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Keywords:

  • poliovirus;
  • oncolytic virus;
  • internal ribosomal entry site;
  • nectin-like molecule 5;
  • CD155;
  • translation;
  • innate antiviral response;
  • interferon;
  • eIF4G

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FUNDING SUPPORT
  5. CONFLICT OF INTEREST DISCLOSURES
  6. REFERENCES

Recently, the century-old idea of targeting cancer with viruses (oncolytic viruses) has come of age, and promise has been documented in early stage and several late-stage clinical trials in a variety of cancers. Although originally prized for their direct tumor cytotoxicity (oncolytic virotherapy), recently, the proinflammatory and immunogenic effects of viral tumor infection (oncolytic immunotherapy) have come into focus. Indeed, a capacity for eliciting broad, sustained antineoplastic effects stemming from combined direct viral cytotoxicity, innate antiviral activation, stromal proinflammatory stimulation, and recruitment of adaptive immune effector responses is the greatest asset of oncolytic viruses. However, it also is the source for enormous mechanistic complexity that must be considered for successful clinical translation. Because of fundamentally different relationships with their hosts (malignant or not), diverse replication strategies, and distinct modes of tumor cytotoxicity/killing, oncolytic viruses should not be referred to collectively. These agents must be evaluated based on their individual merits. In this review, the authors highlight key mechanistic principles of cancer treatment with the polio:rhinovirus chimera PVSRIPO and their implications for oncolytic immunotherapy in the clinic. Cancer 2014;120:3277–3286. © 2014 American Cancer Society.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FUNDING SUPPORT
  5. CONFLICT OF INTEREST DISCLOSURES
  6. REFERENCES

Viruses are difficult vehicles for cancer therapy, because they exert very complex effects on infected tumors that, for the most part, cannot be manipulated. The generic basis for proposed oncolytic virus (OV) platforms—cytotoxicity in select laboratory cell lines and antineoplastic effects in corresponding xenograft models—is insufficient to predict clinical efficacy. This is because the multifaceted, aggregate biologic response to virus infection of cancers rarely lends itself to therapeutic application. Over the past 30 years, a leap in molecular virology has yielded opportunities for genetic manipulation of certain traits, most notably pathogenicity. This made dangerous, lethal pathogens amenable for clinical use in patients. However, other key aspects of the host:virus relationship, in particular those involving the immune system, are difficult to engineer.

Despite this, OVs capable of properly targeting malignancy promise antitumor effects of an extent and range difficult to match with other therapeutic modalities. Viruses can elicit immunogenic forms of tumor cell death, present powerful pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs), induce innate antiviral type 1 interferon (IFNα, IFNβ) responses within the tumor, evoke proinflammatory signals in tumor stroma, and/or recruit adaptive immune responses directed against the infected tumor. However, the demands on a successful OV platform are enormous. Such viruses must: 1) be categorically nonpathogenic, 2) be genetically stable upon intratumoral replication, 3) target/infect notoriously heterogeneous tumors, 4) elicit efficient killing of infected tumor cells, 5) act in the presence of neutralizing antibodies and innate antiviral immune activation, 6) reverse the immune-repressive tumor/stromal microenvironment, and 7) be capable of recruiting immune effector arms against the tumor target. In addition, there are many unique practical and regulatory aspects to consider, such as 1) particle stability (including within the clinically intended delivery apparatus), 2) reliable and efficient manufacture, and 3) public health implications, eg the availability of protective vaccines/antivirals. Combining these features in a single agent is a tremendous challenge. Establishing a proper mechanistic rationale for clinical use of any OV platform requires preclinical research efforts that typically far exceed those for simple, monomechanistic therapeutics.

