Adaptation of transgene mRNA translation boosts the anticancer efficacy of oncolytic HSV1.

BACKGROUND: Transgenes deliver therapeutic payloads to improve oncolytic virus immunotherapy. Transgenes encoded within oncolytic viruses are designed to be highly transcribed, but protein synthesis is often negatively affected by viral infection, compromising the amount of therapeutic protein expressed. Studying the oncolytic herpes simplex virus-1 (HSV1), we found standard transgene mRNAs to be suboptimally translated in infected cells. METHODS: Using RNA-Seq reads, we determined the transcription start sites and 5'leaders of HSV1 genes and uncovered the US11 5'leader to confer superior activity in translation reporter assays. We then incorporated this 5'leader into GM-CSF expression cassette in oncolytic HSV1 and compared the translationally adapted oncolytic virus with the conventional, leaderless, virus in vitro and in mice. RESULTS: Inclusion of the US11 5'leader in the GM-CSF transgene incorporated into HSV1 boosted translation in vitro and in vivo. Importantly, treatment with US11 5'leader-GM-CSF oncolytic HSV1 showed superior antitumor immune activity and improved survival in a syngeneic mouse model of colorectal cancer as compared with leaderless-GM-CSF HSV1. CONCLUSIONS: Our study demonstrates the therapeutic value of identifying and integrating platform-specific cis-acting sequences that confer increased protein synthesis on transgene expression.

Quercetin induces pannexin 1 expression via an alternative transcript with a translationally active 5' leader in rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is a deadly cancer of skeletal muscle origin. Pannexin 1 (PANX1) is down-regulated in RMS and increasing its levels drastically inhibits RMS progression. PANX1 upregulation thus represents a prospective new treatment strategy for this malignancy. However, the mechanisms regulating PANX1 expression, in RMS and other contexts, remain largely unknown. Here we show that both RMS and normal skeletal muscle express a comparable amount of PANX1 mRNAs, but surprisingly the canonical 5' untranslated region (5' UTR) or 5' leader of the transcript is completely lost in RMS. We uncover that quercetin, a natural plant flavonoid, increases PANX1 protein levels in RMS by inducing re-expression of a 5' leader-containing PANX1 transcript variant that is efficiently translated. This particular PANX1 mRNA variant is also present in differentiated human skeletal muscle myoblasts (HSMM) that highly express PANX1. Mechanistically, abolishing ETV4 transcription factor binding sites in the PANX1 promoter significantly reduced the luciferase reporter activities and PANX1 5' UTR levels, and both quercetin treatment in RMS cells and induction of differentiation in HSMM enriched the binding of ETV4 to its consensus element in the PANX1 promoter. Notably, quercetin treatment promoted RMS differentiation in a PANX1-dependent manner. Further showing its therapeutic potential, quercetin treatment prevented RMS in vitro tumor formation while inducing complete regression of established spheroids. Collectively, our results demonstrate the tumor-suppressive effects of quercetin in RMS and present a hitherto undescribed mechanism of PANX1 regulation via ETV4-mediated transcription of a translationally functional 5' leader-containing PANX1 mRNA.

SARS-CoV2-mediated suppression of NRF2-signaling reveals potent antiviral and anti-inflammatory activity of 4-octyl-itaconate and dimethyl fumarate.

Antiviral strategies to inhibit Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2) and the pathogenic consequences of COVID-19 are urgently required. Here, we demonstrate that the NRF2 antioxidant gene expression pathway is suppressed in biopsies obtained from COVID-19 patients. Further, we uncover that NRF2 agonists 4-octyl-itaconate (4-OI) and the clinically approved dimethyl fumarate (DMF) induce a cellular antiviral program that potently inhibits replication of SARS-CoV2 across cell lines. The inhibitory effect of 4-OI and DMF extends to the replication of several other pathogenic viruses including Herpes Simplex Virus-1 and-2, Vaccinia virus, and Zika virus through a type I interferon (IFN)-independent mechanism. In addition, 4-OI and DMF limit host inflammatory responses to SARS-CoV2 infection associated with airway COVID-19 pathology. In conclusion, NRF2 agonists 4-OI and DMF induce a distinct IFN-independent antiviral program that is broadly effective in limiting virus replication and in suppressing the pro-inflammatory responses of human pathogenic viruses, including SARS-CoV2.

Author Correction: SARS-CoV2-mediated suppression of NRF2-signaling reveals potent antiviral and anti-inflammatory activity of 4-octyl-itaconate and dimethyl fumarate.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Induction of an Alternative mRNA 5' Leader Enhances Translation of the Ciliopathy Gene Inpp5e and Resistance to Oncolytic Virus Infection.

Residual cell-intrinsic innate immunity in cancer cells hampers infection with oncolytic viruses. Translational control of mRNA is an important feature of innate immunity, yet the identity of translationally regulated mRNAs functioning in host defense remains ill-defined. We report the translatomes of resistant murine "4T1" breast cancer cells infected with three of the most clinically advanced oncolytic viruses: herpes simplex virus 1, reovirus, and vaccinia virus. Common among all three infections are translationally de-repressed mRNAs, including Inpp5e, encoding an inositol 5-phosphatase that modifies lipid second messenger signaling. We find that viral infection induces the expression of an Inpp5e mRNA variant that lacks repressive upstream open reading frames (uORFs) within its 5' leader and is efficiently translated. Furthermore, we show that INPP5E contributes to antiviral immunity by altering virus attachment. These findings uncover a role for translational control through alternative 5' leader expression and assign an antiviral function to the ciliopathy gene Inpp5e.