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. Author manuscript; available in PMC: 2016 Mar 7.
Published in final edited form as: Discov Med. 2015 May;19(106):359–365.

Oncolytic Immunotherapy Through Tumor-specific Translation and Cytotoxicity of Poliovirus

Michael C Brown 1, Matthias Gromeier 1,*
PMCID: PMC4780852  NIHMSID: NIHMS726603  PMID: 26105699

Abstract

Achieving tumor-specific, robust, and durable effector cytotoxic immune responses is key to successful immunotherapy. This has been accomplished with adoptive cell transfer of ex vivo-expanded autologous tumor-infiltrating or engineered T cells, or with immune checkpoint inhibitors, enhancing inherent T cell reactivity. A natural ability to recruit effector responses makes tumor-targeting (‘oncolytic’) viruses attractive as immunotherapy vehicles. However, most viruses actively block inflammatory and immunogenic events; or, host innate immune responses may prevent immune initiating events in the first place. Moreover, the mechanisms of how virus infection can produce effector responses against host (tumor) neo-antigens are unclear. We are pioneering oncolytic immunotherapy based on poliovirus, which has no specific mechanism to interfere with host immune activation, exhibits lytic cytotoxicity in the presence of an antiviral interferon response and pre-existing immunity, and engages a powerful innate immune sensor implicated in recruiting cytotoxic T cell responses. Central to this approach is a unique confluence of factors that drive tumor-specific viral translation and cytotoxicity.

Introduction

In recent years it has become clear that pathologic mechanisms that enable cancer to escape immune system recognition and targeting can be reversed or overcome. Certain forms of cancer immunotherapy may offer individualized, tumor-specific treatment, tilting the scales away from immune tolerance towards the specific antigens/mutations/distress ligands present within a given tumor (Rosenberg and Restifo, 2015) that are more patient-specific and heterogeneous than most experts fathomed (Lawrence et al., 2013). Among the desirable traits of cancer immunotherapies are the ability to reverse tumor immunosuppression combined with generation of new cytotoxic antitumor immune responses. Hypothetically, activation of intracellular innate immune signaling pathways within a tumor would enhance antigen presentation and co-stimulatory molecule expression, drive a Th1-skewed response, and thus elicit cytotoxic T-cell activation capable of targeting and killing cancer cells. Intracellular pathogens, such as viruses, are capable of such activation and accordingly have gained traction as potential anti-cancer therapeutics.

Oncolytic viruses (OVs) not only are capable of spurring antigen presentation and cytotoxic immune responses but may offer the added benefits of (i) precise affinity for malignant cells; (ii) preferential viral cytotoxicity in neoplasms (possibly due to viral adaptations towards mitotically active tissues); (iii) lytic destruction of tumor cells [potentially involving non-canonical, inflammatory processes of cell death (Kroemer et al., 2013)]; and (iv) an ability to replicate and amplify immunological responses within tumor tissue. Given stark differences in tropism/entry mechanisms, replication strategies (DNA vs. −strand RNA vs. +strand RNA viruses), relations to the innate immune system, lytic potential/relations with host cells, and the mechanisms by which they elicit and counter inflammation, the merits of diverse OV strategies cannot be evaluated collectively. Thus, this review focuses on our experiences in developing a tumor selective, oncolytic poliovirus (PV) currently in Phase-I clinical trials for the treatment of recurrent glioblastoma (GBM).

Why Use PV to Target Cancer?

PV, the prototype of the Enterovirus genus in Picornaviridae, is best known for the severe neurological syndrome poliomyelitis, the result of razor-sharp tropism of PV for spinal cord motor neurons (Bodian, 1955). Poliomyelitis is a rare, ‘unintended’ complication of infection that offers no advantage in terms of spread, because the primary site of PV replication is in the gut. Two successful vaccines are being employed to control PV worldwide: the killed (Salk) and the live-attenuated (Sabin) vaccines. All enteroviruses have their primary replication sites in active epithelia in the gastrointestinal and/or respiratory tracts. Possibly reflecting this preference, they are especially successful at translating and replicating their genomes in cancer cells in vitro (Gromeier et al., 2000a). These infections are invariably and rapidly fatal. It follows that if PV neuronal competence could be ablated sufficiently, it may maintain potency in cancerous cells. Such an agent would require far more sophisticated attenuation, in terms of basic neurovirulent potential and genetic stability, than the (Sabin) live-attenuated vaccines currently in use (Dobrikova et al., 2012).