Descriptions of OV strategies often refer to such agents collectively with common mechanistic implications. Yet the proposed OVs have very little (or nothing) in common with regard to tumor tropism, mechanisms of tumor cell killing, replication strategy, response to innate antiviral immune activation, etc. This makes it difficult to summarize the OV experience in cogent texts or to extrapolate the clinical experience with 1 agent to its peers. Therefore, although we highlight the general aspects of oncolytic virotherapy, we limit the current discussion to our experience with poliovirus and its effects on glioblastoma (GBM). The particular oncolytic agent we are discussing is PVSRIPO, the type 1 (Sabin), live-attenuated poliovirus vaccine that replicates under control of a heterologous internal ribosomal entry site of human rhinovirus type 2 (Fig. 1).[1-4] PVSRIPO is demonstrating early promise in currently accruing phase 1 clinical trials with intratumoral, convection-enhanced delivery in patients with recurrent GBM.[5]

image

Figure 1. The genetic structure of the oncolytic recombinant poliovirus/rhinovirus PVSRIPO is illustrated. Poliovirus has a single-strand, +sense RNA genome of approximately 7400 nucleotide length; overall genome organization is outlined. (A) Poliovirus type 1 (Mahoney), which was isolated in 1941 from stool of healthy children in Cleveland, is the prototype neurovirulent strain. The approximate binding region for the eukaryotic initiation factor 4G (eIF4G)4A:4B translation initiation helicase complex in the internal ribosomal entry site (IRES) is indicated (see Sweeney et al1). UTR indicates untranslated region; ORF, open reading frame; p(A), polyadenylate tract. (B) Serial passage in primate tissue culture/primates yielded the type 1 (Sabin) live-attenuated vaccine (see Sabin and Boulger, 19722). The position of a key attenuating point mutation in the IRES (guanine to adenine substitution at location 480 [G480A]) is indicated (pink circle). (C) PVSRIPO is the type 1 (Sabin) vaccine, which contains a human rhinovirus type 2 IRES (light pink box; a region of major sequence divergence is shaded in dark pink).

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Tumor Targeting and Tropism

Using animal viruses as oncolytic agents in essence requires diverting their natural, ingrained pathogenicity toward a therapeutic effect. Therefore, the outcome of deliberate tumor infection is determined by factors that naturally define the virus:host relationship. The single most important such factor is target cell tropism. We define tropism (gr. τρέπειν [trepein], to turn, to direct, to alter, to change) as the ability of a virus to recognize cell surface features that mediate viral genome entry into the host cell. The term “cell-internal tropism," referring to factors that determine virus susceptibility after host cell entry, is confusing in the context of OVs. Virus entry that does not lead to productive infection can have important biologic consequences, eg, provoking a host response. Therefore, a tight definition is needed to strictly distinguish instances with transfer of viral genetic material into target tumor cells from those without.

For most animal viruses, host cell tropism conveyed by a specific viral receptor(s) shapes their pathogenic properties. Therefore, it is intuitively obvious that an effective OV must have tumor target tropism in patients. Yet the decisive issue of tropism remains vague for many OV platforms. Early clinical investigations included viruses with documented poor cancer tropism, such as adenovirus type 5,[6] which is caused by absent, low, or erratic expression of the coxsackie-adenovirus receptor[7] in human cancers.[8] In some instances, (clinical) cancer tropism of viruses for which the host-cell receptor is unknown was predicted based on virus killing of select laboratory cancer cell lines.[9] This is risky, because viral receptors, many of which are involved in cell adhesion functions, may be induced by the artificial conditions of tissue culture. For example, intercellular adhesion molecule-1 (ICAM-1), a receptor for many human enteroviruses, is induced upon cell adherence/spread on tissue-culture plastic ware.[10] Thus, ex vivo culture of cells from tissues that naturally resist infection by ICAM-1–tropic viruses may make them permissive to infection in vitro. Also, clonal selection of cancer cell lines can yield nonrepresentative frequency of receptor expression. For these reasons, there is no substitute for documenting virus tropism for patients' tumors directly through protein expression studies in tumor tissues.[11]

Obtaining solid, empirical evidence to support viral tropism for a tumor target in the clinic does not suggest that therapeutic efficacy rests on homogeneous targeting/infection of the entire tumor mass. Like any other molecular targets in cancer, viral receptors are unlikely to be expressed homogeneously in all tumor cells, and virus-resistant subpopulations likely exist even if receptor is abundant in the tumor as a whole. This is not of primary concern for devising OV strategies. First, it is inconceivable that all malignant cells can be reached, infected, and eliminated, even with aggressive dosing and invasive administration. Second, the major thrust behind OV strategies in the clinic is their potential for recruitment of host immunogenic responses directed against the tumor. Therefore, tumor tropism is a prerequisite for establishing the conditions for an immunogenic response; it cannot mediate in toto eradication of the tumor mass and its satellites without such a response.

