Induction of viral, 7-methyl-guanosine cap-independent translation and oncolysis by mitogen-activated protein kinase-interacting kinase-mediated effects on the serine/arginine-rich protein kinase.

UNLABELLED: Protein synthesis, the most energy-consuming process in cells, responds to changing physiologic priorities, e.g., upon mitogen- or stress-induced adaptations signaled through the mitogen-activated protein kinases (MAPKs). The prevailing status of protein synthesis machinery is a viral pathogenesis factor, particularly for plus-strand RNA viruses, where immediate translation of incoming viral RNAs shapes host-virus interactions. In this study, we unraveled signaling pathways centered on the ERK1/2 and p38α MAPK-interacting kinases MNK1/2 and their role in controlling 7-methyl-guanosine (m(7)G) "cap"-independent translation at enterovirus type 1 internal ribosomal entry sites (IRESs). Activation of Raf-MEK-ERK1/2 signals induced viral IRES-mediated translation in a manner dependent on MNK1/2. This effect was not due to MNK's known functions as eukaryotic initiation factor (eIF) 4G binding partner or eIF4E(S209) kinase. Rather, MNK catalytic activity enabled viral IRES-mediated translation/host cell cytotoxicity through negative regulation of the Ser/Arg (SR)-rich protein kinase (SRPK). Our investigations suggest that SRPK activity is a major determinant of type 1 IRES competency, host cell cytotoxicity, and viral proliferation in infected cells. IMPORTANCE: We are targeting unfettered enterovirus IRES activity in cancer with PVSRIPO, the type 1 live-attenuated poliovirus (PV) (Sabin) vaccine containing a human rhinovirus type 2 (HRV2) IRES. A phase I clinical trial of PVSRIPO with intratumoral inoculation in patients with recurrent glioblastoma (GBM) is showing early promise. Viral translation proficiency in infected GBM cells is a core requirement for the antineoplastic efficacy of PVSRIPO. Therefore, it is critically important to understand the mechanisms controlling viral cap-independent translation in infected host cells.

Seneca Valley virus replicons are packaged in trans and have the capacity to overcome the limitations of viral transgene expression.

Oncolytic viruses (OVs) promote the anti-tumor immune response as their replication, and the subsequent lysis of tumor cells, triggers the activation of immune-sensing pathways. Arming OVs by expressing transgenes with the potential to promote immune cell recruitment and activation is an attractive strategy to enhance OVs' therapeutic benefit. For picornaviruses, a family of OVs with clinical experience, the expression of a transgene is limited by multiple factors: genome physical packaging limits, high rates of recombination, and viral-mediated inhibition of transgene secretion. Here, we evaluated strategies for arming Seneca Valley virus (SVV) with relevant immunomodulatory transgenes. Specificially in the contex of arming SVV, we evaluated transgene maximum size and stabiltity, transgene secretion, and the impact of transgene inclusion on viral fitness. We find that SVV is not capable of expressing secreted payloads and has a transgene packaging capacity of ∼10% of viral genome size. To enable transgene expression, we developed SVV replicons with greater transgene size capacity and secretion capabilities. SVV replicons can be packaged in trans by virus in co-infected cells to express immunomodulatory transgenes in surrounding cells, thus providing a means to enhance the potential of this therapeutic to augment the anti-tumor immune response.

Development of intravenously administered synthetic RNA virus immunotherapy for the treatment of cancer.

The therapeutic effectiveness of oncolytic viruses (OVs) delivered intravenously is limited by the development of neutralizing antibody responses against the virus. To circumvent this limitation and to enable repeated systemic administration of OVs, here we develop Synthetic RNA viruses consisting of a viral RNA genome (vRNA) formulated within lipid nanoparticles. For two Synthetic RNA virus drug candidates, Seneca Valley virus (SVV) and Coxsackievirus A21, we demonstrate vRNA delivery and replication, virus assembly, spread and lysis of tumor cells leading to potent anti-tumor efficacy, even in the presence of OV neutralizing antibodies in the bloodstream. Synthetic-SVV replication in tumors promotes immune cell infiltration, remodeling of the tumor microenvironment, and enhances the activity of anti-PD-1 checkpoint inhibitor. In mouse and non-human primates, Synthetic-SVV is well tolerated reaching exposure well above the requirement for anti-tumor activity. Altogether, the Synthetic RNA virus platform provides an approach that enables repeat intravenous administration of viral immunotherapy.

