic transformation-dependent toxin (2).




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PVs belong to the genus Parvovirus of the family Parvoviridae. Among the PVs studied for

their anticancer properties, two species are particularly characterized the minute virus of mice

(MVM) and H-1 PV whose natural hosts are mice and rats, respectively. PVs are non-

enveloped icosahedral protein particles 24 nm in diameter containing a single-stranded linear

DNA genome of about 5.1 kb. Their coding capacity is limited to the production of two

capsid proteins (VP1 and VP2) and five nonstructural regulatory polypeptides (NS1, three

NS2 splice variants and SAT). Expression of the NS1/NS2 genes is controlled by the early P4

promoter; that of the VP1/VP2 and SAT gene is programmed by the late (NS1-inducible) P38

promoter (5).

The restricted number (and size) of parvoviral proteins is compensated for, to a certain extent

by the multifunctionality of some of these products. This is exemplified by the NS1 protein

which exerts various functions ranging from viral DNA amplification and transcription

regulation to cytotoxicity. These activities are timely regulated through NS1 post-translational

modification and/or subcellular distribution. In particular, the phosphorylation of distinct NS1

domains controls both enzymatic (ATPase, helicase) and non-enzymatic (DNA and protein

binding) functions of the viral product, and varies during infection (6). Besides being

intrinsically multifunctional, NS1 interacts with a number of cellular partner proteins which

further broaden the range of NS1-mediated activities (see below).

These features make PV strongly dependent on host cell factors. More importantly, PVs

require S-phase factors to start replication and viral protein production. Unlike tumor viruses,

PVs are unable to promote the progression of quiescent cells into S-phase, despite their ability

to enter quiescent cells (5). This dependency restricts PV infections to rapidly proliferating

tissues, and together with other parameters (see Fig. 1), makes tumor cells preferential targets

for PV replication and cytopathic effects.

Cell permissiveness and PV oncotropism

As depicted in Fig. 1, PV oncotropism is due in part to viral dependence on cellular

replication and transcription factors whose synthesis, activity, or both correlate with cell

cycling and oncogenic transformation. Furthermore, the interplay between PVs and

transformation-sensitive cell proteins raises the possibility that transformation might have an

impact on late events of the PV life cycle, e.g. cytopathic effects and virus release. The

presence of these factors is not sufficient, however, to ensure virus propagation. A restriction




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can occur, for instance, at the level of delivery of incoming viral genomes to the nucleus, a

prerequisite to their conversion to double-stranded transcription templates (5). This may

account for the heterogeneity of tumor cells as regards permissiveness towards PV replication.

It represents a target for optimization.

An additional barrier to the PV life cycle is created by the type-I-interferon-mediated antiviral

response, of which PVs were recently shown to be both triggers and targets (7, 8). PV

oncotropism might depend on whether many tumor cells become deficient in the IFN

response or on the capability of PVs to counteract this antiviral defense mechanism in

transformed cells.

In addition to regulation of their synthesis, PV proteins are subject to functional modulations

resulting from posttranslational modifications and alterations of subcellular distribution (6).

These modulations may also contribute to oncotropism, as some of the cellular effector

proteins involved, e.g. NS1-activating PRKCH/PKCη (9), are overexpressed and/or altered in

cancer cells (10). Cell-transformation-dependent modifications of the PV protein NS1 can

thus be considered responsible for its specific toxicity in oncogene-transformed cells at

concentrations that are innocuous to non-transformed cells (3).

Analysis of PV interactions with cell proteins (Fig. 2) has revealed a striking interplay

between PVs and cellular components of various metabolic pathways related to gene

expression or DNA replication and repair. Some of these components are direct binding

partners of PV proteins, as exemplified by the formation of complexes between NS1 and

cellular transcription factors (TBP, TFIIA, SP1/3) or replication factors (RPA) (11-13).