The concept of targeting cancer with poliovirus is largely built on its restrictive tropism for an intriguing cell surface molecule intricately linked to cancer, tumor stroma, and proliferating tumor vasculature.[4] Poliovirus recognizes a single host cell receptor that is necessary and sufficient to convey virus susceptibility.[12] The poliovirus receptor, also known as nectin-like molecule 5 (Necl5) or cluster of differentiation 155 (CD155), is a cell adhesion molecule of the immunoglobulin-like superfamily[13] expressed during embryonic development.[14] The nectin/nectin-like family of genes encode proteins involved in cell:cell adhesions, polarization, differentiation, migration, proliferation, and survival.[13] The physiologic roles of Necl5 are poorly understood, but they involve control over cell movement, eg through participation in leading-edge dynamics in cooperation with αvβ3 integrin and the platelet-derived growth factor receptor[13] (for review, see Takai et al[13]). Accordingly, Necl5 is a major determinant of GBM invasiveness and dissemination in the CNS.[16, 17] Studies in a rat liver injury model suggest that the Necl5 gene is rapidly induced upon acute tissue disruption and is down-regulated once proper cell-to-cell contacts are restored.[18] The same study demonstrated that constitutive, ectopic Necl5 expression upon malignant transformation of hepatocellular carcinoma cells is associated with a loss of Necl5 control.[18] Necl5 is broadly expressed in laboratory GBM cell lines,[4] in primary GBM cell lines grown in culture,[11] in primary (preculture) explant GBM cells obtained from patients at surgery,[19] in GBM biopsy-derived xenotransplants, and in surgically excised GBM tissues.[11] Moreover, widespread ectopic Necl5 expression has been documented in patient tissue samples of many other types of cancers, eg, colorectal carcinoma,[20] lung adenocarcinoma,[21] breast cancers,[22] and melanoma.[23]

An intriguing and possibly relevant factor for therapy is that Necl5 is physiologically expressed in antigen-presenting cells (APCs), ie, macrophages and dendritic cells.[24] Curiously, polio infection of APCs, even with neurovirulent strains, is abortive/nonlethal and does not interfere with their ability to engage T cells (discussed below).[25] PVSRIPO infection of patient peripheral blood mononuclear cells differentiated into macrophage/dendritic lineage generates potent innate and adaptive responses, as reported previously for poliovirus.[25] Tumor-associated macrophages (TAMs), a significant stromal component of GBM,[26] broadly promote tumor progression through proangiogenic, proproliferative/invasive, and prosurvival signals, including the repression of cytotoxic T-cell activity.[27] These properties are the result of TAM polarization effects of the local tumor microenvironment, eg, cytokine/chemokine production or extracellular matrix interactions.[28] Given the key roles of TAMs in tumor support and immune repression, repolarization toward a more proinflammatory, antineoplastic phenotype has been an attractive, if elusive, goal in cancer immunotherapy. Few events may be as effective in TAM repolarization as virus infection. This hypothesis is difficult to test in vivo, because TAM phenotypes are difficult to recapitulate in animal tumor models, and patient explant TAM assays are challenging. However, the plausible in vitro evidence suggests that PVSRIPO targeting of TAM may well contribute to its antineoplastic potential (Fig. 2).

image

Figure 2. Possible host immunogenic responses to immunotherapy with a recombinant oncolytic polio virus (PVSRIPO) are illustrated. (Left) PVSRIPO infects and kills glioblastoma multiforme cells, elicits immunogenic cell death (ICD), and presents danger-associated molecular pattern (DAMP) signals and tumor antigens. ATP indicates adenosine triphosphate; HMGB1, high-mobility group box 1. (Middle) PVSRIPO infection of antigen-presenting cells elicits innate antiviral type 1 interferon (IFN) responses and pathogen-associated molecular pattern (PAMP) signals for proinflammatory stimulation. Mda5 indicates melanoma-derived antigen-5; TNFα, tumor necrosis factor α; IL-12, interleukin 12; M1, type 1 macrophages. (Right) The combination of multiplex direct viral cytotoxicity, DAMP and PAMP presentation, proinflammatory tumor-associated macrophage activation, cytokine release, etc, promote an adaptive antitumor immune effector cell response. Nk/Nk-T cells indicates natural killer/natural killer T cells; CTL, cytotoxic T cells.

