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Macrophages undergo swift changes in mRNA abundance upon pathogen invasion. Herein we describe early remodelling of the macrophage transcriptome during infection by amastigotes or promastigotes of Leishmania donovani . Approximately 10–16% of host mRNAs were differentially modulated in L. donovani -infected macrophages when compared to uninfected controls. This response was partially stage-specific as a third of changes in mRNA abundance were either exclusively driven by one of the parasite forms or significantly different between them. Gene ontology analyses identified categories associated with immune functions (e.g. antigen presentation and leukocyte activation) among significantly downregulated mRNAs during amastigote infection while cytoprotective-related categories (e.g. DNA repair and apoptosis inhibition) were enriched in upregulated transcripts. Interestingly a combination of upregulated (e.g. cellular response to IFNβ) and repressed (e.g. leukocyte activation, chemotaxis) immune-related transcripts were overrepresented in the promastigote-infected dataset. In addition, Ingenuity Pathway Analysis (IPA) associated specific mRNA subsets with a number of upstream transcriptional regulators predicted to be modulated in macrophages infected with L. donovani amastigotes (e.g. STAT1 inhibition) or promastigotes (e.g. NRF2, IRF3, and IRF7 activation). Overall, our results indicate that early parasite stage-driven transcriptional remodelling in macrophages contributes to orchestrate both protective and deleterious host cell responses during L. donovani infection.

Viruses evade the innate immune response by suppressing the production or activity of cytokines such as type I interferons (IFNs). Here we report the discovery of a mechanism by which the SARS-CoV-2 virus coopts an intrinsic cellular machinery to suppress the production of the key immunostimulatory cytokine IFN-β. We reveal that the SARS-CoV-2 encoded nonstructural protein 2 (NSP2) directly interacts with the cellular GIGYF2 protein. This interaction enhances the binding of GIGYF2 to the mRNA cap-binding protein 4EHP, thereby repressing the translation of the Ifnb1 mRNA. Depletion of GIGYF2 or 4EHP significantly enhances IFN-β production, which inhibits SARS-CoV-2 replication. Our findings reveal a target for rescuing the antiviral innate immune response to SARS-CoV-2 and other RNA viruses.

The route of oncolytic virotherapy is pivotal for immunotherapeutic efficacy in advanced cancers. In this preclinical study, an oncolytic reovirus (RC402) is orally administered to induce antitumor immunity. Oral reovirus treatment shows no gross toxicities and effectively suppresses multifocal tumor lesions. Orally administered reovirus interacts with the host immune system in the Peyer’s patch of the terminal ileum, increases IgA + antibody-secreting cells in the lamina propria through MAdCAM-1 + blood vessels, and reshapes the gut microbiome. Oral reovirus promotes antigen presentation, type I/II interferons, and T cell activation within distant tumors, but does not reach or directly infect tumor cells beyond the gastrointestinal tract. In contrast to intratumoral reovirus injection, the presence of the gut microbiome, Batf3 + dendritic cells, type I interferons, and CD8 + T cells are indispensable for orally administered reovirus-induced antitumor immunity. Oral reovirus treatment is most effective when combined with αPD-1(L1) and/or αCTLA-4, leading to complete colon tumor regression and protective immune memory. Collectively, oral reovirus virotherapy is a feasible and effective immunotherapeutic strategy in preclinical studies. Here, the authors show that oral administration of an oncolytic reovirus (RC402) elicits robust suppression of multifocal tumor lesions and antitumor immunity through extensive rewiring of the host immunity and the gut microbiome.

Protein synthesis, or mRNA translation, is one of the most energy-consuming functions in cells. Translation of mRNA into proteins is thus highly regulated by and integrated with upstream and downstream signaling pathways, dependent on various transacting proteins and cis-acting elements within the substrate mRNAs. Under conditions of stress, such as exposure to ionizing radiation, regulatory mechanisms reprogram protein synthesis to translate mRNAs encoding proteins that ensure proper cellular responses. Interestingly, beneficial responses to low-dose radiation exposure, known as radiation hormesis, have been described in several models, but the molecular mechanisms behind this phenomenon are largely unknown. In this review, we explore how differences in cellular responses to high- vs. low-dose ionizing radiation are realized through the modulation of molecular pathways with a particular emphasis on the regulation of mRNA translation control.

The 5’terminal oligopyrimidine (5’TOP) motif is a cis-regulatory RNA element located immediately downstream of the 7-methylguanosine [m7G] cap of TOP mRNAs, which encode ribosomal proteins and translation factors. In eukaryotes, this motif coordinates the synchronous and stoichiometric expression of the protein components of the translation machinery. La-related protein 1 (LARP1) binds TOP mRNAs, regulating their stability and translation. We present crystal structures of the human LARP1 DM15 region in complex with a 5’TOP motif, a cap analog (m7GTP), and a capped cytidine (m7GpppC), resolved to 2.6, 1.8 and 1.7 Å, respectively. Our binding, competition, and immunoprecipitation data corroborate and elaborate on the mechanism of 5’TOP motif binding by LARP1. We show that LARP1 directly binds the cap and adjacent 5’TOP motif of TOP mRNAs, effectively impeding access of eIF4E to the cap and preventing eIF4F assembly. Thus, LARP1 is a specialized TOP mRNA cap-binding protein that controls ribosome biogenesis.