Others are known effectors and/or targets of the PV life-cycle, which are not found in

complexes with PV proteins. Interestingly, a number of these physical and/or functional

partners of PVs get together in specialized areas of infected cells. In particular, many

constituents of the cell DNA replication machinery (RPA, POLA1/POLα, PCNA, RF-C) and

DNA damage response (RPA-P32, γH2AX, NBS1-P, ATM) are recruited to subnuclear PV

replication centers called APAR-bodies (14-16). This redistribution is expected to promote

PV multiplication and contributes to host DNA synthesis shut-off. These various cell factors

are established or potential actors in the PV life cycle, or may contribute to reshaping the

intracellular environment to make it adequate for virus replication and spread. This cell-

conditioning effect is illustrated by the phosphorylation chain of cellular protein kinases that




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is triggered by PV infection and leads to PDK-1 driven activation of PKCλ and PKCη (17).

As stated above, both members of the PKC family are essential for modifying the viral NS1

protein and enabling it to exert its replicative functions. This phosphorylation cascade forms a

positive feedback loop which is thought to be initiated by the PV-induced nuclear export of

PKCλ, as a possible result of the interaction of the viral product NS2 with the cellular

transporter XPO1/CRM1 (Fig. 1) (6).

It is noteworthy that the major metabolic pathways interconnecting with PVs are subject to

modulation through malignant transformation. In particular, overexpression of c-MYC and/or

activated Ha-RAS renders normally resistant cells permissive towards PV propagation and

NS1-induced killing (18). Both oncoproteins are known to have profound effects on the

intracellular environment by dysregulating molecular pathways that are also involved in cell

permissiveness towards PV infection. Factors controlling transcription (E2F, ETS,

ATF/CREB, NF-YA) (19-21) or replication (CCNA1/cyclinA, DNA damage response

effectors) (14-16, 22), and protein kinases (PRKCH/PKCη, PKB/AKT1) [(9); Nüesch

unpublished] are among the cell proteins acting as modulators of the PV life cycle, belonging

to this signaling network, and whose expression or activation is affected by malignant

transformation.

Cell disturbances and PV oncolysis

PV infection of permissive cells leads to shut-off of host macromolecule syntheses, notably

causing early inhibition of cell DNA replication (5, 23), due to the sequestration of

components of the cell DNA replication machinery in viral replication centers (see above).

Stalled replication forks (24) and ssDNA genomes of PV-related adeno-associated viruses

(25) are known to activate the DNA damage response (DDR). Another DDR-activating

mechanism triggered by PVs is NS1-induced production of reactive oxygen species (ROS)

causing DNA damage (26). PV-induced DDR signaling is mediated by activation of ATM but

not ATR, is characterized by phosphorylation of H2AX, NBS1, RPA32, CHK2, and P53, and

results in accumulation of DNA repair proteins in viral replication centers (15, 16).

Consequently, cell cycle arrest after PV infection is observed at the S/G2 transition or G2

phase, depending on the virus and cell type (26, 27). It is associated with upregulation of cell-

cycle control proteins such as CDK1, CCNB1/cyclinB1, CDKN1A/p21/Cip1,

CDKN1B/p27/Kip1, and RLB2/p130 (26, 28, 29). Arrested cells are proposed to offer a more




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favorable terrain for PV replication. Interestingly, expression of the NS1 protein is alone

sufficient to induce cell cycle arrest (26, 28, 29), in keeping with the fact that NS1 is the

major effector of virus cytotoxicity.

Virus-induced cytotoxicity can involve mechanisms ranging from cytostasis (eventually

leading to suicide of the infected/transfected cell) to direct induction of cell death programs.

This is apparent from analysis of the PV-host cell interactome, revealing the interplay of PVs

with cellular stress responses and with regulation of the cell cycle and death (Fig. 2).