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Attenuation and Safety

Ectopic expression of viral receptors like Necl5 means that cancer cells may be susceptible to virus infection and killing. However, tropism for normal target cells (expressing receptors) remains: toxicity stemming from inherent viral pathogenicity is unacceptable in a clinical oncologic setting. Modern molecular virology has had remarkable success in engineering nonpathogenic variants, even of lethal, feared pathogens. For example, genetic recombinants of the neuropathogens poliovirus and herpes simplex virus 1 with neuronal replication deficits in vitro lack neuropathogenicity in relevant nonhuman primates.[29, 30] They also did not cause encephalitis, meningitis, or poliomyelitis after high-titer intratumoral inoculation in patients with GBM.[5, 31]

We devised a uniquely effective way of preventing polio virulence in the normal central nervous system (CNS). Poliovirus binding to Necl5 and ensuing host cell entry lead to perforation of the viral capsid and rapid cytoplasmic release of +strand viral RNA (Fig. 3A). Establishing infection depends on immediate translation of incoming viral RNA (Fig. 3B), because “naked” polio RNA cannot persist, and failure to translate will abort the infectious process. Once initial viral translation has occurred, cytotoxic viral proteins rearrange the host cell cytoplasm and intercept host gene expression to benefit viral replication. Therefore, translation initiation at incoming polio RNA constitutes a rate-limiting step. Both instances in which deliberate administration of live poliovirus to humans met with US Food and Drug Administration approval (the Sabin vaccines and PVSRIPO) relied on interfering with this step specifically in the CNS.

image

Figure 3. Early events are illustrated after the infection of tumor cells with the oncolytic recombinant poliovirus/rhinovirus PVSRIPO. (A) Approximately 15 minutes after virus-receptor docking, empty capsids (indicating viral RNA “uncoating”) can be recovered from infected cells. Necl5 indicates nectin-like molecule 5. (B) The incoming, uncapped, viral RNA serves as a translation template; recruitment of the host cell translation initiation helicase complex eukaryotic initiation factor 4G (eIF4G)4A:4B to the internal ribosomal entry site is required for the initiation of viral protein synthesis (see text). (C) The first non-structural protein released from the nascent viral polyprotein is the viral protease 2A. (D) Initial rounds of viral translation give rise to highly productive viral protein synthesis and genome replication. This is fostered by stimulatory viral polypeptides and the blocking of competing host cell protein synthesis. p(A) indicates polyadenylate tract. (E) Viral progeny is first detected by 4 hours.

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The live-attenuated (Sabin) polio vaccines (types 1 and 3 resulted from serial passage of neurovirulent strains in primate cell cultures/nonhuman primates; type 2 essentially is a field isolate)[2] feature crucial, attenuating point mutations in a cis-acting genetic element responsible for viral protein synthesis: the internal ribosomal entry site (IRES) (Fig. 1B).[32] Later investigations linked the Sabin-attenuating IRES mutations to compromised neuronal translation initiation.[33-35] PVSRIPO was engineered by exchange of the entire cognate IRES with the corresponding segment from a distant poliovirus relative: human rhinovirus type 2 (HRV2) (Fig. 1C).[2] Although PVSRIPO attenuation may involve a mechanism similar to that of the Sabin IRESes, its neuronal incompetence is more pronounced,[33] it has a far broader genetic footprint, and, most important, it is genetically stable upon replication in the intended target.[36] The reasons for this become apparent when considering the molecular mechanism for translation initiation at enterovirus IRESes (Fig. 1A).