CReP mediates selective translation initiation at the endoplasmic reticulum.

Eukaryotic protein synthesis control at multiple levels allows for dynamic, selective responses to diverse conditions, but spatial organization of translation initiation machinery as a regulatory principle has remained largely unexplored. Here we report on a role of constitutive repressor of eIF2α phosphorylation (CReP) in translation of poliovirus and the endoplasmic reticulum (ER)-resident chaperone binding immunoglobulin protein (BiP) at the ER. Functional, proximity-dependent labeling and cell fractionation studies revealed that CReP, through binding eIF2α, anchors translation initiation machinery at the ER and enables local protein synthesis in this compartment. This ER site was protected from the suppression of cytoplasmic protein synthesis by acute stress responses, e.g., phosphorylation of eIF2α(S51) or mTOR blockade. We propose that partitioning of translation initiation machinery at the ER enables cells to maintain active translation during stress conditions associated with global protein synthesis suppression.

Regulation of Hypoxia-Inducible Factor 1α during Hypoxia by DAP5-Induced Translation of PHD2.

Death-associated protein 5 (DAP5) is an atypical isoform of the translation initiation scaffolds eukaryotic initiation factor 4GI (eIF4GI) and eIF4GII (eIF4GI/II), which recruit mRNAs to ribosomes in mammals. Unlike eIF4GI/II, DAP5 binds eIF2ß, a subunit of the eIF2 complex that delivers methionyl-tRNA to ribosomes. We discovered that DAP5:eIF2ß binding depends on specific stimuli, e.g., protein kinase C (PKC)-Raf-extracellular signal-regulated kinase 1/2 (ERK1/2) signals, and determines DAP5's influence on global and template-specific translation. DAP5 depletion caused an unanticipated surge of hypoxia-inducible factor 1α (HIF-1α), the transcription factor and master switch of the hypoxia response. Physiologically, the hypoxia response is tempered through HIF-1α hydroxylation by the oxygen-sensing prolyl hydroxylase-domain protein 2 (PHD2) and subsequent ubiquitination and degradation. We found that DAP5 regulates HIF-1α abundance through DAP5:eIF2ß-dependent translation of PHD2. DAP5:eIF2-induced PHD2 translation occurred during hypoxia-associated protein synthesis repression, indicating a role as a safeguard to reverse HIF-1α accumulation and curb the hypoxic response.

Validation of an Immunohistochemistry Assay for Detection of CD155, the Poliovirus Receptor, in Malignant Gliomas.

CONTEXT: - The oncolytic polio-rhinovirus recombinant (PVSRIPO) has demonstrated promise in currently ongoing phase I/II clinical trials against recurrent glioblastoma and was granted breakthrough therapy designation by the Food and Drug Administration/Center for Biologics Evaluation and Research. A reliable clinical assay to document expression of the poliovirus receptor, CD155, in routinely available patient tumor samples is needed for continued clinical development of PVSRIPO oncolytic immunotherapy in primary brain tumors and beyond. OBJECTIVES: - To validate a novel anti-CD155 antibody for immunohistochemistry and develop a robust, reliable, and specific protocol for detecting CD155 expression in glioblastoma formalin-fixed, paraffin-embedded (FFPE) tissue samples. To characterize the expression of CD155 in human glioblastoma cells as well as to evaluate the influence of CD155 expression levels on tumor cell susceptibility to PVSRIPO infection and killing. DESIGN: - Immunohistochemical staining on glioblastoma FFPE tissue sections and immunoblot of corresponding frozen tissues were performed. Positive controls were confirmed sites of poliovirus propagation, spinal cord anterior horn, and tonsils; negative controls were vascular smooth muscle in patient samples and FFPE sections from a confirmed CD155-negative Burkitt lymphoma line (Raji). RESULTS: - We succeeded in developing a reliable assay to specifically detect CD155 by immunohistochemistry in glioblastoma FFPE sections. Our data suggest widespread, virtually universal expression of CD155 in glioblastoma cells at levels commensurate with susceptibility to PVSRIPO infection and killing. CONCLUSIONS: - Anti-CD155 antibody D3G7H achieves monospecific detection of CD155 in immunoblots of tumor homogenates and immunohistochemistry of tumor FFPE sections. Our assay has utility in defining appropriate use of PVSRIPO in oncolytic immunotherapy against malignant glioma and other cancer histotypes.