Whatever the nature of the initiating event, cell death and eventual lysis are the final outcome

when a PV infects permissive malignant cells. Depending on the cell type and metabolic

status, PV-infected cells can die through different mechanisms (see Fig. 1). H-1PV is reported

to induce apoptotic cell death in various cancer cell lines (26, 30-32), and this is reflected in

the statistically very significant interaction of PVs with components of the apoptosis-

regulating pathway (Fig. 2). Interestingly, NS1 expression is sufficient to trigger caspase-9-

and caspase-3-mediated apoptosis via oxidative stress and DNA damage (26). Alternatively,

PV infection can induce a lysosomal type of death involving multiplication and

permeabilization of acidic vesicles and repression of cathepsin inhibitors, resulting in the

cytosolic accumulation of functional cathepsins to levels the cell cannot tolerate. It is

noteworthy that by mobilizing this pathway, PVs can circumvent the mechanism of resistance

to apoptosis developed by many tumor cells, and thereby kill cancer cells that dodge pro-

apoptotic treatments (33).

The mechanisms by which PVs affect cell growth and cell death pathways are still elusive but

likely to be multiple, including viral usurpation of, and interference with the cellular gene

expression and DNA replication machineries (Fig. 2). Furthermore, the NS1 protein appears

to exert at least part of its toxic activity in the cytosol. This has been traced back to the

illegitimate phosphorylation of cell substrates by a complex containing NS1 and the catalytic

subunit of a cellular protein kinase (CKIIα in the case of MVM NS1) (34). PV-infected cells

and cells expressing the NS1 protein alone undergo morphological changes that typically lead

to their shrinkage and rounding (35). This has been ascribed to targeting of cytoskeletal

microfilaments. Actin filaments are destroyed through NS1/CKIIα-mediated phosphorylation

and activation of the severing protein gelsolin in conjunction with repression of the

polymerization protein N-WASP (36, 37). This may have a greater impact on cancer cells,

which lack the most rigid actin fibers. Filamentous tropomyosin is also a direct target of the




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NS1/CKIIα complex. Differential NS1/CKIIα-driven phosphorylation of tropomyosins 2 and

5 leads to perinuclear restructuring and decay of these filaments (38). These alterations are

likely to be especially deleterious to cancer cells, where tropomyosin 1 is underrepresented

(39). Interestingly, artificial polypeptides combining the targeting domain of NS1 with CKIIα

(or a binding site thereof) have been found to cause the death of transformed cells (38). This

has led us to propose that the toxic activity of the NS1 protein depends at least partly on its

ability to act as a cytosolic adaptor allowing a cellular kinase or kinases to phosphorylate

illegitimate substrates.

It is also worth mentioning that timely induction of cell death is necessary after PV infection

in order to allow virus multiplication prior to cell collapse. There is evidence suggesting that

premature death of PV-infected cells is prevented by NS1-mediated activation of the PDK-

1/PKB survival pathway [(17); Bär et al., in prep], providing enough time for the formation of

progeny virions and their eventual release through active vesicular transport to the cell

surface. This transport appears important for the maturation of progeny particles and the

eventual lysis of infected tumor cells (Bär et al., in prep).

Direct killing of tumor cells is not the only way oncolytic viruses can contribute to cancer

suppression. That they might pass the job on to the immune system is actually expected, since

a productive lytic viral infection of cancer cells should promote the display or release of both

tumor-associated antigens and immunostimulating viral or cellular molecular patterns.

Adoptive transfer, rechallenge, and immunodepletion experiments indicate that the host

immune system takes part in PV-mediated oncosuppression (40, 41). The active role of PVs

in inducing this anti-cancer immune response is further supported by the observation that PV

infection of tumor cells enhances their ability to serve as an autologous vaccine (41, 42) and

to stimulate both natural killer (43) and dendritic (44) cells with which they come into

contact. Interestingly, the vesicular egress of progeny PVs may contribute to exposing

intracellular tumor-associated antigens (37). Altogether, these data point to synergies between

virus- and immune-cell-mediated oncolysis in PV-initiated tumor suppression.