Reflecting genetic austerity and minimal requirements for (+) strand RNA virus replication, the PV lifecycle is exceedingly simple and swift (Molla et al., 1991) (Figure 1). This is particularly attractive from an immunotherapy standpoint, because it conveys relative insensitivity to type 1 interferon (IFN) responses elicited by the innate immune picornavirus RNA sensor, melanoma-derived antigen 5 (MDA5) (Kato et al., 2006). Intriguingly, PV/enteroviruses retain robust replicative capacity and cytotoxicity in the presence of an active antiviral IFN response, due to immediate early host protein synthesis shut-off (Morrison and Racaniello, 2009) (Figure 1). This is advantageous for clinical application, because it may allow several rounds of viral replication even in the presence of a productive IFN response, to amplify immune-stimulating viral cytotoxicity.

Figure 1.

Figure 1

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Early events in PV infection. (1) Virus contacting its sole receptor CD155 facilitates entry and release of (2) viral +strand genomic RNA into the cytoplasm. (3) The viral RNA genome harbors an IRES within its 5′UTR that drives m7G-cap-independent translation of the viral polyprotein by recruiting the eIF4G:4A translation initiation helicase. The polyprotein contains all proteins needed for viral RNA and production/packaging of viral progeny. (4) Autoproteolytic cleavage yields the highly cytotoxic protease 2A, the first non-structural viral protein released. 2A cleaves host proteins including (5) eIF4G (Etchison et al., 1982) and nuclear pore complex components (Gustin and Sarnow, 2001), leading to rapid host cell protein synthesis shut-off, stimulation of viral translation/RNA replication, and, ultimately, lytic destruction of the infected cell

Taming PV for Immunotherapy: PVSRIPO

Our group has developed a highly attenuated oncolytic PV, termed PVSRIPO (Brown et al., 2014c). PVSRIPO was derived from PV serotype 1 (Sabin) by replacing the cognate PV internal ribosomal entry site (IRES) with its counterpart from human rhinovirus type 2 (HRV2), a related enterovirus with exclusive respiratory-tract tropism (Gromeier et al., 1996). This substitution renders the virus stably neuro-incompetent, but retains cancer cell cytotoxicity (Campbell et al., 2005; Dobrikova et al., 2008; Gromeier et al., 1996; 2000a; Yang et al., 2009). PVSRIPO effectively targets malignant cells without scathing normal tissues [reviewed in (Goetz et al., 2011)]: (i) it lacks neuropathogenicity following direct intrathalamic injection in M. fascicularis, the WHO-standard primate model for neurovirulence testing of live-attenuated PV vaccines (Dobrikova et al., 2012); (ii) it exhibits tumor-specificity in a cell culture-based release assay in GBM cells vs. the neuroblastoid HEK293 cell line (Campbell et al., 2005; Yang et al., 2009); (iii) the principal determinants for neuronal incompetence (Merrill et al., 2006; Merrill and Gromeier, 2006) and tumor cell type-specificity of the HRV2 IRES (Brown et al., 2014a; 2014b; Goetz et al., 2010) have been mechanistically defined (see below).

How Does Polio Target Cancer?

Two elemental aspects of oncolytic PV immunotherapy define its anti-neoplastic potential: receptor binding and translation of the viral genome. A notorious Achilles heel of OVs is the required specific tropism for delivering the immune-activating and lytic viral payload to the intended tumor target (Brown et al., 2014c). This in turn ensures that tumor-specific antigens can be released with adjuvancy of viral pathogen- and host cell danger-associated patterns (P/DAMPs)/co-stimulatory antigen-presenting cell (APC)-activation following innate antiviral activation in situ. Preceding events that enable such ‘inflammatory’ cell killing of cancer cells by PVSRIPO, the virus must first contact its receptor, CD155; also known as PV receptor (PVR) (Mendelsohn et al., 1989) and Nectin-like molecule 5 (Necl-5) (Takai et al., 2008). CD155 is a transmembrane protein in the immunoglobulin superfamily that, apart from PV, serves as a ligand for the DNAM-1 (CD226) activating receptor on natural killer (NK) cells and a subset of T cells. The CD155-DNAM-1 relationship has been implicated in NK-cell mediated tumor cell destruction (Bottino et al., 2003) and may be actively manipulated by tumors to counter NK and T cell directed anti-cancer immunity (Carlsten et al., 2009; Stanietsky et al., 2009). CD155 is widely overexpressed in solid tumors (Takai et al., 2008), e.g., GBM (Merrill et al., 2004), where it may be a determinant of invasiveness and dispersion (Sloan et al., 2004; 2005). CD155 upregulation has been linked to tissue injury/repair (**Erickson et al., 2006) and the DNA damage response (Fionda et al., 2015; Tang and Gasser, 2013). CD155 is expressed in APCs, rendering them susceptible to PV infection. Curiously, this is limited in scope, non-lethal, does not interfere with their effector functions, and leads to the release of pro-inflammatory cytokines (Wahid et al., 2005). Targeting of tumor-associated APCs by PVSRIPO may elicit pro-inflammatory stromal effects, such as M1 polarization of tumor-associated macrophages (Wahid et al., 2005). Given the staggering heterogeneity of solid tumors it is unlikely that every cancer cell within a tumor expresses CD155. Since PVSRIPO drives tumor regression primarily by recruiting an anti-neoplastic immune response and not through lysis of bulk tumor (Toyoda et al., 2011; 2007), limited cytolysis of some tumor may be sufficient for therapy (Brown et al., 2014c).