Conventional protein synthesis occurs when the eukaryotic initiation factor 4E (eIF4E) binds the canonical 7-methyl-guanidine “cap” modification at the 5′ end of messenger RNAs.[37] eIF4E recruits the large-scaffold eIF4G, which assembles the eIF4G:4A:4B helicase complex (responsible for scanning and unwinding the 5′-untranslated region [UTR] until the initiation codon is identified) and binds eIF3 (the bridge to the small ribosomal subunit).[37] Poliovirus RNA, which is uncapped,[38] cannot bind eIF4E. Therefore, it uses an unorthodox translation mechanism by recruiting the eIF4G:4A:4B helicase complex directly, independent of the 5′ end or eIF4E (Fig. 1A).[1] Binding to eIF4G:4A:4B is enabled by the IRES, an approximately 500-nucleotide structure in the 5′UTR of the viral genome (Fig. 1A).[39] Thus, the rate-limiting step in initial translation of incoming poliovirus RNA depends on attracting eIF4G:4A:4B to the IRES (Fig. 3B). By substituting the HRV2 IRES, we eliminated the ability of PVSRIPO to recruit eIF4G to the viral genome in normal CNS cells.[40] The part of the HRV2 IRES responsible for this defect (approximately 33% of the distal IRES comprising stem-loop domains v and vi) (Fig. 1C)[41] is the most genetically divergent (from poliovirus)[41] and contains the “footprint” for eIF4G:4A:4B binding.[1, 34] The Sabin IRES mutations (Fig. 1B) and the heterologous HRV2 IRES in PVSRIPO (Fig. 1C) may interfere similarly with host cell translation factor recruitment in the CNS. However, in the former, attenuation rests on artificial (tissue culture-generated) single point mutations that spontaneously revert to the wild-type sequence.[42] In PVSRIPO, attenuation is caused by a functionally integrated, naturally occurring, intact IRES, which is inherently deficient for eIF4G:4A:4B recruitment in the CNS. We believe that this distinction explains the superior attenuation/genetic stability profile of PVSRIPO compared with its Sabin precursor.[36]

The neuronal deficits in PVSRIPO for recruiting host translation factors are likely caused by: 1) host cell (neuronal) RNA-binding proteins that interact with the HRV2 IRES and interfere with eIF4G recruitment[40, 43, 44]; and/or 2) absent Raf/extracellular signal-related kinase 1 and 2 (Erk1/2)/mitogen-activated protein kinase (MAPK) signals in the normal CNS, restricting ribosome recruitment to the viral IRES. This could involve effects on eIF4G:4A:4B[45, 46] or signals to the MAPK-interacting kinase (Mnk) that affect IRES competence.[47] It is noteworthy that, despite its profound CNS incompetence, PVSRIPO is fully translation-proficient in tumor cells (see below), indicating a truly tumor-specific viral translation/cytotoxicity/killing phenotype.[4]

Tumor Cytotoxicity and Cell Killing

Genetic engineering can yield nonpathogenic virus variants that are safe for use in cancer patients. Ideally, this does not affect the major attraction of OVs: their tumor cytocidal potential. Because host immune responses to OV tumor targeting may be key mediators of clinical efficacy,[48] there is a debate about the relative contribution of direct viral cytotoxicity to therapy. Traditional views stipulate primary (direct cytotoxic) effects that may evoke, but are functionally separate from, secondary (host-inflammatory) responses. However, tumor cell killing and the host immunologic response may be closely integrated mechanistically and biologically. For example, an attractive prospect for therapy is the propensity of viruses to elicit immunogenic cell death (ICD), a noncanonical form of cell demise characterized by release of the proinflammatory cytokine high-mobility group box 1 (HMGB1), adenosine-triphosphate (ATP), and preapoptotic exposure of calreticulin.[49] ICD in essence posits the dying cancer cell as a potent immune stimulus; thus, OV efficacy may occur as a result of intertwined cytotoxicity and host immunogenic responses.

Many animal viruses exhibit replication success that leads to cell killing in clonal laboratory cancer cell lines. This is unsurprising, because common impediments to viral growth in normal living tissues (eg, lacking tropism, intact innate antiviral defenses, an inactive cell cycle in terminally differentiated cells, silent mitogenic signaling pathways, etc) may be lifted in such hosts. However, this is not representative of exceedingly heterogeneous cancers laced with (nonmalignant) stromal components in patients. Therefore, to provide empirical support for clinical investigations, it is of utmost importance to identify the molecular mechanisms of viral cytotoxicity in vitro for correlation with the clinical target.