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Clinical-Translation Advances

Implications for cancer parvovirotherapy

As discussed above, both activation of cellular helper functions and defects in cell defense

mechanisms contribute to the permissiveness of cancer cells towards PV replication and

toxicity. In contrast, the mildness of PV attack against normal tissues in adult animals results

in clinically unapparent PV infections (45). This tolerance is assumed also to exist in humans

and is the subject of a current clinical study. This trial involves patients with glioblastoma

multiforma (GBM), the most malignant brain tumors against which standard treatments,

including surgery and radio-chemotherapy, are inefficient. Preclinical evidence of strong

cytotoxic and oncosuppressive effects of H-1PV against glioma cells led to the launch, in

October 2011, of a first phase I/IIa study of H-1PV in patients with recurrent GBM (ParvOryx

01 – ClincialTrials.govIdentifier:NCT01301430). ParvOryx 01 is an open, non-controlled,

dose escalation, single center trial (46). H-1PV is administered before resection by either

intratumoral or intravenous route, and again into the walls of the tumor cavity during tumor

removal. The objectives of the trial are related primarily to local/systemic safety and

tolerability, and secondarily to proof of concept.

Activation of (proto)oncogenes leads to changes in the intracellular environment which boost

the parvoviral life cycle, as documented for c-myc (18). Overexpression of MYC drives

malignant transformation in Burkitt’s lymphoma, medulloblastoma, and a large proportion of

breast cancers with poor prognosis (47). In GBM stem cells, MYC is involved in regulating

malignant progenitor cell differentiation, self-renewal, and tumorigenic potential (48).

Parvovirus H-1PV efficiently suppresses these MYC-dependent tumors in preclinical models

[(49, 50); Lacroix, in prep]. In response to PV infection, MYC expression is repressed in

various cancer cell lines of different origins [(30, 51); Lacroix et al.,in prep]. These facts

strongly suggest that cancers associated with MYC alterations are good targets for PV-

induced oncolysis, and argue in favor of evaluating MYC as a predictive marker associated

with tumor responsiveness to H-1PV treatment. In the absence of maternal immunity, PVs can

infect rodent embryos, where they propagate in a variety of cell types, while maternal tissues

remain unaffected (45). This has prompted us to hypothesize that malignant cells having

embryonic features may show similar permissiveness. Accordingly, neuroblastoma and




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pediatric medulloblastoma cell lines prove to be very susceptible to H-1PV killing in vitro,

and we have observed suppression of xenotransplants in animal models [(32); Lacroix, in

prep]. Besides these hypothesis-based suggestions, needs-driven consideration of cancers with

desolate prognosis should also influence the choice of tumor targets for subsequent PV

clinical trials. In this regard, it is noteworthy that H-1PV synergizes with gemcitabine to kill

pancreatic carcinoma cells, and improves gemcitabine-based therapy of pancreatic tumors in

animal models (52)

Within a given cancer entity, the heterogeneity of tumors calls for individualized treatments

based on prior identification of patients most likely to respond. It is therefore important to

discover biomarkers that can serve as predictors of individual tumor sensitivity to PV

infection. For PDAC, a first candidate is the transcription factor SMAD4, which controls P4

promoter activity and hence nonstructural protein expression and toxicity (53). Other

candidates should emerge from “omic” approaches allowing the correlative analysis of PV

sensitivity and interactome status in tumors from large cohorts of patients.

Future developments

Although PVs can cure cancers in animals and open new prospects for cancer therapy in

humans, optimizing their oncosuppressive capacity is likely required in order to reach

effectiveness in humans. The mere fact that some PVs have been isolated from human tumor

xenotransplants indicates that their oncolytic activity is not always sufficient to arrest tumor

growth and induce remission. This may be due to limitations on PV infectiousness,

cytotoxicity, spread, and/or immunostimulation. A portfolio of second-generation therapeutics

based on replication-competent PVs is under development to circumvent these limitations.

Different complementary strategies might be used to achieve this goal. Virus variants with an

enhanced ability to replicate lytically and spread in tumor cell populations are being isolated

through natural selection or site-directed mutagenesis (54, 55). To minimize PV sequestration

by normal tissues, PV capsids are first detargeted and then retargeted to cancer cells through

the display of specific peptide ligands (56). The ability of PVs to act as adjuvants to boost the

host anti-tumor response can also be increased by arming them with immunostimulatory

molecular patterns (42). Furthermore, PVs can be combined with ionizing radiation (57), the

cytostatic drug gemcitabine (52), the cytokine interferon γ (40) or oncolytic reovirus (58) to




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achieve additive or even synergistic therapeutic effects. Indeed, the non-PV component of

these combination treatments can be administered so as to minimize interference with PV

replication and instead to potentiate PV oncolytic or immunomodulating activities. These

various improvements of replicative PV-based treatments should pave the way for future

clinical testing of anticancer efficacy, after the hoped-for confirmation of H-1PV safety in the

current clinical trial.




