Astrocytes and the tumor microenvironment inflammatory state dictate the killing of glioblastoma cells by Smac mimetic compounds.

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.

Tepilamide Fumarate as a Novel Potentiator of Virus-Based Therapy.

Oncolytic virotherapy, using viruses such as vesicular stomatitis virus (VSVΔ51) and Herpes Simplex Virus-1 (HSV-1) to selectively attack cancer cells, faces challenges such as cellular resistance mediated by the interferon (IFN) response. Dimethyl fumarate (DMF) is used in the treatment of multiple sclerosis and psoriasis and is recognized for its anti-cancer properties and has been shown to enhance both VSVΔ51 and HSV-1 oncolytic activity. Tepilamide fumarate (TPF) is a DMF analog currently undergoing clinical trials for the treatment of moderate-to-severe plaque psoriasis. The aim of this study was to evaluate the potential of TPF in enhancing the effectiveness of oncolytic viruses. In vitro, TPF treatment rendered 786-0 carcinoma cells more susceptible to VSVΔ51 infection, leading to increased viral replication. It outperformed DMF in both increasing viral infection and increasing the killing of these resistant cancer cells and other cancer cell lines tested. Ex vivo studies demonstrated TPF's selective boosting of oncolytic virus infection in cancer cells without affecting healthy tissues. Effectiveness was notably high in pancreatic and ovarian tumor samples. Our study further indicates that TPF can downregulate the IFN pathway through a similar mechanism to DMF, making resistant cancer cells more vulnerable to viral infection. Furthermore, TPF's impact on gene therapy was assessed, revealing its ability to enhance the transduction efficiency of vectors such as lentivirus, adenovirus type 5, and adeno-associated virus type 2 across various cell lines. This data underscore TPF's potential role in not only oncolytic virotherapy but also in the broader application of gene therapy. Collectively, these findings position TPF as a promising agent in oncolytic virotherapy, warranting further exploration of its therapeutic potential.

SARS-CoV-2 antigen-carrying extracellular vesicles activate T cell responses in a human immunogenicity model.

Extracellular vesicles (EVs) are entering the clinical arena as novel biologics for infectious diseases, potentially serving as the immunogenic components of next generation vaccines. However, relevant human assays to evaluate the immunogenicity of EVs carrying viral antigens are lacking, contributing to challenges in translating rodent studies to human clinical trials. Here, we engineered EVs to carry SARS-CoV-2 Spike to evaluate the immunogenicity of antigen-carrying EVs using human peripheral blood mononuclear cells (PBMCs). Delivery of Spike EVs to PBMCs resulted in specific immune cell activation as assessed through T cell activation marker expression. Further, Spike EVs were taken up largely by antigen-presenting cells (monocytes, dendritic cells and B cells). Taken together, this human PBMC-based system models physiologically relevant pathways of antigen delivery, uptake and presentation. In summary, the current study highlights the suitability of using human PBMCs for evaluating the immunogenicity of EVs engineered to carry antigens for infectious disease therapeutics.

Repression of mRNA translation initiation by GIGYF1 via disrupting the eIF3-eIF4G1 interaction.

Viruses can selectively repress the translation of mRNAs involved in the antiviral response. RNA viruses exploit the Grb10-interacting GYF (glycine-tyrosine-phenylalanine) proteins 2 (GIGYF2) and eukaryotic translation initiation factor 4E (eIF4E) homologous protein 4EHP to selectively repress the translation of transcripts such as Ifnb1, which encodes the antiviral cytokine interferon-ß (IFN-ß). Herein, we reveal that GIGYF1, a paralog of GIGYF2, robustly represses cellular mRNA translation through a distinct 4EHP-independent mechanism. Upon recruitment to a target mRNA, GIGYF1 binds to subunits of eukaryotic translation initiation factor 3 (eIF3) at the eIF3-eIF4G1 interaction interface. This interaction disrupts the eIF3 binding to eIF4G1, resulting in transcript-specific translational repression. Depletion of GIGYF1 induces a robust immune response by derepressing IFN-ß production. Our study highlights a unique mechanism of translational regulation by GIGYF1 that involves sequestering eIF3 and abrogating its binding to eIF4G1. This mechanism has profound implications for the host response to viral infections.

Loss of Dnajc21 leads to cytopenia and altered nucleotide metabolism in zebrafish.

Mutations in the DNAJC21 gene were recently described in Shwachman-Diamond syndrome (SDS), a bone marrow failure syndrome with high predisposition for myeloid malignancies. To study the underlying biology in hematopoiesis regulation and disease, we generated the first in vivo model of Dnajc21 deficiency using the zebrafish. Zebrafish dnajc21 mutants phenocopy key SDS patient phenotypes such as cytopenia, reduced growth, and defective protein synthesis. We show that cytopenia results from impaired hematopoietic differentiation, accumulation of DNA damage, and reduced cell proliferation. The introduction of a biallelic tp53 mutation in the dnajc21 mutants leads to the development of myelodysplastic neoplasia-like features defined by abnormal erythroid morphology and expansion of hematopoietic progenitors. Using transcriptomic and metabolomic analyses, we uncover a novel role for Dnajc21 in nucleotide metabolism. Exogenous nucleoside supplementation restores neutrophil counts, revealing an association between nucleotide imbalance and neutrophil differentiation, suggesting a novel mechanism in dnajc21-mutant SDS biology.

Octyl itaconate enhances VSVΔ51 oncolytic virotherapy by multitarget inhibition of antiviral and inflammatory pathways.