Tumor-specific Translation of PVSRIPO

While CD155 is key to directing PVSRIPO cytotoxicity to neoplasia, it is also present in a non-malignant site that categorically must be spared from viral damage: the CNS (Gromeier et al., 2000b). For this reason, we are exploiting PV’s reliance on an unorthodox mode of protein synthesis initiation to achieve tumor-selective viral translation, replication, and cytotoxicity (Brown et al., 2014c). PV has a (+) strand RNA (i.e., mRNA) genome and relies on initiation of viral translation immediately upon entry (Figure 1). Since viral genomic RNA is inherently unstable, failure to translate will abort the infection. Conventionally, eukaryotic translation is initiated by ribosome recruitment via the 5? 7-methyl-guanosine (m7G)-cap/eukaryotic initiation factor (eIF) 4E to the 5? terminus of mRNAs (Sonenberg et al., 1978). eIF4E binds the eIF4G:4A:4B translation initiation helicase, which scans/unwinds the 5? untranslated region to find the initiation AUG (Gingras et al., 1999). Picornaviral RNAs lack an m7G-cap (Lee et al., 1977) and have a viral protein covalently linked to their 5? end instead (Nomoto et al., 1976). This prevents conventional, cap-dependent translation of viral genomic RNAs. Therefore, picornaviruses use their IRESes to attract ribosomal subunits independent of a 5? end, the m7G-cap, or eIF4E (Pelletier and Sonenberg, 1988) (Figure 2). This process occurs by recruiting the eIF4G:4A:4B translation initiation helicase complex directly to the IRES (de Breyne et al., 2009; Sweeney et al., 2014) and requires the cooperation of certain host factors, termed ‘IRES trans-acting factors (ITAFs)’: poly(rC) binding protein 2 (PCBP2) (Blyn et al., 1996; Sweeney et al., 2014) and Ser-Arg rich protein 20 (SRp20) (Bedard et al., 2007) (Figure 2).

Figure 2.

Figure 2

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Hypothetical schema of MNK-mediated events that control SRPK′s role in modulating m7G-cap-independent translation via the IRES RNP (Brown et al., 2014a; 2014b). (1) MNK is activated through upstream MAPK signals to specifically stimulate mTORC1. (2) This in turn leads to indirect inhibition of mTORC2, potentially through phosphorylation of Sin1 and Rictor (mTORC2 components) by p70 ribosomal protein S6 kinase (downstream of mTORC1) (Dibble et al., 2009). (3) Reduced mTORC2 activity diminishes phosphorylation of AKT (S473) (Sarbassov et al., 2005), an event required for full activation of AKT. (4) This prevents the activation of SRPK by AKT. (5) With regards to PVSRIPO translation, SRp20 likely is responsible for SRPK-mediated effects, since SRp20 has been shown to be an important ITAF by associating with PCBP2 and enhancing translation/ribosomal recruitment (Bedard et al., 2007). Thus, it follows that SRPK activation (via AKT) prevents SRp20 from associating with viral and/or ribosomal RNA to mediate IRES-mediated translation and viral cytotoxicity.