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37. Bar S, Daeffler L, Rommelaere J, Nuesch JP. Vesicular egress of non-enveloped lytic parvoviruses depends on gelsolin functioning. PLoS Pathogens. 2008;4:e1000126. 38. Nuesch JP, Rommelaere J. A viral adaptor protein modulating casein kinase II activity induces cytopathic effects in permissive cells. Proceedings of the National Academy of Sciences of the United States of America. 2007;104:12482-7. 39. Bhattacharya B, Prasad GL, Valverius EM, Salomon DS, Cooper HL. Tropomyosins of human mammary epithelial cells: consistent defects of expression in mammary carcinoma cell lines. Cancer Research. 1990;50:2105-12. 40. Grekova S, Aprahamian M, Giese N, Schmitt S, Giese T, Falk CS, et al. Immune cells participate in the oncosuppressive activity of parvovirus H-1PV and are activated as a result of their abortive infection with this agent. Cancer Biology & Therapy. 2011;10:1280-9. 41. Raykov Z, Grekova S, Galabov AS, Balboni G, Koch U, Aprahamian M, et al. Combined oncolytic and vaccination activities of parvovirus H-1 in a metastatic tumor model. Oncology Reports. 2007;17:1493-9. 42. Raykov Z, Grekova S, Leuchs B, Aprahamian M, Rommelaere J. Arming parvoviruses with CpG motifs to improve their oncosuppressive capacity. International Journal of Cancer. 2008;122:2880-4. 43. Bhat R, Dempe S, Dinsart C, Rommelaere J. Enhancement of NK cell antitumor responses using an oncolytic parvovirus. International Journal of Cancer. 2011;128:908-19. 44. Moehler MH, Zeidler M, Wilsberg V, Cornelis JJ, Woelfel T, Rommelaere J, et al. Parvovirus H-1-induced tumor cell death enhances human immune response in vitro via increased phagocytosis, maturation, and cross-presentation by dendritic cells. Human Gene Therapy. 2005;16:996-1005. 45. Itah R, Tal J, Davis C. Host cell specificity of minute virus of mice in the developing mouse embryo. Journal of Virology. 2004;78:9474-86. 46. Geletneky K, Huesing J, Rommelaere J, Schlehofer JR, Dahm M, Krebs O, et al. Phase I/IIa study of intratumoral/intracerebral or intravenous/intracerebral administration of Parvovirus H-1 (ParvOryx) in patients with progressive primary or recurrent glioblastoma multiforme: ParvOryx01 protocol. Biomed Central Cancer. 2012;12:99. 47. Ben-Porath I, Thomson MW, Carey VJ, Ge R, Bell GW, Regev A, et al. An embryonic stem cell-like gene expression signature in poorly differentiated aggressive human tumors. Nature Genetics. 2008;40:499-507. 48. Zheng H, Ying H, Yan H, Kimmelman AC, Hiller DJ, Chen AJ, et al. p53 and Pten control neural and glioma stem/progenitor cell renewal and differentiation. Nature. 2008;455:1129-33. 49. Angelova AL, Aprahamian M, Balboni G, Delecluse HJ, Feederle R, Kiprianova I, et al. Oncolytic rat parvovirus H-1PV, a candidate for the treatment of human lymphoma: In vitro and in vivo studies. Molecular Therapy. 2009;17:1164-72. 50. Van Pachterbeke C, Tuynder M, Brandenburger A, Leclercq G, Borras M, Rommelaere J. Varying sensitivity of human mammary carcinoma cells to the toxic effect of parvovirus H-1. European Journal of Cancer. 1997;33:1648-53. 51. Li J, Werner E, Hergenhahn M, Poirey R, Luo Z, Rommelaere J, et al. Expression profiling of human hepatoma cells reveals global repression of genes involved in cell proliferation, growth, and apoptosis upon infection with parvovirus H-1. Journal of Virology. 2005;79:2274-86. 52. Angelova AL, Aprahamian M, Grekova SP, Hajri A, Leuchs B, Giese NA, et al. Improvement of gemcitabine-based therapy of pancreatic carcinoma by means of oncolytic parvovirus H-1PV. Clinical Cancer Research. 2009;15:511-9. 53. Dempe S, Stroh-Dege AY, Schwarz E, Rommelaere J, Dinsart C. SMAD4: a predictive marker of PDAC cell permissiveness for oncolytic infection with parvovirus H-1PV. International Journal of Cancer. 2010;126:2914-27. 54. Faisst S, Faisst SR, Dupressoir T, Plaza S, Pujol A, Jauniaux JC, et al. Isolation of a fully infectious variant of parvovirus H-1 supplanting the standard strain in human cells. Journal of Virology. 1995;69:4538-43.




