ONCOLYTIC VIROTHERAPY

MIR-508 Advanced Topic in Immunology Spring 2017 April 27, 2017

Oncolytic virotherapy mechanism of action: viral replication, cell killing, virus release and spread within cancer tissue but not normal tissues

oncolytic virotherapy

armed oncolytic virotherapy

ARMED

DNA viruses: herpes simplex virus, vaccinia virus RNA viruses: vesicular stomatitis virus

Limitations of Oncolytic Virotherapy Inefficient systemic delivery of the virus to tumors

Inefficient replication in tumor cells and poor intratumoral spread Virus replication in normal cells Toxicity Immune clearance may reduce oncolytic effects

Insufficient single agent potency due to limited understanding of the viral interaction with different elements within

the context of tumor stroma

Future Goals: Improve clinical tolerability and efficacy

Improve tumor selectivity with tropism-modifying strategies Improve systemic delivery for treatment of metastatic cancer (minimize virus sequestration, protect form neutralizing antibodies, increase cellular

penetration) Attack and kill cancer stem cells and chemoresistant tumor cells Induce innate and adaptive antitumor immunity

Woller et al., Frontiers in Oncology, 2014, 4:1

Immunogenic cell death (ICD)

RNA: RNA pol III, RIG-I/MDA5 - Signaling molecules: MAVS, TBK1, IKK, NFκB, IRF3 DNA: cGAS, IFI16/p204 - Signaling molecules: STING, TBK1, IKK, IRF3, NFκB LRRFIP1 - Signaling molecules: β-catenin, p300, IRF3 DAI - Signaling molecules: TBK1, IRF3 RIP1, RIP3, IKK, NFκB, IRF3 Ku70 - Signaling molecules: IRF1, IRF7, IFNλ1 CpG: TLR9, DHX9, DHX36 - Signaling molecules: MyD88, IKK, NFκB, IRF7

Cytosolic Sensors of Nucleic Acids Regulating Type I IFN Induction

RIG-I-like receptors-MAVS-mediated signal transduction pathway

RIG-I-like receptors (RLRs) have been identified as important cytoplasmic viral RNA sensors that recognize the genomic RNA and/or RNA replication intermediates of numerous viruses. The two RLR members retinoic acid-inducible gene-I protein (RIG-I) and melanoma differentiation-associated protein 5 (MDA5) are kept inactive in uninfected cells through the phosphorylation of their caspase activation and recruitment domains (CARDs) and carboxy-terminal domains (CTDs). In addition, RIG-I adopts a 'closed' auto-inhibited conformation. Following viral infection, RIG-I recognizes cytoplasmic viral short double-stranded RNA (dsRNA) that contains a 5′-triphosphate or 5′-diphosphate moiety, whereas MDA5 detects long dsRNA structures. Following the binding of RNA, RIG-I and MDA5 are dephosphorylated by PP1α or PP1γ, which induces the formation of a signalling-active CARD conformation. RIG-I is further activated by the Lys63-linked ubiquitylation of its CARDs that is mediated by tripartite motif protein 25 (TRIM25). Riplet, another E3 ubiquitin ligase, mediates the Lys63-linked ubiquitylation of the CTD of RIG-I, which is also crucial for the activation of RIG-I. Lys63-linked ubiquitylation induces the tetramerization of RIG-I (the signalling-active form of RIG-I), which subsequently interacts with the adaptor mitochondrial antiviral signalling protein (MAVS) on mitochondria, mitochondria-associated membranes (MAMs) or peroxisomes (not shown). The mitochondrial-targeting chaperone protein 14-3-3ε is essential for the translocation of RIG-I to mitochondrial MAVS. In the case of MDA5, binding to long dsRNA induces MDA5 filament formation, which subsequently enables MDA5 to bind to MAVS. MAVS activates TBK1 or IκB kinase-ε (IKKε) as well as the IKKα–IKKβ–IKKγ complex, which activate interferon (IFN) regulatory factor 3 (IRF3) and IRF7, and nuclear factor-κB (NF-κB), respectively, through phosphorylation events. IRF3 and/or IRF7 and NF-κB together with activator protein 1 (AP1; not illustrated) induce the gene expression of type I IFNs (mainly IFNα subtypes and IFNβ), type III IFNs (IFNλ subtypes), and many other pro-inflammatory cytokines and chemokines, to establish an antiviral state. Solid arrows indicate direct signalling events. Dashed arrows indicate indirect signalling events. DENV, dengue virus; HBV, hepatitis B virus; HCV, hepatitis C virus; IAV, influenza A virus; JEV, Japanese encephalitis virus; K63-Ub, Lys63-linked ubiquitylation; P, phosphate; PACT, protein kinase R activator; Ub, ubiquitin; WNV, West Nile virus. Chan, YK & Gack, MU, Nat. Rev.