PVSRIPO has a simple, short-lived, and extremely toxic relationship with infected cancer cells. A scant coding capacity and primitive life cycle do not allow parasitic, long-term relations with the host. Successful infection, which depends on the rate-limiting step of initial translation of incoming viral RNA (see above), produces very rapid, irreversible, and lethal alterations of host cell physiology in tumor cells.[4] If initial viral protein synthesis succeeds, then the first non-structural viral protein to be released is the protease 2Apro, which is cleaved autocatalytically from the nascent viral polyprotein (Fig. 3C). 2Apro intercepts host cell gene expression as early as approximately 60 minutes after infection, eg, through degradation of eIF4G[50] and nuclear pore proteins.[51] eIF4G cleavage selectively shuts off host cell protein synthesis; a C-terminal eIF4G fragment capable of forming fully functional eIF4G:4A:4B helicase complexes is sufficient for the translation of poliovirus genomes.[32]

Unlike in the CNS, PVSRIPO drives rampant viral translation and cytotoxicity in cancer cells. This is because they offer an enormous advantage to the virus by allowing wanton viral, IRES-mediated translation.[4] This is not specific to the HRV2 IRES in PVSRIPO, but it relates to the initiation mechanism operational at all enterovirus IRESes.[33] Repression of HRV2 IRES-mediated translation in the CNS (see above) is virtually absent in malignant cells.[44] Neuron-like cells that recapitulate the CNS incompetence of PVSRIPO become fully susceptible to viral translation upon activation of Raf-Erk1/2 MAPK signals.[47] Erk1/2 MAPK networks converge on translation machinery, ie, eIF4G and its binding partners involved in 5′UTR scanning/unwinding (eIF4B), ribosome recruitment (eIF3), RNA-binding (eIF4G:4A:4B helicase complex), the MAPK interacting kinase (Mnk) and other less obvious factors with influence over translation control.[45, 46, 52-54] Efforts to mechanistically unravel MAPK signaling networks are ongoing, but we have conclusive evidence that PVSRIPO's tumor cytotoxicity rests on catalytic activity of the Erk1/2 downstream kinase Mnk.[47] Thus, the oncolytic capacity of PVSRIPO relies on a key, central oncogenic signaling network centered on Raf-Erk1/2-Mnk signals.

Oncolytic Viruses and Innate Antiviral Immunity in Cancer

Viral infection invariably triggers antiviral defenses stemming from tumor or stromal components. The first line of defense is activation of an innate antiviral response to nonself PAMPs, such as viral genetic material.[55] For example, viral RNA (structurally distinct from host messenger RNA) is detected by the cytoplasmic “RIG-I”-like receptors RIG-I (retinoic acid-inducible gene-I) and MDA-5 (melanoma-derived antigen-5),[55] which induce type 1 interferons (IFNs) that mediate autocrine/paracrine induction of hundreds of IFN-stimulated genes (Fig.4A). Most animal viruses have evolved with means for evading, blocking, or counteracting antiviral defenses in their preferred host species. Thus, the pathogenic process results from an “arms race” between the host's intent to detect/eliminate infected cells versus the virus' intent to deceive/establish infection. It is obvious that the virus:innate defense interplay is a major factor in oncolytic virotherapy, either in a restrictive role (preventing viral cytotoxicity, replication, spread) or, a far more exciting possibility, in a supportive role.