The presence of heterogeneity in responses to oncolytic virotherapy poses a barrier to clinical effectiveness, as resistance to this treatment can occur through the inhibition of viral spread within the tumor, potentially leading to treatment failures. Here we show that 4-octyl itaconate (4-OI), a chemical derivative of the Krebs cycle-derived metabolite itaconate, enhances oncolytic virotherapy with VSVΔ51 in various models including human and murine resistant cancer cell lines, three-dimensional (3D) patient-derived colon tumoroids and organotypic brain tumor slices. Furthermore, 4-OI in combination with VSVΔ51 improves therapeutic outcomes in a resistant murine colon tumor model. Mechanistically, we find that 4-OI suppresses antiviral immunity in cancer cells through the modification of cysteine residues in MAVS and IKKß independently of the NRF2/KEAP1 axis. We propose that the combination of a metabolite-derived drug with an oncolytic virus agent can greatly improve anticancer therapeutic outcomes by direct interference with the type I IFN and NF-κB-mediated antiviral responses.

Treatment of gliomas with combination of viral hemolytic virus and novel small molecule inhibitor / 中华神经医学杂志

Objective To study the in vitro killing effect of novel small molecule inhibitors, ribosomal S6 kinase1 (RSK1) inhibitor (BI-D1870) and polo-like kinase 1 (PLK1) inhibitor (BI2536), combined with recombinant attenuated vesicular stomatitis virus VSVΔM51 on various glioma cells. Methods (1) In vitro cultured GL261, CT2A and HS68 cells were divided into control group, rapamycin group, BI-D1870 group, BI-2536 group, VSVΔM51 group, rapamycin +VSVΔM51 group, BI-D1870+VSVΔM51 group, and BI2536+VSVΔM51 group; pretreatments with 100 nmol/L rapamycin, 10 μmol/L BI-D1870, and 100 nmol/L BI-2536 for 2 h were given to the cells from the above groups, respectively, and then, they were infected with VSVΔM51 virus at 0.1 mutiplicity of infection (MOI); at 72 h after treatments, the cell survival rate was determined by Alarma Blue method; VSV△M51 virus was infected at 10 MOI one h after pretreatment with the above drugs, apoptosis of GL261 cells was detected by cleaved caspase-3 staining 24 h after that; the expression of apoptotic protein polyadp-ribosomal polymerase (PARP) was detected by Western blotting; Annexin V-FITC/propidium iodide double staining was used to detect the cell apoptosis. (2) GL261 and CT2A cells were divided into VSVΔM51 group, rapamycin+VSVΔM51 group, BI-D1870+VSVΔM51 group, and BI2536+ VSVΔM51 group; VSV△M51 virus was infected at 0.1 MOI one h after pretreatment with the above drugs,; 48 h after treatments, fluorescence microscope was used to detect the expression of green fluorescent protein (GFP); IVIS200 in vivo imaging system was used to detect the changes of cell virus luciferase in the 4 groups. (3) Fifteen CT2A intracranial implanted glioma model mice were divided into VSVΔM51 group, BID-1870+VSVΔM51 group and BI2536+VSVΔM51 group according to random number table method (n=5); mice in the latter two groups were intraperitoneally injected with BI-1870 (100 mg/kg) or intravenously injected with BI-2536 (20 mg/kg); 24 h after that, mice in the three groups were intravenously injected with virus VSVΔM51; virus luciferase was detected by IVIS200 in vivo imaging system 24 and 72 h after treatments; the grouping and treatments of GL261 intracranial glioma model mice were the same as above, the expression of virus GFP was observed under fluorescence microscope 48 h after treatments, and virus titers of these mice were detected by virus plaque assay. Results (1) As compared with the control group, rapamycin group, BI-D1870 group, BI-2536 group, and VSVΔM51 group, the rapamycin+VSVΔM51 group, BI-D1870+VSVΔM51 group, and BI2536+VSVΔM51 group had significantly lower cell survival rate (P<0. 05); cleaved Caspase-3 staining showed no cell apoptosis in the control group, a small amount of apoptotic corpuscles in the rapamycin group, BI-D1870 group, BI-2536 group, and VSVΔM51 group, but obvious increased amount of apoptotic corpuscles in the rapamycin+VSVΔM51 group, BI-D1870+VSVΔM51 group, and BI2536+ VSVΔM51 group; Western blotting indicated that GL261 and CT2A cells from the control group, rapamycin group, BI-D1870 group, BI-2536 group, and VSVΔM51 group had lower cleaved PARP expression level than those from the rapamycin+VSVΔM51 group, BI-D1870+VSVΔM51 group, and BI2536+VSVΔM51 group. The results of Annexin V-FITC/propidium iodide double staining were consistent with those of cleaved Caspase-3 staining. (2) As compared with VSVΔM51 group and rapamycin+VSVΔM51 group, BI-D1870+VSVΔM51 group and BI2536+VSVΔM51 group had significantly increased GFP expression and statistically higher intensity of virus luciferase (P<0.05). (3) CT2A cells in the VSVΔM51 group, BID-1870+VSVΔM51 group and BI2536+VSVΔM51 group had increased intensity of virus luciferase successively, with significant differences (P<0.05); GL261 cells in the VSVΔM51 group, BID-1870+VSVΔM51 group and BI2536+VSVΔM51 group had increased virus titers successively, with significant differences (P<0.05). Conclusion Both small molecule inhibitors promote the replication of VSVΔM51 virus and enhance the killing effect on glioma cells, and its synergistic effect is obviously better than rapamycin.