Small-molecule inhibitor of apoptosis (IAP) antagonists, called Smac mimetic compounds (SMCs), sensitize tumours to TNF-α-induced killing while simultaneously blocking TNF-α growth-promoting activities. SMCs also regulate several immunomodulatory properties within immune cells. We report that SMCs synergize with innate immune stimulants and immune checkpoint inhibitor biologics to produce durable cures in mouse models of glioblastoma in which single agent therapy is ineffective. The complementation of activities between these classes of therapeutics is dependent on cytotoxic T-cell activity and is associated with a reduction in immunosuppressive T-cells. Notably, the synergistic effect is dependent on type I IFN and TNF-α signalling. Furthermore, our results implicate an important role for TNF-α-producing cytotoxic T-cells in mediating the anti-cancer effects of immune checkpoint inhibitors when combined with SMCs. Overall, this combinatorial approach could be highly effective in clinical application as it allows for cooperative and complimentary mechanisms in the immune cell-mediated death of cancer cells. Smac mimetics sensitize cancer cells to the extrinsic cell death pathway and stimulate anti-tumour immunity. In this study, the authors show that Smac mimetics can synergize with immune checkpoint inhibitors to control tumour growth in mouse cancer models, including aggressive CNS tumours, in a cytotoxic CD8 + T-cell- and TNFα-dependent manner.

Metformin has been reported to lower cancer incidence among type Il diabetics. Metformin exhibits antiproliferative and antineoplastic effects associated with inhibition of mammalian target of rapamycin complex 1 (mTORC1), but the mechanisms are poorly understood. We provide a unique genome-wide analysis of translational targets of canonical mTOR inhibitors (rapamycin and PP242) compared with metformin, revealing that metformin controls gene expression at the level of mRNA translation to an extent comparable to that of canonical mTOR inhibitors. Importantly, metformin's antiproliferative activity can be explained by selective translational suppression of mRNAs encoding cell-cycle regulators via the mTORCI /eukaryotic translation initiation factor 4E-binding protein pathway. Thus, metformin selectively inhibits translation of mRNAs encoding proteins that promote neoplastic proliferation, which should facilitate studies on metformin and related biguanides in cancer prevention and treatment.

Smac mimetic compounds (SMCs) are small molecule drugs that sensitize cancer cells to TNF-α-induced cell death and have multiple immunostimulatory effects through alterations in NF-κB signaling. The combination of SMCs with immunotherapies has been reported to result in durable cures of up to 40% in syngeneic, orthotopic murine glioblastoma (GBM) models. Herein, we find that SMC resistance is not due to a cell-intrinsic mechanism of resistance. We thus evaluated the contribution of GBM and brain stromal components to identify parameters leading to SMC efficacy and resistance. The common physiological features of GBM tumors, such as hypoxia, hyaluronic acid, and glucose deprivation were found not to play a significant role in SMC efficacy. SMCs induced the death of microglia and macrophages, which are the major immune infiltrates in the tumor microenvironment. This death of microglia and macrophages then enhances the ability of SMCs to induce GBM cell death. Conversely, astrocytes promoted GBM cell growth and abrogated the ability of SMCs to induce death of GBM cells. The astrocyte-mediated resistance can be overcome in the presence of exogenous TNF-α. Overall, our results highlight that SMCs can induce death of microglia and macrophages, which then provides a source of death ligands for GBM cells, and that the targeting of astrocytes is a potential mechanism for overcoming SMC resistance for the treatment of GBM.

Targeting mTORC1 is a highly promising strategy in cancer therapy. Suppression of mTORC1 activity leads to rapid dephosphorylation of eIF4E-binding proteins (4E-BP1–3) and subsequent inhibition of mRNA translation. However, how the different 4E-BPs affect translation during prolonged use of mTOR inhibitors is not known. Here we show that the expression of 4E-BP3, but not that of 4E-BP1 or 4E-BP2, is transcriptionally induced during prolonged mTORC1 inhibition in vitro and in vivo . Mechanistically, our data reveal that 4E-BP3 expression is controlled by the transcription factor TFE3 through a cis -regulatory element in the EIF4EBP3 gene promoter. CRISPR/Cas9-mediated EIF4EBP3 gene disruption in human cancer cells mitigated the inhibition of translation and proliferation caused by prolonged treatment with mTOR inhibitors. Our findings show that 4E-BP3 is an important effector of mTORC1 and a robust predictive biomarker of therapeutic response to prolonged treatment with mTOR-targeting drugs in cancer. The eIF4E-binding proteins (4E-BPs) are critical repressors of cap-dependent translation via mTOR, a pathway frequently hyperactivated in cancer. Here the authors show that 4E-BP3 specifically mediates the cap-dependent translation repression and antiproliferative effects of prolonged pharmacological mTOR inhibition.

The large coding potential of vaccinia virus (VV) vectors is a defining feature. However, limited regulatory switches are available to control viral replication as well as timing and dosing of transgene expression in order to facilitate safe and efficacious payload delivery. Herein, we adapt drug-controlled gene switches to enable control of virally encoded transgene expression, including systems controlled by the FDA-approved rapamycin and doxycycline. Using ribosome profiling to characterize viral promoter strength, we rationally design fusions of the operator element of different drug-inducible systems with VV promoters to produce synthetic promoters yielding robust inducible expression with undetectable baseline levels. We also generate chimeric synthetic promoters facilitating additional regulatory layers for VV-encoded synthetic transgene networks. The switches are applied to enable inducible expression of fusogenic proteins, dose-controlled delivery of toxic cytokines, and chemical regulation of VV replication. This toolbox enables the precise modulation of transgene circuitry in VV-vectored oncolytic virus design. The large coding potential of vaccinia virus (VV) vectors is a defining feature. Here the authors present vaccinia virus vector-adapted chemogenetic switches enabling temporal and dose control of viral replication and transgene expression, implementation of complex tunable transgene circuitry, and generation of safer, more efficacious vectors.