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55. Daeffler L, Horlein R, Rommelaere J, Nuesch JP. Modulation of minute virus of mice cytotoxic activities through site-directed mutagenesis within the NS coding region. Journal of Virology. 2003;77:12466-78. 56. Allaume X, Andaloussi NE, Leuchs B, Bonifati S, Kulkarni A, Marttila T, et al. Retargeting of rat parvovirus H-1PV to cancer cells through genetic engineering of the viral capsid. Journal of Virology. 2012. 57. Geletneky K, Hartkopf AD, Krempien R, Rommelaere J, Schlehofer JR. Improved killing of human high-grade glioma cells by combining ionizing radiation with oncolytic parvovirus H-1 infection. Journal of Biomedicine & Biotechnology. 2010;2010:350748. 58. Alkassar M, Gartner B, Roemer K, Graesser F, Rommelaere J, Kaestner L, et al. The combined effects of oncolytic reovirus plus Newcastle disease virus and reovirus plus parvovirus on U87 and U373 cells in vitro and in vivo. Journal of Neuro-Oncology. 2011;104:715-27. 59. Szklarczyk D, Franceschini A, Kuhn M, Simonovic M, Roth A, Minguez P, et al. The STRING database in 2011: functional interaction networks of proteins, globally integrated and scored. Nucleic Acids Research. 2011;39:D561-8. 60. Huang da W, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nature Protocols. 2009;4:44-57.




16

Figure legends

Figure 1. Parvovirus-induced cell disturbances. Viral genome replication and gene

expression take place in the nucleus and depend strictly on S-phase-associated cellular factors.

Conversion of the single-stranded viral DNA to a double-stranded transcription template is

achieved by cellular replication factors (RF) under the control of cyclin A {1}. After

conversion, S-phase (E2F) and transformation-sensitive (ATF/CREB, ETS, NF-Y)

transcription factors (TF) activate the parvovirus early promoter P4 controlling expression of

the nonstructural proteins NS1 and NS2 {2}. NS1 activates the late parvovirus promoter P38

programming capsid gene expression, and drives viral DNA amplification as an initiator

protein and helicase (not illustrated in the Figure). NS1 also interferes with cell survival at

many levels. Direct interactions of NS1 with components of the DNA replication (RPA 1-3)

and transcription (TBP, TFIIA, SP1) machineries play a role both in viral DNA replication

and transcription and in deregulating DNA/RNA metabolic processes in infected cells {3, 4}.