Microbiol. 14, 2016

In the cytoplasm of infected cells, cyclic GMP–AMP synthase (cGAS) recognizes double-stranded DNA (dsDNA) from DNA viruses or dsDNA that is produced by retroviruses through the reverse transcription of their RNA genomes. Following the binding of DNA, cGAS synthesizes the second messenger cyclic GMP–AMP (cGAMP), which then binds to and activates stimulator of interferon (IFN) genes (STING) on the endoplasmic reticulum. STING is further activated by dimerization and Lys63-linked ubiquitylation that is mediated by tripartite motif protein 32 (TRIM32) and TRIM56. Furthermore, STING is phosphorylated by TBK1. The sensor IFNγ-inducible protein 16 (IFI16) senses viral dsDNA in both the cytoplasm and the nucleus. Following the binding of viral DNA, IFI16 multimerizes and then signals through STING in the cytoplasm. The activation of STING induces the expression of type I IFN genes and other pro-inflammatory cytokines through the TBK1–IFN regulatory factor 3 (IRF3) axis and nuclear factor-κB (NF-κB). Solid arrows indicate well-established signalling events. Dashed arrows indicate signalling events that are indirect or that have not yet been fully elucidated. EBV, Epstein–Barr virus; HBV, hepatitis B virus; HCMV, human cytomegalovirus; HSV-1, herpes simplex virus 1; K63-Ub, Lys63-linked ubiquitylation; KSHV, Kaposi sarcoma-associated herpesvirus; P, phosphate; Ub, ubiquitin.

Signaling mediated by cGAS and IFI16 through STING

Chan, YK & Gack, MU, Nat. Rev. Microbiol. 14, 2016

Virally-induced STING-dependent signaling in cancer cells that leads to induction of phagocytosis by dendritic cells

Xia et al., Cell Reports 14, 2016

STING-dependent signaling is frequently suppressed in a variety of cancers. Inhibition of STING signaling commonly involves silencing of the synthase cGAS. DNA-damage-induced cytokine production is lost in STING defective tumors. Loss of STING function may predict the outcome of DNA-virus-mediated oncolytic activity

Ultra-sensitive FISH technique

An overview on critical components to be included in multimodale virotherapy-based therapies that work like prime-boost strategies

The efficacy of OVs is dependent on the magnitude of direct oncolysis and is also underpinned by induction of type I IFNs. Understanding the cellular and molecular mechanisms associated with viral infection of metastatic and/or drug-resistant tumor cells will help to identify patients who can benefit from the oncolytic virotherapy alone or in combination with other anti-cancer treatments rather than the standard regimen.

Conclusions

Suggested review articles: Bartlett, D., Liu, Z., Sathaiah, M., Ravindranathan, R., Guo, Z., He, Y. & Guo, Z.S. Oncolytic viruses as therapeutic cancer vaccines. Cancer 12, 103-119, (2013). Chan, Y. K. & Gack, M. U. Viral evasion of intracellular DNA and RNA sensing. Nature Rev. Microbiol. 14, 360-73, (2016). Research article for discussion: Xia, T., Konno, H., & Barber, G. N. Recurrent Loss of STING Signaling in Melanoma Correlates with Susceptibility to Viral Oncolysis . Cancer Research 76(22), 6747-6759, (2016).

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