image

Figure 4. The effect of innate antiviral activation on PVSRIPO oncolysis in vitro is illustrated. (A) This is a schematic of retinoic acid-inducible gene-I (Rig-I)-like receptors, melanoma-derived antigen-5 (Mda-5) activation, and interferon (IFN) induction by polio RNA. TLR3 indicates toll-like receptor 3; ssRNA, single-stranded RNA; dsRNA, double-stranded RNA; Necl5, nectin-like molecule 5; pU/UC, polyuridine/polpyrimidine tract; 5′pppNt, 5′-triphosphate nucleotide; 5′Vpg, 5′ viral protein genome-linked; AP-1, adapter protein complex 1; p, short arm; q, long arm; IRF3, interferon regulatory factor 3; NF-κB, nuclear factor κB; INFNAR, interferon receptor α; Jak, Janus kinase; Stat1, signal transducer and activator of transcription 1. (B) DM443 and (C) DM440 cell cultures were infected with PVSRIPO at a multiplicity of infection (MOI) of 0.1. (B) In DM443 cells, stable levels of Stat1 phosphorylation (phosphor-Stat1) indicate failure of type 1 IFN signaling. (C) DM440 cells responded with vigorous Stat1 phosphorylation (red arrowheads). Viral cell killing (indicated by eukaryotic initiation factor 4G [eIF4G] cleavage) and expression of viral proteins (2C) were indistinguishable in both cell lines. The relatively protracted appearance of viral proteins/eIF4G cleavage was caused by the low MOI. GAPDH indicates glyceraldehyde-3-phosphate dehydrogenase.

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A frequent argument in favor of tumor specificity and cytotoxicity of OVs is the purportedly reduced state of the innate antiviral response in cancers. Viruses that are neutralized by intact innate defenses are unable to establish successful infections in normal tissues and, thus, generally lack pathogenicity. However, such viruses may replicate in and kill tumor cells with deficient innate defenses. On the basis of this paradigm, OV platforms were devised in which tumor selectivity and cytotoxicity depended on hindered innate antiviral defenses in malignant cells.[56, 57] Applying this concept in the clinic is problematic for several reasons. First, there is the notorious tumor heterogeneity. It is true that many laboratory tumor cell lines exhibit deficits in innate antiviral IFN responses.[58] Such cells are permissive to killing by viruses that are otherwise blocked by intact antiviral defenses.[57] However, the manifestation of this phenomenon in patient tumors is far from clear. Ex vivo cultivation and passage of any type of cell, eg, the nonmalignant African Green Monkey kidney (Vero) explant cell line, can select for profound innate antiviral defense defects.[59] Innate antiviral defects in laboratory cell lines may primarily reflect serial in vitro propagation/tissue culture adaptation rather than malignant transformation.

The problem is easily recognizable when screening cancer cell lines for the innate response to virus infection (Fig. 4B,C). DM440 and DM443 are 2 melanoma cell lines (chosen at random), derived from similar biopsy material of distinct patients with metastatic disease (courtesy of Dr. H. Seigler, Duke University Medical Center).[60] DM440 (passage 43) vigorously responds to PVSRIPO infection with phosphorylation of signal transducer and activator of transcription 1 (STAT1) (indicating an IFN response) (Fig. 4A,C). DM443 (passage 25) does not (Fig. 4B). No information about the tumor of origin that could shed light on this issue is available; however, considering the heterogeneity of advanced, metastatic, and/or treatment-refractory cancers, it is safe to assume that such tumors harbor cells with a wide range of innate antiviral competency. Indeed, this has recently been demonstrated for the status of the innate antiviral defense and RIG-I in hepatocellular carcinoma.[61] OVs that were characterized in cell lines/animal tumor models with homogeneous innate antiviral deficits may exert very different biologic effects in heterogeneous target tumors in patients. Second, concepts of targeting innate immune deficiency may miss out on promising modes of antineoplastic efficacy. Tumor cytotoxicity, eg, the coveted “immunogenic” kind,[49] may result from the innate defense:virus arms race rather than unilateral toxic effects of the virus or its products alone.[62] Harming/killing of defenseless cells with a blunted innate antiviral response may lack this potentially important quality. In other words, an ability to successfully overcome active defenses in tumor cells, ie, killing in the presence of a functional innate antiviral response may be important for therapy. Third, IFN responses have significant proinflammatory and immune-stimulatory effects that may help to recruit immune effector cells and mediate adaptive antitumor immunity. For viruses whose oncolytic capacity relies on absent antiviral responses, this issue is tricky, because the (desirable) costimulatory effects of the innate antiviral response occur at the expense of viral cytotoxicity and spread.