The node of immediate early IRES-mediated translation is where PV is most vulnerable post-entry, being at the mercy of host protein synthesis machinery. Once viral translation takes hold, toxic viral enzymes remodel host cytoplasm in favor of highly productive, viral protein synthesis (Mattern and Daniel, 1965). Thus, a key step in mediating tissue type-specific cytotoxicity and focusing immunogenic cell killing on the tumor occurs at the level of viral translation initiation (Gromeier et al., 2000a). It is our overarching hypothesis that enteroviral IRESes evolved to channel viral infection to mitotically active cell populations in respiratory or gastrointestinal epithelia. Principles of translation control in such cells have not been deciphered, but m7G-cap independent translation is broadly favored in neoplasia (Braunstein et al., 2007).

HRV2 IRES-mediated Translation Is Hampered in the CNS

Tests in neuron:glioma heterokarya suggest that trans-dominant, neuronal factors suppress HRV2 IRES function (Merrill et al., 2006). A screen for such factors indicated that — besides positive ITAFs such as PCBP2/SRp20 (see above) — (host) RNA-binding proteins may associate with viral IRESes to impede translation (Merrill et al., 2006; Merrill and Gromeier, 2006; Neplioueva et al., 2010). Specifically, our investigations show that the double-stranded (ds) RNA binding protein 76 binds the HRV2 IRES in PVSRIPO (Merrill et al., 2006) and blocks eIF4G:4A:4B-mediated ribosome recruitment (Merrill and Gromeier, 2006). Intriguingly, such a function of DRBP76 has been described as a general innate antiviral mechanism (Harashima et al., 2010; Patel et al., 1999). DRBP76’s isoform distribution and subcellular partitioning and, thus, RNA-binding capacity, is sharply tissue type-specific (Neplioueva et al., 2010). We postulate that neuron-specific expression, partitioning, and RNA-binding of DRBP76 forms ribonucleo-protein complexes (RNPs) at the PVSRIPO IRES that are incompatible with ribosome recruitment (Brown et al., 2014b; Merrill et al., 2006; Merrill and Gromeier, 2006). A particularly useful tissue-culture model for mirroring neuron-specific PVSRIPO replication deficits in vitro is the HEK293 cell line (Campbell et al., 2005), a neuro-blastoid line transformed with sheared adenovirus DNA (Shaw et al., 2002).

Viral IRES-mediated Translation in Cancer Is Determined by the Ser-Arg-rich Protein Kinase, SRPK

Our studies suggest that PVSRIPO tumor-specificity not only rests on neuron-specific IRES incompetence (due to a translation-incompatible IRES RNP), but is favored by constitutive mitogenic stimuli in cancerous cells. HEK293 cells, which are resistant to PVSRIPO translation (Campbell et al., 2005), become susceptible to PVSRIPO cytotoxicity upon transformation with oncogenic H-Ras (G12V) (Goetz et al., 2010). While this may involve H-Ras-induced changes to DRBP76, our investigations point toward a direct, Ras-Raf-ERK1/2-driven process supporting viral IRES performance (Goetz et al., 2010).

Raf-ERK1/2-mediated stimulation of PVSRIPO translation and cytotoxicity are due to the ERK1/2 downstream substrate, MAPK-interacting kinase (MNK) (Brown et al., 2014a; Goetz et al., 2010). Surprisingly, MNK induces PVSRIPO translation independent of its well-known effects on translation factors, e.g., eIF4G-binding and eIF4E phosphorylation (Pyronnet et al., 1999; Shveygert et al., 2010; Waskiewicz et al., 1997), suggesting a previously unrecognized function (Brown et al., 2014a). To unravel this possibility, we confirmed a recently proposed regulatory relationship of MNK with SRPK (Hu et al., 2012), which is at the core of PVSRIPO translation competence in neoplastic cells (Figure 2). Our findings are consistent with a suppressive role for SRPK in viral, cap-independent translation (Brown et al., 2014a; 2014b) (Figure 2). It is unlikely that MNK controls SRPK directly, because no substrate(s) that could account for this have been identified. Rather, the available evidence suggests indirect regulatory effects of MNK on SRPK via AKT (Brown et al., 2014b). It has been reported that AKT activation leads to SRPK release from inhibitory interactions with heat shock proteins (Zhou et al., 2012), which control SRPK activity (Zhong et al., 2009). We observed that inhibiting MNK exerts similar effects on SRPK activity as selective activation of AKT (Brown et al., 2014b). Thus, MNK may counteract AKT-mediated activation of SRPK thereby enhancing PVSRIPO translation (Figure 2). Concordantly, we found that MNK activates mTORC1 signaling while opposing mTORC2-mediated phosphorylation of AKT (S473) (Brown et al., 2014b). Thus, MNK indirectly inhibits SRPK signaling by tempering AKT activity through mTORC1 activation (see full mechanism in Figure 2). Such feedback activation of AKT through mTORC1 inhibition is consistent with the biological effects of the mTOR components PRAS40, Deptor, and the mTORC1 inhibitor rapamycin (Peterson et al., 2009; Wan et al., 2007).