Other virus protein-cell protein interactions such as NS1-CKII {5} or NS2-XPO1 {6}

interfere, respectively, with host cell signaling and nuclear export. These events affect the

host cell dramatically by causing oxidative stress, DNA damage, cell cycle arrest,

cytoskeleton structure re-arrangements, mitochondrial membrane depolarization, and/or

lysosome permeabilization. Cell death ensues, and ectopic NS1 expression alone is sufficient

to cause it. Productive PV infections are characterized by virus-induced cytolysis followed by

virus dissemination and subsequent rounds of infection. Besides providing permissiveness

factors, transformed cells fail to mount an efficient anti-PV type I interferon response, which

also contributes to the oncotropism of these agents. The oncoselectivity of PVs is thus

attributed to both features of cancer cells: the presence of positive permissiveness factors and

the inability to antagonize viral infection, as indicated with asterisks in the Figure.

Abbreviations: CKII, casein kinase II alpha; ds, double-stranded; GSN: gelsolin;

GTF2A/TFIIA: general transcription factor 2A; NS1, PV nonstructural protein 1; NS2, PV

nonstructural protein 2; PV, parvovirus; ROS, reactive oxygen species; RPA: replicator

protein A; SP1/3: Sp1/3 transcription factor; TBP; TATA binding protein; RF, replication

factors; TF, transcription factors; TPM2/5: tropomyosin 2/5; VP, PV capsid proteins; XPO1

exportin 1 (CRM1 yeast homolog).




17

Figure 2. Parvovirus-host cell interplay. (A) The PV interactome. Cellular factors involved

in PV propagation and spread and/or subject to modulation upon PV infection are represented

as a network. The interconnections between the different genes are based on direct (physical)

and indirect (functional) protein interactions according to STRING analysis (59). Most of

these factors belong to known cellular pathways, as indicated by color codes. (B) Cellular

metabolic pathways whose targeting by PVs is of highest significance (P-values). Pathway

analysis enrichment was performed using the gene functional classification tool of DAVID

(60). Resulting pathways are marked in different colors referring to the associated proteins

identified under (A). PV impacts on the host cell are indicated on the right.




© 2012 American Association for Cancer Research

Parvoviral particle Cell membrane

NS1

NS1

NS1

NS1

LysosomeMitochondrion

GSN

NS1

TPM2/5

Cytoskeleton

LysisCell membrane

RPA

VPs

P38P4

NS

TBP, GTF2A, SP1/3

3

14

PV-genome

Virus/cell genetranscription

2

XPO1Nuclear trafficking

Expression

DNA replication/repair

Cell DNA

Cell cyclearrest

ROS

Permeabilization Depolarization Rearrangement

DNA damage

PV-ds DNA

Nucleus

NS2

NS1

NS1

NS1

NS1

TF

RF

Other viralcomponents

Virus/cell DNAamplification

Target cellularproteins

5

6CKII

CKII

Antiviral response




© 2012 American Association for Cancer Research

PV-cell interactomeA

B

PIF1

RPA3

RPA1

RPA2

CDKN1B

YWHAQ

PCNA

CCNB1

POLA1

YBX1

GMEB2

CSTB

SMAD1

PDPK1

PRKCI

SP3

SMN1

XPO1

ID1

ATF1

CSNK2A1

SGTANFYA

EZR MSN

RDX

HMGB1

GTF2A1

TPM2

CASP3

CTSB

CASP9

POLD1

H2AFXPRKCH

Involvement in:

Response to DNAdamage stimulus

DNA metabolicprocesses andtranscription

regulation

Regulation ofcell cycle

Regulation ofapoptosis

Dysregulationof transcription

Cell death

Cell cyclearrest

Dysregulationof replication

Activation of cellstress responses

1.00E+00 1.00E-02 1.00E-04 1.00E-06 1.00E-08

(P-value)

Response to DNA damage stimulus

Cellular response to stress

DNA replication

DNA metabolic process

Regulation of cell cycle

Regulation of apoptosis

Regulation of transcription

Pathways analysis and major perturbances for the host cell

Otherpathways

E2F1

CDK1

CD CCNA1

MYC

CREB1 SP1

JUNTBP




Published OnlineFirst May 7, 2012.Clin Cancer Res Jürg P.F. Nüesch, Jeannine Lacroix, Antonio Marchini, et al. PROSPECTS FOR CLINICAL CANCER TREATMENTRODENT PARVOVIRUSES: MECHANISMS OF ONCOLYSIS AND

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