Poliovirus stands out among its peers with regard to the innate antiviral defense: 1) the main (and perhaps only) PAMP is viral RNA; 2) unlike other RNA viruses, poliovirus (and the picornaviridae family) are exclusively detected by MDA-5 (Fig. 4A)[63]; and 3) poliovirus lacks a specific strategy to block or counter host innate defenses. Rather, it is the virus' remarkably efficient cytotoxicity that permits host cell killing in the presence of innate immune activation.[64] Therefore, polioviruses uniquely exhibit almost undisturbed viral replication and cell killing in IFNα-treated cells.[64] Assays in DM440 and DM443 cells confirm this scenario (Fig. 4B,C). We deliberately infected these cells with PVSRIPO at a multiplicity of infection (MOI) of 0.1. This means that, initially, only ∼10% of cells in the culture are infected, enabling amplification of IFN signaling to both infected and uninfected cells by delaying cell death (in DM440 cells) (Fig. 4C). This response could interfere with subsequent rounds of infection, cytotoxicity, and cell killing of the remaining ∼90% of cells in the culture. Yet host cytotoxic effects (eg, eIF4G cleavage) and viral translation occur at a similar rate in DM440 and DM443 cells (Fig. 4B,C). This demonstrates that primary PVSRIPO cytotoxicity (first round of infection) and subsequent cell killing and spread are not significantly affected by intact antiviral defenses. Thus, for PVSRIPO, triggering the innate antiviral response in tumor/tumor stroma does not primarily impede oncolytic efficacy.

The Role of the Host Immune System

The arguably most intriguing, mechanistically complex, and least explored aspect of OVs is their potential to elicit host immunogenic responses directed against the tumor.[48] Because viruses generate responses to viral cytotoxicity in autologous tumor, they may be regarded as in situ vaccines that are specific to the antigenic profile of individual tumors. A long list of events may conspire in oncolytic immunotherapy.[48] For PVSRIPO, these include: 1) primary tumor cytotoxicity that releases tumor antigens; 2) DAMPs, ICD, and their proinflammatory stimuli; 3) PAMP exposure/MDA-5 activation within the tumor; 4) innate antiviral-mediated recruitment of natural killer cell responses; 5) virus-mediated alterations of intratumoral cytokine profiles; 6) viral-targeting, PAMP-exposure, proinflammatory stimulation of APCs; 7) TAM repolarization/reversal of the immune-suppressive cancer microenvironment; and 8) recruitment of adaptive antitumor humoral and cytotoxic T-cell responses. These possibilities are depicted schematically in Figure 2.

Conclusion

Targeting cancer with viruses has obvious attractions that motivated efforts to quickly move prototype OV constructs toward clinical investigation. As with other areas of experimental cancer research, the empirical basis for clinical use of OVs is more complicated than anticipated. The common problem of matching preclinical paradigms established in experimental tumor models with the patient situation is particularly acute with OVs. This is because of the exceedingly complex, multilayered relations with their malignant host tissues, normal tumor stroma, and the host organism. The recent promise of oncolytic virotherapy in the clinic, eg, our observations with PVSRIPO, demonstrate that relentless preclinical, mechanistic research addressing all aspects of the virus:host relationship can yield promising clinical results in the most difficult oncologic indications that resist all available conventional therapy.

FUNDING SUPPORT

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FUNDING SUPPORT
  5. CONFLICT OF INTEREST DISCLOSURES
  6. REFERENCES

This work was supported by Public Health Service Awards R01 CA124756 (M.G.), R01 CA140510 (M.G.), and P50 NS20023 (D.D.B.). PVSRIPO preclinical development, IND-directed toxicology, and IND submission were supported in part by the Rapid Access to Intervention Development (RAID) and the Experimental Therapeutics (NExT) Programs of the National Cancer Institute Division of Cancer Treatment and Diagnosis, Developmental Therapeutics Program.

CONFLICT OF INTEREST DISCLOSURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FUNDING SUPPORT
  5. CONFLICT OF INTEREST DISCLOSURES
  6. REFERENCES

Drs. Desjardins, Sampson, Friedman, Bigner, and Gromeier are co-owners of the intellectual property related to the technology discussed herein, for which they have a patent pending. Dr. Gromeier holds 5 other, unspecified patents.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FUNDING SUPPORT
  5. CONFLICT OF INTEREST DISCLOSURES
  6. REFERENCES