How Does SRPK Oppose IRES-mediated Translation?

Understanding how SRPK affects cap-independent translation is of central importance to decipher PVSRIPO’s oncolytic potential (Brown et al., 2014a; 2014b). It is probable that SRPK affects IRES competence through its major substrate and ITAF, SRp20 (Brown et al., 2014b). PCBP2 and SRp20 bind to each other (Bedard et al., 2007), bind to IRESes (Bedard et al., 2007; Blyn et al., 1996), and may associate with the eIF4G:4A:4B helicase complex (unpublished data) and/or ribosomal subunits, but their precise role in the IRES RNP and in m7G-cap-independent translation remain enigmatic. SR proteins regulate constitutive and alternative splicing, but continuous, dynamic nucleo-cytoplasmic shuttling of some SR proteins (including SRp20) give them roles in post-transcriptional mRNA metabolism, including broadly supportive functions in protein synthesis (Blaustein et al., 2005; Caceres et al., 1998; Sanford et al., 2005; 2004). SRPK’s main physiologic role is control of SR proteins via phosphorylation of their C-terminal SR dipeptide repeats. This leads to their dissociation from mRNA and favors nuclear import of shuttling SR proteins (Sanford et al., 2005). Active SRPK may divert shuttling SR proteins away from cytoplasmic roles, e.g., in translation, towards nuclear activities, e.g., in splicing. Thus, PVSRIPO m7G-cap-independent translation may respond to a critical link of protein synthesis with signal transduction networks that regulate the SRPK/SR protein system.

Viral Cap-independent Translation Is Broadly Favored in Malignancy

At first glance, the prevalence of aberrant PI3K-AKT activation in cancer seems to contradict a role for MNK-mediated AKT inhibition in viral IRES competence. There is much evidence, however, that AKT is carefully balanced even in cancer cells with very high, constitutive AKT activity (e.g., PTEN loss or PIK3CA mutation) (Brown et al., 2014a). This homeostatic balance is necessary to avoid toxic runaway AKT activity (Nogueira et al., 2008). A primary role of MNK, complementing countless other functional links connecting Raf-ERK1/2 to PI3K-AKT signaling, is to oppose constitutively active AKT, and thus SRPK activity (Brown et al., 2014a). This balancing function of MNK occurs within a complex, integrated signaling network that broadly enables IRES competency. Thus, PVSRIPO’s tumor-specific cytotoxicity and oncolytic potential are favored by a basic homeostatic mechanism of the malignant state.

Conclusions

Targeting cytotoxicity specifically to malignancy is the first and most obvious of many challenges in designing safe and efficacious OVs. PVSRIPO exhibits natural, multi-layered targeting of malignancy by virtue of ectopic CD155 in most solid cancers, and through virtually universal viral tumor-specific cytotoxicity mediated by unfettered IRES activity. This is coupled with pronounced HRV2 IRES incompetence in the CNS. SRPK is a major roadblock to PVSRIPO translation, possibly affects (viral) IRES-mediated initiation in general, and points towards ITAFs (e.g., PCBP2/SRp20) as critical determinants of cell type-specific IRES competence. Unhinged mitogenic signaling networks that lift physiologically tight control over post-transcriptional gene regulation, e.g., m7G-cap-independent protein synthesis, enable PVSRIPO’s inflammatory cancer cytotoxicity and relative insensitivity to innate antiviral defenses (Brown et al., 2014c). Ongoing investigations are focused on the relative contributions of PVSRIPO cancer cytotoxicity and innate immune activation to the recruitment of effector immune responses.

Acknowledgments

This work was supported by Public Health Service Grants R01 CA124756 and P50 CA190991 (M.G.), a grant from the Southeastern Brain Tumor Foundation, the Slomo and Cindy Silvian Foundation, and the Blast Glioblastoma Foundation.

Footnotes

Disclosure

M.G. is a co-Inventor of intellectual property related to the technology discussed.

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